Contents Section I: Biochemistry Chapter 1. Acid-Base Equilibrium and Buffering ........................... 3 Chapter 2...
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Contents Section I: Biochemistry Chapter 1. Acid-Base Equilibrium and Buffering ........................... 3 Chapter 2. Proteins, Enzymes, and Coenzymes ........................... 7 Chapter 3. Bioenergetics ............................................ 23 Chapter 4. Carbohydrates ........................................... 37 Chapter 5. Lipids .................................................. 63 Chapter 6. Amino Acid Metabolism ................................... 89 Chapter 7. Summary of Metabolism .................................. 103
Section II: Molecular Biology Chapter 1. Nucleic Acids ........................................... 115 Chapter 2. The Nucleus, the Cell Cycle, and Meiosis ..................... 123 Chapter3. DNA Replication and Repair ............................... 129 Chapter 4. Transcription ............................................ 135 Chapter 5. Protein Synthesis ........................................ 143 Chapter 6. Regulation of Gene Expression ............................. 151 Chapter 7. Genetic Engineering ...................................... 159
I KAPLAlf med lea
vii
Section III: Cell Biology Chapter 1. Overview of Cell Types .................................... 177 Chapter 2. Nucleus and Nucleolus ................................... 183 Chapter 3. Subcellular Organelles .................................... 187 Chapter 4. Cytoskeleton ........................................... 199 Chapter 5. Cellular Adhesion and Extracellular Matrix ..................... 205
Section IV: Genetics Chapter 1. Mendelian Inheritance .................................... 213 Chapter 2. Chromosomal Abnormalities ............................... 223 Chapter 3. Linkage ............................................... 237 Chapter 4. Population Genetics ...................................... 243
Section V: Microbiology Chapter 1. Introduction ............................................ 251 Chapter 2. Bacteriology Overview .................................... 255 Chapter 3. Bacteriology: Gram-Positive Cocci .......................... 267 Chapter 4. Bacteriology: Gram-Positive Bacilli .......................... 277 Chapter 5. Bacteriology: Gram-Negative Organisms ..................... 283 Chapter 6. Bacteriology: Anaerobes .................................. 299 Chapter 7. Bacteriology: Mycobacteria and Actinomycetes ................ 303 Chapter 8. Bacteriology: Rickettsiae and Chlamydiae .................... 309 Chapter 9. Bacteriology: Spirochetes ................................. 315 Chapter 10. Bacteriology: Mycoplasma and Ureaplasma ................. 321
viii
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----------- ----- - - - - - - -
Chapter 11. Virology .............................................. 323 Chapter 12. Mycology ., ........................................... 343 Chapter 13. Protozoa .............................................. 351 Chapter 14. Helminths ............................................ 359 Chapter 15. Antimicrobial Agents .................................... 365
Section VI: Immunology Chapter 1. Basic Immunology ....................................... 405 Chapter 2. Clinical Immunology ..................................... 449 Index ............. , .. , .......... " ...... , .. , .. " .......................... 483
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ix
SECTION I
Biochemistry
Acid-Base Equilibrium and Buffering Homeostasis requires that the pH of biologic fluids be maintained at a constant and characteristic value. The control of pH in body fluids and subcellular compartments is achieved by a number of biologic buffers. The most important buffers in physiologic systems are bicarbonate, phosphate, and proteins. Buffers consist of a weak acid and its conjugate base that are in equilibrium and are able to donate and accept protons from the surrounding medium. The relationship between pH, pKa, and the concentration of the conjugate acid and base of a buffering system is described by the HendersonHasselbalch equation. Disturbances in the acid-base balance can lead to acidosis (a plasma pH less than 7.35) or alkalosis (a plasma pH greater than 7.45). This chapter reviews the concepts underlying acid-base equilibrium, the pH of various body fluids, and the properties of the buffer systems that resist fluctuations in pH.
ACID-BASE EQUILIBRIUM The most appropriate biochemical definition for acids and bases is that acids are proton donors and bases are proton acceptors. Many important compounds found in biologic fluids, including amino acids, lactic acid, and acetoacetic acid, are weak acids. A. Properties of weak acids. Weak acids, in contrast to strong acids such as Hel or H 2S04, are not completely dissociated under the conditions that exist within biologic systems. The undissociated form (HA) that retains the proton is known as the conjugate acid, whereas the corresponding deprotonated form (A-) is a conjugate base that can accept a proton to recreate the acid. The reaction that describes the dissociation of a weak acid is shown in Figure I-l-l.
HA
~,============~'
conjugate acid
H+
+ conjugate base
Figure 1-1-1. Dissociation of a weak acid.
KAPLlIf . ._ I me 1C8
3
Biochemistry
B. The Henderson-Hasselbalch equation shown in Figure 1-1-2 describes the relationship
between pH and the concentration of a weak acid and its conjugate base. In this equation, pH is defined as the negative log of [H+], and pKa is defined as the negative log of the . . constant Ka' were h Ka = [H+] [A-] . For any weak'd K'IS a constant t h at · d IssoClatlOn [HA] aCl, th epa
Note
is characteristic of that particular ionizing group. The Henderson-Hassselbach equation is
A corollary of the equation: When the pH is greater than the pKa, the base (A-) form is dominant; when the pH is less than the PKa, the acid (HA) form is dominant.
useful because it provides a meaningful definition of pKa and permits calculation of the amounts of the undissociated acid and its conjugated base at any pH.
Figure 1-1-2. Henderson-Hasselbalch equation.
1. A functional definition of pKa can be deduced from the Henderson-Hasselbalch equation. The pKa of any ionizing group can be defined as the pH where the concentrations of the acid (HA) and its conjugate base (A-) are equal. When the pH is equal to the pKa of the acid, the concentrations of HA and A-are equal. Inspection of the equation indicates that when [HA] = [A-J, the last term of the equation becomes zero, and the equation reduces to pH = pKa. 2. The logarithmic nature of the Henderson-Hasselbalch equation indicates that small changes in pH will bring about large changes in the relative concentrations of HA and A-. These effects are summarized in Table I-I-I, where lactic acid, having a pKa of 3.8, is considered as an example of a weak acid. The same principles apply for any weak acid, i.e., a change of 1 pH unit results in a lO-fold change in the ratio of conjugate acid to conjugate base. Table 1-1-1. Effect of pH on the relative amounts of lactic acid and lactate. pH 1.8 12.8 pKa 3.8 4.8 5.8
[Lactic Acid] I [Lactate-] 100 101 1 0.1 0.01
TITRATION AND BUFFERING PROPERTIES OF WEAK ACIDS Titration can be defmed as the addition of a strong base to a weak acid (or a strong acid to a weak base) for the purpose of buffering the solution, i.e., maintaining a constant pH. A. Titration curve. The addition of a strong base such as sodium hydroxide to a weak acid may be written as:
4
KAPLA~. I meulca
Acid-Base Equilibrium and Buffering
As NaOH is added, the protons released by the weak acid combine with hydroxide to form water. The continued addition of sodium hydroxide progressively decreases the [H+J and increases the pH. A graph of the titration of a weak acid with a strong base is shown in Figure 1-1-3.
---------- ~--z-~--~h7'77T
Buffering range
HA
o
0.5
1.0
NaOH (equivalents) Figure 1-1-3. Titration curve for a weak acid.
At the midpoint in the titration, half of the acid has been neutralized and, therefore, [HAJ = [A-J. The Henderson-Hasselbalch equation shows that when [HAJ = [A-J, the pH of the solution is equal to the pKa of the weak acid. B. Buffering capacity. Buffers are mixtures of weak acids and their conjugate bases. The capac-
ity of a buffer, to resist change in pH is dependent on two factors: the concentration of the buffer, and the pH at which it is used. A buffer is most effective when it is used in a pH range near the pKa. It is clear from Figure 1-1-3 that in the pH range defined by 1.0 pH unit above and below the pKa' the addition of either strong acid or strong base will result in minimal change in the pH.
Note The best buffering occurs when pKa = pH, resulting in 50% of acid and 50% of base. This system can then handle either an addition of base or an addition of acid.
PHYSIOLOGICALLY IMPORTANT BUFFERING SYSTEMS The three most important buffering systems in biologic fluids are proteins, bicarbonate, and phosphate. The conjugate acid-base pairs and the pKa values for these buffers are summarized in Table 1-1-2. The major buffer in plasma and interstitial fluid is bicarbonate, whereas protein and organic phosphate esters are the major buffers of intracellular fluid. A. Protein buffering systems. The cytosol of cells contains high concentrations of proteins with amino acid side chains that are weak acids and bases. These side chains impart great buffering capacity to proteins. The acidic amino acids (glutamic and aspartic acids) and the basic amino acids (lysine, histidine, and arginine) all contain ionizable side chains (discussed in the next chapter). However, histidine is the only amino acid with good buffering capacity at physiologic pH. The imidazole side chain of histidine has a pKa that ranges from 5.6-7.0, depending on its microenvironment within the protein.
Bridge to Physiology Histidine is frequently found in the catalytic site of enzymes where the imidazole ring can donate or accept protons in the formation and breaking of bonds.
meCtical
5
Biochemistry
Table 1-1-2. Properties of major physiologic buffers. Buffering System Protein systems: Histidine side chains
5.6-7.0
Bicarbonate system: HC03-ICOz
6.1
Phosphate systems: HP042-/HzP04-
6.8
Organic phosphate esters
6.5-7.5
The buffering reaction for proteins is shown in Figure 1-1-4, in which the imidazole side chain of histidine can reversibly donate and accept protons. The presence of multiple histidine side chains within each protein molecule contributes to the buffering capacity of the protein. Histidine plays a key role in making hemoglobin an excellent buffer in red blood cells.
O rotein
Bridge to Physiology Acidosis, a plasma pH below 7.35, can result from the accumulation of acids such as lactic acid, acetoacetic acid, and B-hydroxybutyric acid (resulting in a metabolic acidosis), or from hypoventilation that results in the accumulation of CO 2 (respiratory acidosis). Alkalosis, a plasma pH above 7.45, can result from a loss of stomach acids due to excessive vomiting (metabolic alkalosis) or from hyperventilation (respiratory alkalosis). Acidosis and alkalosis are discussed in greater detail in the Respiratory Physiology and Renal Physiology sections.
6
KAPLAN"d_
me lea
I
CH
I
NH
=
CH
I
+ NH
H
+
+
orotein CH = I NH
"~
"
CH
CH
CH
I
N
~
Figure 1-1-4. Protein buffering system. B. Bicarbonate buffering system. The bicarbonate-C0 2 system is the most important buffer in
maintaining the pH of blood plasma and interstitial fluid at its normal value of 7.4. Figure I -1-5 summarizes the properties of this buffering system. Carbonic acid (H 2 C03 ) is the proton donor, bicarbonate anion (HC0 3-) is the proton acceptor, and the pKa for this reaction is 6.1. The strength of this buffering system lies in the ability of carbonic acid to be converted to carbon dioxide.
pKa = 6.1
Figure 1-1-5. The bicarbonate/C0 2 buffering system.
C. Phosphate buffering system. Intracellular fluids contain high concentrations of inorganic
phosphate and many organic phosphate esters that contribute significantly to the buffering power of the cytosol. The phosphate buffering system is of little importance in plasma and interstitial fluid because of the low concentrations of phosphates in extracellular fluids. The phosphate buffering system consists of H 2 P0 4- as the proton donor and HPoi- as the proton acceptor. The pKa of 6.8 is sufficiently close to the normal intracellular pH to make it an ideal buffer in those fluids that contain high concentrations of phosphates, such as red blood cells and kidney tubules.
- - - - - - - - - -
-------
- - - -
Proteins, Enzymes, and Coenzymes Proteins are linear polymers of amino acids, each having distinct chemical and structural properties. They are the largest class of molecules found in living systems and are responsible for most of the diverse functions of the cell. They act as transport proteins, storage proteins, hormone receptors, structural proteins, regulatory proteins, and enzymes. Each protein has a unique amino acid sequence that determines its precise three-dimensional conformation and specifies its function. The most versatile class of proteins is enzymes, which act as biologic catalysts, enhancing the rate of chemical reactions occurring in cells. Most enzymes catalyze only one reaction, and their catalytic activities may be stringently regulated, allowing the cell to control and coordinate its metabolic pathways. The kinetic parameters, Km and Vmax, are constants that describe the catalytic properties of enzymes. The function of many enzymes requires a cofactor-a metal or a small organic coenzyme derived from a vitamin precursor. This chapter reviews the structure and properties of amino acids that are found in proteins, the kinetic and regulatory properties of enzymes, and the relationship between coenzymes and their vitamin precursors.
AMINO ACIDS: THE BUILDING BLOCKS OF PROTEINS A. Amino acid structure. The building blocks for proteins are the 20 common amino acids that are encoded in the DNA of the cell. 1. General structure. Nineteen of the twenty common amino acids can be represented by the structure shown in Figure 1-2-1. They all have a central carbon atom attached to a carboxyl group, an amino group, and a hydrogen atom. The amino acids differ from one another only in the chemical nature of the side chain (R). The only amino acid that is not described by this structure is proline, an imino acid in which the side chain forms a cyclic structure with the amino group (Figure 1-2-2). NH2
I R-Ca-COOH I H
In a Nutshell Amino Acids with Hydrophobic Side Chains
Figure 1-2-1. General structure of a-amino acids.
2. Classification. The amino acids can be classified as either hydrophobic or hydrophilic, depending on the ease with which their side chains interact with water. This particular method of classification is especially useful when considering amino acids as the building blocks of proteins. The properties of amino acid side chains influence how the protein folds into more compact structures. In general, proteins fold so that the hydrophobic side
Aliphatic Ala Val Leu lie Pro
Aromatic Phe Tyr Trp
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7
Biochemistry
In a Nutshell
chains are in the interior of the molecule, where they are protected from water, and the hydrophilic side chains are on the surface.
Amino Acids with Hydrophilic Side Chains Positive Negative Lys Asp Arg Glu His
a. Hydrophobic amino acids have side chains with aliphatic groups or aromatic ring structures (Figure 1-2-2).
Neutral Ser
b. Hydrophilic amino acids have side chains that contain 0, N, or S atoms. Some of the hydrophilic side chains are charged at physiologic pH. The acidic amino acids (aspartic and glutamic acids) have carboxyl groups that are negatively charged, whereas the basic amino acids (lysine, arginine, and histidine) have nitrogen atoms that are positively charged. Other hydrophilic amino acids with uncharged side chains contain hydroxyl groups (serine and threonine), a sulfur atom (cysteine), or amide groups (asparagine and glutamine). The structures of the R groups for the hydrophilic amino acids are shown in Figure 1-2-3. Methionine, tyrosine, cysteine, and glycine may be found either in the interior or on the surface of globular proteins.
Thr Cys Met Asn Gin
Nonpolar, Aliphatic Side Chains
coo· + I H3N -C-H
GJ Glycine
coo· I
H3N+-C --ti
coo· I
H3N+-C-H
~ Alanine
CH2
Leucine
+
H3N~-H
CH CH3/
'CH3
Valine
H3N -C -H
COO· +
I
H3N -C-H
oQ OH
Phenylalanine
I
I
CH3 Isoleucine
Proline
Figure 1-2-2. The hydrophobic amino acids.
8
I
H3~-C-H
CH
"CH 3
coo·
I
I
H-C-CH3
CH 3/
coo·
coo·
CH2
I
Aromatic Side Chains
liP LIN"iIme leaI
- - - - - - - - -
Tyrosine
Tryptophan
Proteins, Enzymes, and Coenzymes
Positively charged R groups
Polar, uncharged R groups
coo +
coo
I
+
H3 N - C -H
I
H3N -C-H
I
I
CH 2
H-C-OH
CH 2
CH 3
I
I
I
CH 2
I
Serine
Threonine
Cysteine
Methionine
Asparagine
Glutamine
NH
I
+
C=NH 2
I
NH2 Lysine
Arginine
Histidine
Negatively charged R groups
coo +
I
H3 N - C-H
I CH 2
I
COO
Aspartate
Glutamate
Figure 1-2-3. The hydrophilic amino acids.
3. Modified amino acids. In addition to the 20 common amino acids that are encoded by DNA, proteins often contain other amino acids that are generated by modification of the common amino acids after the protein has been synthesized. This process is called posttranslational modification. a. Cystine is formed in proteins by the reaction of two cysteine side chains to form a disulfide linkage. It is found most frequently in extracellular proteins where the disulfide bond stabilizes the three-dimensional structure of the protein. b. Hydroxyproline is formed in an oxygen-dependent hydroxylation reaction that occurs in t1broblasts. It is found in collagen, where it stabilizes the triple helical structure. c. Phosphotyrosine, phosphoserine, and phosphothreonine are formed by transferring
phosphate from ATP to the hydroxyl group of serine, tyrosine, or threonine. These amino acids are found in many enzymes and proteins, where they serve as regulatory signals.
In a Nutshell • S-S bridges:
Cystine
• Collagen triple helix:
Hydroxyproline
Phosphotyrosine • Signal transduction: Phosphoserine Phosphothreonine
meCtical
9
Biochemistry
B. Properties of amino acids. The properties of proteins are influenced by the properties of
Note There are other amino acids, like ornithine and citrulline, that play important roles in the cell but that are not building blocks for protein synthesis.
their constituent amino acids. In particular, the stereochemistry and ionic properties of amino acids have an impact on the structure and/or properties of the protein. 1. Stereochemistry. The a-carbon atom of all amino acids except glycine is linked to four
different chemical groups, making the a-carbon atom an asymmetric center. As shown in Figure 1-2-4, an asymmetric center has two stereoisomers (enantiomers) that are mirror images of each other and are designated as D- and L-amino acids. Only L-amino acids are incorporated into proteins.
coo-
cooI
I
H-C- NH3+
I
+H N-C-H
I
3
CH3
CH 3 D-alanine
L-alanine
Figure 1-2-4. Stereoisomers of an a-amino acid.
2. Ionic properties. Depending on the pH, an amino acid may have no net charge, or it may have either a positive or a negative charge. The pH at which a molecule is electrically neutral is defined as the isoelectric point (pI). The equilibrium between the ionic forms of an amino acid is shown in Figure 1-2-5. The pKa values for the a-carboxyl and a-amino groups are approximately 2.1 and 9.8, respectively. At a pH below the pKa' the protonated species predominates, whereas at a pH above the pKa' the deprotonated species predominates. The pI for the amino acid shown in Figure 1-2-5 is the average of the two pKa values. At neutral pH, the species that has both positive charge and negative charge is called a zwitterion.
+NH3
+NH3 NH2 pK a = 2.1 I pK a 9.8 \ ....R -CH -COO ~ ...==~"" R-CH-COOR -CH -COOH ...
I
Net charge = + 1 pH = 1
Net charge = 0 pH = 7
Net charge = -1 pH = 11
Figure 1-2-5. The ionic equilibrium for an a-amino acid.
The acidic and basic amino acids also have ionizing groups in their side chains. Aspartic and glutamic acids have side chain carboxyl groups with a pKa of approximately 4.0 and are negatively charged at physiologic pH. Lysine and arginine have side chains with protonated nitrogen atoms having pKa values of approximately 10 and 12, respectively, and are positively charged at physiologic pH. Histidine has an imidazole side chain with a pKa of approximately 6.0, a value sufficiently close to the physiologic pH to allow some of the histidine side chains to be positively charged, whereas others have no charge. The ratio of positively charged histidine side chains to uncharged side chains at any pH can be calculated from the Henderson-Hasselbalch equation (described in the previous chapter).
10
KAPLAlfdme leaI
Proteins, Enzymes, and Coenzymes
PROTEINS Each protein has a unique three-dimensional structure dictated by its amino acid sequence and is responsible for the highly specific function of the protein. A. Structural features. The amino acids in a protein are linked together by peptide bonds in which the a-carboxyl of one amino acid is linked to the a-amino group of another amino acid (Figure 1-2-6). The peptide (amide) bond has partial double-bond character, making it planar and rigid, thereby imposing limitations on higher orders of structural organization in a protein. 1. Primary structure. The sequence in which the amino acids occur in the polypeptide is
defined as the primary structure. This sequence is encoded by the DNA, and it determines how the protein folds into a more compact three-dimensional structure. 2. Native conformation. The final structure that a particular protein assumes proceeds through several levels of organization. In contrast to the primary structure, which is stabilized by strong covalent bonds, higher orders of structure are stabilized by numerous weak non covalent interactions.
+
H[JR I
H N-C 3
I
R
II
C-NH
0
I
II
CHlEJNH CH-C-COO-
II
I
0
R
Figure 1-2-6. The peptide bond.
a. Secondary structure in proteins is organized around the polypeptide backbone and is stabilized by large numbers of hydrogen bonds formed between the amide hydrogen atom of one peptide bond and the carbonyl oxygen atom of another. Proteins contain two major types of secondary structure: a-helical structure, which is stabilized by intrachain hydrogen bonds, and 13-sheet structure, which is stabilized by interchain hydrogen bonds. In some fibrous proteins, the highest order of structure is secondary. These proteins are elongated asymmetrical proteins that frequently function as structural components of cells and tissues. Simple combinations of a few secondary structural elements are called motifs and are often repeated multiple times in proteins. An example of a motif would be a 13-a-13 loop motif that can be used to connect two parallel13 strands. The a-helix in the middle of the two 13 strands allows for the reversal of direction. b. Tertiary and quaternary structure. Segments of secondary structure in globular proteins associate with one another and fold into a tertiary structure that is stabilized by non covalent interactions between amino acid side chains. The fundamental unit of tertiary structure is the domain, a part of the polypeptide chain that folds into a stable tertiary structure and is encoded by an exon in DNA. Proteins that are composed of more than one polypeptide chain have quaternary structure. Both tertiary and quaternary structures are stabilized by ionic and hydrophobic interactions and by hydrogen bonding between side chains. 3. Denaturation is defined as the loss of native conformation, resulting in a random coil that has little, if any, of the biologic activity of the native protein. Denaturation may be caused by heat, extremes in pH, or detergents.
In a Nutshell 1° structure: • Amino acid sequence
2° structure: • a-helix
• 13-sheet 3° structure: • Overall protein
structure • How a-helix and 13-sheet fold with respect to each other 4° structure: • Mutiple polypeptide chains
• How chains fold with
respect to each other
meClical
11
Biochemistry
B. Function. Proteins perform most of the important functions of the cell. Many of the pro-
teins in skin, bone, muscle, and hair perform structural roles. Other proteins carry out the dynamic functions of the cell. Many plasma proteins transport small molecules such as iron or oxygen; immunoglobulins and clotting factors participate in defense functions; hormones, hormone receptors, and transcription factors play critical regulatory roles in the cell; and enzymes function as biologic catalysts.
ENZYMES Virtually all biologically important reactions are catalyzed by enzymes. Most of these reactions occur under very mild conditions of temperature and pH that are compatible with living organisms. In the absence of enzymes, reactions in the cell would proceed at insignificant or undetectable rates. Enzymes differ from inorganic catalysts in their specificity, catalytic efficiency, and regulatory properties. A. Classification. Enzymes are divided into six different classes on the basis of the types of reactions they catalyze (Table 1-2-1). All of the thousands of different reactions occurring in cells fall into one of these six types of reactions. B. Specificity. The high specificity of enzyme-catalyzed reactions occurs because enzymes have
active sites composed of a small number of amino acid side chains. The side chains come together to form a three-dimensional site on the surface of the enzyme that is complementary to the structure of the substrate. Some of the amino acid side chains in the active site participate in binding the substrate to the enzyme; others act as catalytic groups and enhance the rate of the reaction by acting as acids, bases, or nucleophiles.
Table 1-2-1. The six classes of enzymes.
12
KAPLAlfdme leaI
Class
Type of Reaction
Oxidoreductases
Oxidation-reduction reactions Frequently use coenzymes NAD+, FAD, NADP+' or O 2 as electron acceptors (dehydrogenase, oxidase, reductase)
Transferases
Transfer of a chemical group from a donor to an acceptor Groups transferred include amino, carboxyl, acyl, glycosyl, phosphoryl (transaminase, kinase)
Hydrolases
Cleavage of a bond between carbon and some other atom by the addition of water (protease, phosphatase, amylase)
Lyases
Nonhydrolytic cleavage of carbon-carbon, carbon-sulfur, and some carbon-nitrogen bonds (aldolase, decarboxylase, dehydratase)
Isomerases
Interconversion of isomers (epimerase, mutase)
Ligases
Formation of bonds between carbon and oxygen, nitrogen, or sulfur atoms in reactions that require energy (carboxylase, thiokinase)
Proteins, Enzymes, and Coenzymes
C. Catalytic properties. The energy profile of a chemical reaction, as it proceeds from substrate
to product, is shown in Figure 1-2-7. The highest point on the curve is the energy of the transition state, an intermediate whose properties resemble both the substrate and the product. The energy barrier created by the transition state is also known as the activation energy (Act) of the reaction. To initiate the reaction, energy must be expended to overcome the energy barrier. The rate of a reaction is inversely proportional to the magnitude of the activation energy. As shown by the dotted line in Figure 1-2-7, enzymes increase the rate of a reaction by decreasing the activation energy barrier. They have no effect on the equilibrium constant for the reaction, which is related to ~G, the energy difference between the products and substrates.
Transition state
~-.-__ ~_ ~ minus enzyme
>OJ .....
\3s
Q)
~
~-------
" -------------- - --
.... ;
Q)
c
~G* = G TS - G s
/",-
Q)
"
LL
~G
plus enzyme
= Gp-G s
Reaction progress Figure 1-2-7. Energy profile for a catalyzed and uncatalyzed reaction.
D. Kinetic properties of enzymes. Kinetics is the study of the rate at which chemical reactions occur. 1. Factors affecting the rate of enzyme-catalyzed reactions include temperature, pH, enzyme concentration, and substrate concentration. Typical rate responses to these factors are shown in Figure 1-2-8. a. Temperature. The rate of most reactions increases approximately twofold with a 1O.ODC increase in temperature. For enzyme-catalyzed reactions, however, there is an optimum temperature beyond which the rate rapidly decreases due to denaturation of the enzyme. b. pH. The optimal activity of most enzymes occurs between pH 5 and 9. The shape of the rate-versus-pH curve reflects different ionization states for specific amino acid side chains that are required for substrate binding or for catalysis.
mectical
13
Biochemistry
Mnemonic STEP up the reaction rate: Substrate concentration Temperature Enzyme concentration
c. Enzyme concentration. The rate of an enzyme-catalyzed reaction is directly proportional to the concentration of enzyme provided the substrate is present in concentrations sufficient to saturate the binding sites. d. Substrate concentration. At very low concentrations of substrate, first -order kinetics are observed, with the rate being directly proportional to [S]. When the concentration of substrate is sufficiently high that all of the binding sites are occupied, zero-order kinetics are seen, with the rate being independent of [5]. 2. The Mic:haelis-Menten equation is an expression that quantifies the relationship between the rate of an enzyme-catalyzed reaction and the substrate concentration. In deriving an equation for the curve shown in Figure 1-2-SD, it is assumed that the limiting rate observed at an infinitely high substrate concentration is due to a finite number of sites on the enzyme and that when all of the sites are occupied, the maximum rate is obtained. The Michaelis-Menten expression for velocity is illustrated by the following equation:
pH
In this expression, Vmax and Km are constants that are characteristic of the enzyme. They are defined as follows: a. The maximum velocity, Vmax' is the rate obtained when all of the enzyme is present as an E·S complex, with substrate bound to the active site. The Vmax increases as the concentration of enzyme increases.
In a Nutshell • Vmax: theoretical rate when all enzymes are working
• Km: substrate concentration at which the reaction is running at half of the maximum rate (where V= 1/2 Vmax>
b. The Km is the substrate concentration that is required to achieve half of the maximum velocity and is independent of the enzyme concentration. These relationships are shown in Figure 1-2-9. Rough estimates of the Vmax and Km values can be obtained from this figure. However, values for both Vmax and Km obtained from this type of graph are inherently subject to large errors because of the shape of the curve; the value for Vmax must be extrapolated from an infinitely large value for [S]. Much more accurate values of these two kinetic parameters can be obtained from secondary plots, as described below.
A.
>
~
B.
0
~
Temp C.
pH
D.
zer~ order
> Q)
iii '-
~order Enzyme Concentration
Substrate Concentration
Figure 1-2-8. Factors affecting the rate of enzyme-catalyzed reactions.
14
KAPLA~.
I
meulCa
Proteins, Enzymes, and Coenzymes
Vm~r-------------------~====~
t
>
0.5
Vm~ 1----7/'
[Sl~
Figure 1-2-9. Dependence of rate on substrate concentration.
3. Lineweaver-Burk plot. A more useful graph for obtaining values of Km and Vmax can be obtained by taking the reciprocal of the Michaelis-Menten equation and rearranging it to give the Lineweaver-Burk equation: 1
Km
1
1
V
Vmax
5
Vmax
Note Remember that the equation for a straight line is:
-=--e-+--
As shown in Figure I-2-lO, this equation describes a straight line when IN is plotted against 1/[S]. The slope of the line is equal to KmNmax, the intercept of the line on the y-axis is equal to l/Vmax' and the intercept on the x-axis is equal to -l/Km.
y=mx+b, where m = slope and b = y-i ntercept.
In a Nutshell Lineweaver-Burk Plot x-axIs: y-axIs: slope: x-intercept: y-intercept:
o
1
[8]
Note • Km
Figure 1-2-10. Lineweaver-Burk plot.
4. Significance of Km and Vmax' Each of these kinetic constants reflects an intrinsic property of the enzyme.
l/[S] 1/V = l/rate Km/Vmax -l/Km l/Vmax
oc
l/affinity
. .i Km ~ i
substrate
affinity Km ~ .i substrate affinity
· i
meClical
15
Biochemistry
a. Km is related to the affinity of the enzyme for the substrate. The lower the value for Km , the higher the affinity of the enzyme for the substrate, and vice versa. Frequently, the Km value of an enzyme for its substrate is approximately equal to the substrate concentration found in the cell. b. Vmax is related to the efficiency with which substrate is converted to product when all of the active sites are occupied. Some enzymes, such as catalase, are highly efficient, with the rate of product formation approximating the rate of substrate binding; others, like RNA polymerase, are much less efficient at converting substrate to product. E. Inhibitors of enzyme activity. Much of toxicology and pharmacology is based on the principles of enzyme inhibition. Many metabolic poisons, such as insecticides and nerve gases, are enzyme inhibitors. Similarly, many drugs are inhibitors of key enzymes in metabolic pathways and often have structures similar to the normal enzyme substrate. There are two large classes of enzyme inhibitors: reversible and irreversible. 1. Reversible inhibitors alter the kinetic properties of an enzyme by binding noncova-
In a Nutshell Competitive Inhibition
• Vmax is unchanged • Slope increases • y-intercept is unchanged
lently to the enzyme through multiple interactions with amino acid side chains. The effect of a reversible inhibitor is removed by its dissociation from the enzyme. There are three types of reversible inhibitors: competitive, noncompetitive, and uncompetitive. a. Competitive inhibitors are structural analogs of the substrate and compete with the substrate for binding to the active site. The effect of a competitive inhibitor can be overcome by increasing the concentration of substrate. The Km for the substrate is increased (i.e., because both inhibitor and substrate are competing for the same site, more substrate is needed to reach half-maximum velocity). Vmax remains unchanged. Competitive inhibitors are easily identified by Lineweaver-Burk plots (Figure 1-2-11), in which the slope is increased but the intercept on the 1IV axis is unchanged. The intercept on the x-axis is shifted closer to zero (corresponding to an increase in Km).
• x-intercept shifts to the right
In a Nutshell Noncompetitive Inhibition • Km is unchanged
• -tV max • Slope increases • y-intercept shifts upward • x-intercept is unchanged
16
meclical
o
1
[8]
Figure 1-2-11. Lineweaver-Burk plot of competitive inhibition.
b. Noncompetitive inhibitors bind to some site other than the active site and are not structural analogs of the substrate. The Vmax of the reaction is decreased, but the Km for the substrate remains unchanged. The effect of a noncompetitive inhibitor cannot be overcome by increasing the substrate concentration. In a Lineweaver-Burk plot (Figure 1-2-12), the intercept on the 1IV axis is increased (corresponding to a decrease in Vmax ), and the intercept on the 11[5] axis is unchanged.
Proteins, Enzymes, and Coenzymes
In a Nutshell Uncompetitive Inhibition •
Km and Vmax are changed
• Slope remains the same • x-intercept shifts to the left
o
1
• y-intercept shifts upward
[8] Figure 1-2-12. Lineweaver-Burk plot of noncompetitive inhibition.
c. Uncompetitive inhibitors bind directly to the enzyme substrate complex but not to
free enzyme. This binding causes a conformational change at the active site that renders the enzyme inactive. Both Vmax and Km are changed. Both Vmax and Km decrease, but the slope of the inhibited reaction is parallel with the slope of the uninhibited reaction (Km/Vmax)' Lineweaver-Burk plots at various concentrations of the inhibitor will be a series of parallel lines. 2. Irreversible inhibitors covalently bind to the enzyme, resulting in permanent inactivation of the enzyme. Some irreversible inhibitors are referred to as "mechanism-based inhibitors" or "suicide substrates" because they bind to the active site and mimic the enzyme by beginning to undergo catalysis; however, the catalytic cycle is never completed because they become covalently linked to the enzyme. The effect of irreversible inhibitors on the kinetic parameters of an enzyme is identical to that of a noncompetitive inhibitor. F. Regulation of enzyme activity. Homeostasis requires that the rate of metabolic pathways be carefully regulated and coordinated with one another. This is typically achieved by the regulation of one or two key enzymes in each pathway. The key enzymes that are regulated usually catalyze either the rate-limiting reaction in the pathway or a reaction that is essentially irreversible. There are three major mechanisms for controlling the activity of enzymes. 1. Allosteric regulation. The activity of allosteric enzymes is regulated by the reversible bind-
ing of an effector molecule to a site other than the active site. Substrate saturation curves for allosteric enzymes are usually sigmoidal (Figure 1-2-13). Allosteric effectors can be either positive (activators) or negative (inhibitors) and act by altering either the K m , Vmax' or both. Activators either decrease the Km or increase the Vmax' whereas inhibitors increase the Km or decrease the Vmax' Common types of effectors include end products of pathways (heme, cholesterol, purine, and pyrimidine nucleotides) or molecules that reflect the energy state of the cell (ATP, ADP, AMP, NADH, NAD+, acetyl-CoA).
Clinical Correlate Lead toxicity is caused by lead's inhibitory effect on a number of enzymes having essential sulfhydryl groups in their active sites. The sulfhydryl groups react covalently with lead, resulting in irreversible inactivation of the enzyme. Two enzymes in the pathway of heme synthesis (aminolevulinic acid dehydratase and ferrocheletase) are inactivated by low levels of lead, resulting in decreased synthesis of heme and hemoglobin and increased excretion of aminolevulinic acid. Treatment with chelators such as penicillamine decreases the toxicity by forming nontoxic complexes with lead.
In a Nutshell Allosteric Regulation • Activators: J, Km or i Vmax
i
Km or
J, Vmax
KAPLA~. I meulca
17
• Inhibitors:
Biochemistry
Clinical Correlate Growth factors (i.e., hormones) activate receptors via tyrosine phosphorylation; the newly phosphorylated tyrosine can induce signal transduction in the cell. In many cancers, this pathway is often disregarded and the growth signal is always on, causing unregulated cell proliferation.
v
Figure 1-2-13. Substrate saturation curve for an allosteric enzyme.
Clinical Correlate Patients with chest pain who are suspects for acute myocardial infarction (AMI) are evaluated by EKG and serial measurements of serum creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) isoenzymes. The CPKMB isoenzyme is a marker for cardiac tissue damage. In patients with AMI, the tissue damage results in a transient increase of CPK-MB measurable 6-8 hours after the infarction, peaking between 12 and 24 hours, and returning to normal within 48-72 hours. If the patient is admitted 24 hours after the AMI, LDH isoenzyme analysis is performed. Following an AMI, the total serum LDH is elevated and the relationship between LDH1 and LDH2 is inverted, so that LDH1 > LDH2. The change in LDH isoenzymes occurs within 24-36 hours post-AMI.
18
meClical
2. Covalent modification. The activity of many enzymes is regulated by a phosphorylation/dephosphorylation cycle in which a specific serine, threonine, or tyrosine side chain becomes modified (Figure 1-2-14). The addition and removal of the phosphate group are catalyzed by a family of protein kinases and protein phosphatases that respond to hormonal stimulation of cells. Phosphorylation can increase or decrease the activity of an enzyme, or it can alter the regulatory properties.
Protein kinase
0
00-p~=
0H
Altered activity Protein phosphatase
Figure 1-2-14. Regulation of enzyme activity by covalent modification.
3. Induction and repression of enzyme synthesis. Because enzyme activity is directly proportional to the amount of enzyme present, one way to regulate enzyme-catalyzed reactions is to alter the rate at which enzymes are synthesized. This type of regulation is frequently mediated by steroid or thyroid hormones that act in the nucleus to increase or decrease the rate of transcription, and, secondarily, protein synthesis. G. Isoenzymes. Isoenzymes are different proteins that catalyze the same reaction but have different catalytic and regulatory properties and frequently differ in tissue and/or organelle specificity. The appearance of tissue-specific isoenzymes in plasma is of diagnostic value in identifying sites of tissue damage.
Proteins, Enzymes, and Coenzymes
COENZYMES AND VITAMINS Most enzymes that catalyze transfer reactions or oxidation/reduction reactions require a coenzyme that serves as an intermediate carrier of some specific functional group. Coenzymes are small organic molecules that are more stable than proteins. Coenzymes are derived from vitamins. Vitamins cannot be synthesized de novo by human tissues and must therefore be supplied by the diet. In addition to dietary sources, some vitamins are also synthesized by bacteria normally present in the intestinal flora. Vitamins may be classified as either water soluble or fat soluble. All of the water-soluble vitamins and some of the fat-soluble vitamins serve as precursors for coenzymes. Other fat-soluble vitamins control the rate of transcription of specific genes, thereby influencing the rate of enzyme-catalyzed reactions by altering the amount of enzyme present in the cell. A. Water-soluble vitamins 1. Niacin, also known as vitamin B3 or nicotinic acid, is present in whole grains, meat, and
nuts. Up to 50% of the body's niacin supply can be derived from the amino acid tryptophan. Niacin is converted to nicotinamide, which is then incorporated into the coenzymes NAD+ and NADP+. These coenzymes are very important in both lipid and carbohydrate metabolism, where they act as carriers of hydride ions (two electrons and a proton) in oxidation and reduction reactions. NAD+ is generally involved in oxidative, catabolic pathways and is more concentrated in mitochondria than in cytosol; NADPH is used in reductive, anabolic pathways and is found primarily in the cytosol. 2. Riboflavin, vitamin Bz, is present in organ meat, whole grains, and dairy products. The two coenzymes derived from riboflavin are FMN and FAD, both of which act as carriers of hydrogen atoms in oxidation and reduction reactions. These coenzymes are important in the oxidation of carbohydrates, lipids, and amino acids and are found primarily in the mitochondria. Human riboflavin requirements increase with increased protein use during growth periods, pregnancy, lactation, and wound healing. Patients with a deficiency of riboflavin develop lesions of the lips, mouth, skin, and genitalia. 3. Thiamine, vitamin Bl> is present in meat, beans, peas, and grains. The coenzyme derived
from thiamine is thiamine pyrophosphate, which functions in oxidative decarboxylation of a-ketoacids. The four major enzymes requiring thiamine pyrophosphate are pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, branched-chain a-keto acid dehydrogenase, and transketolase. 4. Pyridoxine, vitamin B6, is present in many foods, including most meats and vegetables. The coenzyme derived from this vitamin is pyridoxal phosphate, which acts as a carrier of amino groups in transamination, decarboxylation, racemization, and dehydration reactions. Pyridoxine deficiency may develop during pregnancy, alcoholism, with prolonged exposure to isoniazid or penicillamide therapy, and in women on oral contraceptives. Both a deficit and excess of pyridoxine may lead to peripheral neuropathy and dermatitis.
Clinical Correlate Side effects of exogenous niacin use include vasodilatation, flushing, and itching. Deficiency results in pellagra, producing the clinical triad of the three Os: diarrhea, dermatitis, and dementia. (If untreated, a fourth possible "0" is death.)
Clinical Correlate Mild thiamine deficiency leads to peripheral neuropathy (dry beriberi). More severe vitamin depletion leads to high-output cardiac failure (wet beriberi) and the dementia, ataxia, and ophthalmoplegia of the W~nicke-Korsakoff syndr~ll}e
frequently seen in chronic alcoholics.
5. Pantothenic acid is widely distributed in foods, particularly in meats and grains. The coenzyme derived from this vitamin is coenzyme A, which acts as a carrier of acyl groups and is particularly important in lipid metabolism. The key feature of this coenzyme is a sulfhydryl group that forms a high-energy thioester linkage with the carboxyl group of fatty acids. Pantothenic acid deficiency is rare. 6. Biotin is present in many foods, including liver, grains, and eggs. It is also synthesized by intestinal bacteria. The conversion to a coenzyme simply requires that it be covalently linked to the appropriate enzymes. Biotin acts as a carrier of "activated carboxyl" groups for three key enzymes that catalyze carboxylation reactions. The enzymes and the pathways they participate in are: pyruvate carboxylase (gluconeogenesis), acetyl-CoA
meClical
19
Biochemistry
Clinical Correlate Folic acid deficiency is most commonly seen in pregnancy and alcoholism. Deficiency states can result in megaloblastic anemia and neural tube defects. Supplementation is recommended before and during pregnancy.
Clinical Correlate Vitamin C deficiency results in scurvy, characterized by poor wound healing and bone formation, gum changes, and petechiae.
carboxylase (fatty acid synthesis), and propionyl-CoA carboxylase (branched-chain amino acid catabolism). Biotin deficiency is rare. Excessive consumption of raw egg impairs biotin absorption because of the presence of a biotin-binding protein, avidin, in egg whites. Antibiotics that alter the intestinal flora can also lead to biotin deficiency. Symptoms of deficiency include alopecia, skin and bowel inflammation, and muscle pain. 7. Folic acid is present in liver, fresh fruit, and leafy green vegetables. The coenzyme form of this vitamin is tetrahydrofolic acid (THF), which acts as a carrier of one-carbon fragments in metabolism. THF carries one-carbon units at all stages of oxidation except carboxyl groups. The coenzyme is important in amino acid catabolism and in the synthesis of purines and pyrimidines. The donors of the one-carbon units are serine, histidine, glycine, and tryptophan; the acceptors are intermediates in the pathways of purine and pyrimidine nucleotide synthesis. 8. Vitamin C, or ascorbic acid, is found in fresh fruits and vegetables. This vitamin exists in both oxidized and reduced forms, with the reduced form being the active form. Vitamin C facilitates iron absorption from the gut and is also required in a number of hydroxyl ation reactions, including proline and lysine hydroxylation for collagen synthesis and dopamine hydroxylation in catecholamine synthesis. Vitamin C also serves as an antioxidant in cells. 9. Vitamin B12, or cobalamin, is synthesized exclusively by microorganisms but is conserved in animal tissues, where it is found in high concentrations in the liver and kidney. It is required as the coenzyme for two reactions in human biochemistry: the methylation of homocysteine to methionine, and the conversion of methylmalonyl-CoA to succinylCoA. The absorption of vitamin B12 requires intrinsic factor, a protein synthesized by parietal cells of the gastric mucosa. A deficiency of vitamin B12 is rare, with the most common cause being a defect in absorption. A deficiency of vitamin B12 leads to megaloblastic anemia, with or without an associated neuropathy due to widespread demyelination. B. Fat-soluble vitamins
Clinical Correlate
1. Vitamin A is supplied by several sources, including yellow and orange vegetables, liver, milk, and eggs. Vitamin A exists in three forms: retinal, retinol, and retinoic acid. Retinal acts as a cofactor for the protein opsin to form a rhodopsin complex, which acts as the light receptor in the visual process. Retinol and retinoic acid are required for growth, differentiation, and maintenance of epithelial cells; they bind to nuclear receptors and regulate the rate of transcription of specific genes, including the gene for keratin. Patients with fat malabsorption or celiac disease may become vitamin A deficient, producing night blindness and dryness of the conjunctiva, cornea, and skin (hyperkeratosis). Excessive amounts of this vitamin are also toxic to humans, producing joint pain, headache, and long-bone thickening.
A deficiency of vitamin D occurs with lack of sunshine or renal failure and leads to rickets in children and osteomalacia in adults. Excessive amounts of vitamin D lead to hypercalcemia and widespread calcification of soft tissues.
2. Vitamin D is found in high concentrations in fish oils, liver, and fortified milk. It can also be synthesized in human skin by ultraviolet irradiation of sterols. The vitamin is metabolically activated by sequential hydroxylation in the liver and kidney to produce the active form of the vitamin 1,25-(OH)z-Vit D. One of the major roles of vitamin D is to increase intestinal calcium and phosphate absorption. It binds to nuclear receptors and increases the rate of transcription of the gene coding for a protein that transports calcium from the lumen into the intestinal mucosal cell. It also acts in concert with parathyroid hormone to mobilize calcium from bone.
Bridge to Pharmacology Isotretinoin, a form of retinoic acid, is used in the treatment of acne. It is teratogenic and is therefore used with caution in women of child-bearing age.
3. Vitamin K is synthesized by intestinal bacteria and is supplied by leafy green vegetables. Vitamin K acts as a coenzyme for glutamate carboxylase, an enzyme that catalyzes the carboxylation of glutamic acid side chains in several of the clotting factors (factors II, VII, IX, and X). A deficiency of vitamin K results in an accumulation of preprothrombin, a
20
meClical
Proteins, Enzymes, and Coenzymes
deficiency in prothrombin, and an increase in clotting time. Newborns are vitamin K deficient because their intestinal tract is still sterile. Antibiotic sterilization of the gut or malabsorption of fat can lead to deficiency and bleeding complications. 4. Vitamin E, also known as tocopherol, is found in leafy vegetables, vegetable oils, and grains. The major function of vitamin E is as an antioxidant, where its first line of defense is against peroxidation of polyunsaturated fatty acids found in cellular membranes. Fat malabsorption may lead to vitamin E deficiency. In newborns, symptoms include hemolytic anemia; in adults, sensory ataxia due to spinocerebellar degeneration may occur. In humans, vitamin E deficiency is seen frequently in premature infants and malabsorption syndromes.
Bri~Je to .~~~rma~()I()fl' Warfarin, an oral anticoagulant, acts as an antagonist to vitamin K and interferes with the synthesis of vitamin K-dependent clotting factors. Vitamin K can be administered to reverse the effects of warfarin. Warfarin is discussed in greater detail in the HematologicfLymphoreticular Pharmacology chapter of Organ Systems Book 1 (Volume III).
KAPLAlf I med1C8
21
Bioenergetics
Cells extract energy from food by the oxidation of carbohydrates, proteins, and fats to CO2 and H20. Catabolic (degradative) pathways involve oxidation reactions that release energy, most of which is captured in the high-energy phosphate bonds of ATP. In contrast, anabolic (synthetic) pathways involve reduction reactions that require the input of energy, with the reducing power and energy being supplied by NADPH and ATP, respectively. The pathways for catabolism of the metabolic fuels converge at acetyl-CoA, a common intermediate in the degradation of carbohydrates, proteins, and fats. Acetyl-CoA is oxidized in the mitochondria via the citric acid cycle. The released energy is conserved as electron pairs that are transferred from acetyl-CoA to NADH and FADH2. The electrons are then transferred sequentially from NADH and FADH2 to O2, resulting in the formation of H20. The oxidation of NADH and FADH2 by molecular oxygen, a process catalyzed by the electron transport chain, is a highly exergonic process. The energy released is used to drive the phosphorylation of ADP to form ATP, an endergonic reaction. Most of the ATP supply in the cell is derived from oxidative phosphorylation. The regulation of oxidative phosphorylation and the citric acid cycle are closely linked-they are both dependent upon the availability of molecular oxygen. This chapter reviews the principles of thermodynamics and oxidation-reduction reactions that form the basis for energy metabolism.
METABOLIC SOURCES OF ENERGY Energy is extracted from food via oxidation, resulting in the end products of carbon dioxide and water. This process occurs in four stages (Figure 1-3-1). In the first stage, metabolic fuels are hydrolyzed to a diverse set of monomeric building blocks, including glucose, amino acids, and fatty acids. In the second stage, the building blocks are degraded by various pathways to a common metabolic intermediate, acetyl-CoA. Most of the energy contained in metabolic fuels is conserved in the chemical bonds (electrons) of acetyl-CoA. In stage three, the citric acid (Krebs, or tricarboxylic acid, TCA) cycle oxidizes acetyl-CoA to CO 2, and the electron pairs present in the carbon-carbon and carbon-hydrogen bonds are transferred to the electron carriers NADH and FADH 2 . The final stage in the extraction of energy from food is oxidative phosphorylation, where the energy in the electron pairs of NADH and FADH2 is released via the electron transport chain (ETC) and is used to synthesize ATP.
I KAPLAlf medlea
23
Biochemistry
Stage
Carbohydrate
Protein
Gluctse
AminJ Acids
~pyruvate
t
Fat Fatt}Acids
________
-------------Acetyl-Co~
II
III
IV
Figure 1-3-1. Extraction of energy from metabolic fuels.
THERMODYNAMIC PRINCIPLES The principles underlying energy metabolism are based on the thermodynamics of chemical reactions. Some of the abbreviations and corresponding definitions involved are: G = free energy (energy available to do work)
= enthalpy (heat content of a compound) S = entropy (randomness of a system) H
T = absolute temperature (measured in OK) R = gas constant (l.987 cal/mol·degree) F = Faraday's constant (23 kcal/volt·mol) A. Free energy change (ilG) of reactions. The free energy change of a system is the portion of the total energy that is available for useful work. For any chemical reaction, the ilG is equal to the difference in energy between the products and the reactants. The free energy change predicts the direction in which a reaction will proceed spontaneously. ilG = ilH - TilS = Gproducts - Greactants O
The standard free energy change (ilG is a constant for any given reaction. (The superscript indicates "standard conditions:' which are pH 7, 25°C, and all reactants and products at 1.0 M concentration.) The ilGo is related to the equilibrium constant, Keq. For the reaction )
A+B - - - - - - i..~ C + D
24
KAPLA~.
meulCa
I
Bioenergetics
the equilibrium constant is defined as:
[C] [D] Keq = [A] [B]
LlGo = -RT In Ke q = -2.3 RT log Keq
Therefore, if the concentrations of reactants and products at equilibrium are known, the Ke q and the LlGo for the reaction can be calculated. B. Exergonic and endergonic reactions are distinguished on the basis of whether the LlG of the
reaction is positive or negative. Exergonic reactions have a negative LlG and will proceed spontaneously in the direction written. In contrast, endergonic reactions have a positive LlG and require the input of energy to proceed in the direction written. If LlG = 0, the reaction is at equilibrium, and the rates of the forward and the reverse reactions are equal. C. Coupled reaction systems. Endergonic reactions in metabolism frequently proceed by being
coupled to an exergonic reaction. The requirement for a coupled reaction system is that the product of the first reaction must be the substrate for the second reaction. Many enzymecatalyzed reactions that use ATP are examples of coupled reactions, where the energy released by the hydrolysis of ATP is used to drive an endergonic reaction. For example, consider the phosphorylation of glucose to glucose-6-phosphate, a reaction that occurs in all cells and is catalyzed by hexokinase.
Glucose + Pi
----il .....
Glucose-6-phosphate
LlGo
= +3.3 kcalimol
Because this reaction is endergonic, it will not proceed spontaneously in the direction of glucose-6-phosphate formation unless energy is supplied. However, if the formation of glucose-6-phosphate is coupled to the hydrolysis of ATP, the sum of the two reactions is exergonic.
ATP+HzO Glucose + Pi Sum:
Glucose + ATP
---I..... ---I..... ---I....
ADP + Pi Glucose-6-phosphate Glucose-6-phosphate + ADP
In a Nutshell • LlG < 0 ---7 spontaneous reaction • LlG > 0 ---7 nonspontaneous reaction • LlG = 0 ---7 equilibrium
Note Cells often couple energetically unfavorable reactions with ATP hydrolysis to force the reaction to proceed.
=-7.3 kcalimol = +3.3 kcalimol LlGo = -4.0 kcalimol LlGo LlGo
The function of hexokinase, therefore, is to couple these two reactions so that the formation of glucose-6-phosphate is thermodynamically favorable. D. Central role of adenine nucleotides in energy transduction. Some of the common
phosphate-containing compounds found in cells and the energy released by hydrolysis of their phosphate bonds under standard conditions are shown in Table 1-3-1.
KAPLAN'iIme leaI
25
Biochemistry
Table 1-3-1. Energy of hydrolysis of phosphate compounds. ~GO
Compound
(kcallmol)
Phosphoenolpyruvate
-14.8
1,3-Bisphosphoglycerate
-11.8
Creatine phosphate
-10.3
Pyrophosphate
-8.0
ATP Glucose-l-phosphate
1-7.31 -5.0
Fructose-6-phosphate
-3.8
AMP
-3.4
Glucose-6-phosphate
-3.3
Glycerol-3-phosphate
-2.2
The positioning of these compounds in the table illustrates important concepts in energy transfer reactions. Note the structure of ATP (Figure 1-3-2).
000 II II II
Adenine-rib0-;Jse --0 -~
-0
0-
ester bond (low energy)
\~O(~ 0-
-0-
0-
anhydride bonds (high energy)
Figure 1-3-2. Structure of ATP.
The energy in the terminal anhydride bond of ATP is greater than the energy found in the compounds listed after it in Table 1-3-1. Therefore, the energy released by hydrolysis can be used to drive the synthesis of these compounds. Conversely, the compounds listed before ATP ATP formation via substrate-level have more energy in their phosphate bonds than does ATp, thus allowing these compounds phosphorylation occurs with: to transfer their phosphate groups to ADP with the formation of ATP. The conversion of ADP • PhosPhOenOlPyrUvate} . glycolysIs to ATP by the use of high-energy phosphate metabolites is known as substrate-level phosphorylation. As shown in Table 1-3-1, there are three phosphorylated intermediates in • l,3-Bisphosphoglycerate cells with sufficient energy to participate in substrate-level phosphorylation. Phospho• Creatine phosphate ~ enolpyruvate and 1,3-bisphosphoglycerate are intermediates in glycolysis, and muscle creatine phosphate serves as a reservoir of high-energy phosphate bonds in muscle.
In a Nutshell
E. Other high-energy carriers. Many chemical groups that are transferred in metabolic reactions require "high-energy carriers" for the reaction to be exergonic. Examples of some groups that are transferred, their high-energy carriers, and the types of reactions and/or pathways in which they participate are summarized in Table 1-3-2.
26
KAPLAlfdI me lea
------------~
.---~~
Bioenergetics
Table 1-3-2. High-energy carriers of chemical groups in metabolism. Group p.
Carrier
Pathways/reactions
ATP
Kinase reactions
Sugars
UDP-sugar
Polysaccharide synthesis
Acetate
Acetyl-CoA
Fatty acid synthesis
Fatty acids
Acyl-CoA
Triacylglycerol synthesis
Amino acids
AMP-amino acid
Protein synthesis
Methyl
SAM*
Methylation reactions
Carboxyl
Carboxy-biotin
Carboxylation reactions
Sulfate
PAPst
Sulfation reactions
1
*S-adenosylmethionine; t phosphoadenosine phosphosulfate
OXIDATION-REDUCTION REACTIONS The principles of oxidation and reduction are an integral part of energy conservation in cells. The energy contained in metabolic fuels is released through a series of oxidation-reduction (redox) reactions that occur mainly in the mitochondria. Approximately 40% of this energy is conserved as ATP. A. General principles. Oxidation-reduction reactions involve the transfer of electrons between a donor and an acceptor. 1. Definitions. Oxidation is defined as the loss of electrons, and reduction is defined as the gain of electrons. Every oxidation is accompanied by a reduction, each of which is considered to be a half-reaction. 2. The standard reduction potential, E", is a constant that describes the tendency of a compound to act as a reducing agent (lose electrons). It is expressed in volts and is measured under standard conditions defined as 2SoC, pH 7, and at concentrations of 1.0M for electron donors and acceptors. Values of standard reduction potentials for some common half-reactions are shown in Table 1-3-3. Stronger electron donors have a more negative reduction potential. Thus, under standard conditions, NADH with an E' of -0.32 volt will reduce pyruvate (or any other compound with a less negative E').
Mnemonic LEO the lion says GER: loss of electrons = oxidation gain of electrons = reduction OIL RIG: oxidation is loss (of electrons) reduction is gain (of electrons)
Table 1-3-3. Standard reduction potentials for common redox pairs. * E'(V)
Half-reaction NAD+ + 2e- + H+
~
NADH
-0.32
Pyruvate + 2e- + 2H+
~
Lactate
-0.19
Oxaloacetate + 2e- + 2H+~
Malate
-0.17
FAD + 2e- + 2H+
~
FADH2
-0.06
CoQ + 2e- + 2H+
~
COQH2
+0.10
Fumarate + 2e- + 2H+
~ ~
Succinate Cyt a (Fe2+)
+0.13
Cyt a (Fe3 +) + e-
+0.29
112 O2 + 2e- + 2H+
~
H 2O
+0.82
*Note that all of the reactions are written as reductions.
KAPLAlf I meillea
27
Biochemistry
Note If the reduction potential is given, reverse the sign to find the oxidation potential:
3. Relationship between ABO and LiGO. The change in the standard reduction potential for an oxidation-reduction reaction is related to the standard free energy change (LiGO) for the reaction by the expression shown below, where n is the number of electrons transferred, F is the Faraday constant, and LiEO = EO electron acceptor - E' electron donor' LiG = -nFLiEo O
E'red = -0.32 ~ E'QX = +0.32
From this relationship, it is clear that for an oxidation-reduction reaction to be exergonic and to proceed spontaneously, LiEO must be a positive value. 4. Example. These thermodynamic principles can be illustrated by considering the transfer of electrons from NADH to molecular oxygen. In this example, NADH is the electron donor (it has the more negative standard reduction potential), and oxygen is the electron acceptor. The overall oxidation-reduction reaction can be written as the sum of two half-reactions. Oxidation of NADH: NADH ----~..~ NAD+ + H+ + 2eReduction of 0: - - - - - - l.. ~ H 20 Overall reaction:
NADH + 1/2 O 2 + H+ ~ NAD+ + H 20
LiEO LiEo
= +0.32 volt = +0.82 volt
LiEo = + 1.14 volt
The positive value of LiEO indicates that the reaction will proceed spontaneously. As defined above, the change in standard free energy can be calculated (n = 2 electrons being transferred, and F = 23 kcal/volt) as follows: AGO = -nFLiEo AGO = -(2)(23 kcallvolt)(1.14 volt) = -52.4 kcal Because AGO is negative, the oxidation of NADH by oxygen is an exergonic reaction. This exergonic reaction is the overall reaction catalyzed by the electron transport chain. The electrons in NADH are transferred sequentially through a series of carriers to oxygen, where each electron carrier is reduced by the preceding carrier. B. Major electron carriers. A wide variety of dehydrogenases participate in the oxidation of
In a Nutshell • Electron acceptors
~
• Electron donors
~
NAD+ FAD NADPH NADH
metabolic fuels. Most of these enzymes use either NAD+ or FAD as electron acceptors. The major carrier of electrons in reductive biosynthetic reactions is NADPH . 1. NAD+ is the electron accceptor in reactions involving oxidation of hydroxylated carbon
atoms (Figure 1-3-3). NAD+ accepts a hydride ion, H- (two electrons and a proton), to form NADH, and the hydroxyl group is oxidized to a carbonyl group.
OH
I
o II
R-C-COOH + NAD+ ~ R-C-COOH + NADH + H+
I
H Figure 1-3-3. The reduction of NAD+ to NADH.
2. FAD is the electron acceptor in reactions involving the oxidation of two adjacent carbons, resulting in the formation of a carbon-carbon double bond (Figure 1-3-4). A hydrogen atom is removed from each carbon atom and is transferred to FAD to form FADH2•
28
KAPLA~. I meulca
Bioenergetics
H I
H I
I
I
R-C-C-R' + FAD
~
R-C =C -R' + FADH
H H
I H
I H
2
Figure 1-3-4. The reduction of FAD to FADH 2 •
3. NADPH is the major source of reducing power for biosynthetic pathways. In contrast to NADH, which is generated and used primarily in the mitochondria, most of the NADPH is formed and used in extramitochondrial reactions (described in Chapter 4 of this section).
CITRIC ACID CYCLE The citric acid cycle, also known as the tricarboxylic acid (TCA) cycle or the Krebs cycle, is localized in the mitochondria. A. Functions. The pathway is amphibolic, playing important roles in both catabolic and anabolic pathways. 1. Catabolic function. The major catabolic function of the citric acid cycle is to transfer
electron pairs (potential energy) from acetyl-CoA to NAD+ and FAD. With every turn of the cycle, two carbon atoms enter as acetyl-CoA, two carbon atoms leave as COl> and the four pairs of electrons in the carbon-hydrogen and the carbon-carbon bonds are transferred to NAD+ and FAD. The overall reaction for the oxidation of acetyl-CoA is shown in Figure 1-3-5.
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi -----. 2 C02 + 3 NADH + FADH2 + GTP + CoA Figure 1-3-5. Stoichiometry of the citric acid cycle.
Oxidation of acetyl-CoA to CO 2 results in the production of one high-energy phosphate bond (GTP) by substrate level phosphorylation. Assuming adequate oxygen is available, subsequent oxidation of NADH and FADH2 will result in the synthesis of 11 molecules of ATP by oxidative phosphorylation (described below). Therefore, the complete oxidation of one molecule of acetyl-CoA to CO 2 and H 20 results in the synthesis of 12 ATP equivalents. 2. Anabolic functions. Intermediates in the citric acid cycle are used as substrates for vari-
ous biosynthetic pathways. a. Citrate is a substrate for fatty acid synthesis. b. Oxaloacetate is the first intermediate in gluconeogenesis. c. Succinyl-CoA is required for the synthesis of heme.
Note Some amino acids can be converted to a-ketoglutarate and can enter the citric acid cycle at this intermediate.
d. Oxaloacetate and a-ketoglutarate are substrates for amino acid synthesis.
I KAPLAlf med lea
29
Biochemistry
B. Reactions of the citric acid cycle. The reactions by which acetyl-CoA is oxidized to CO 2 are
shown in Figure 1-3-6. AcetylCoA
~Citr"e~
NADHfo.cel.,e
c;o-\.,e
L-M~late
Isocltrate
[7
3
Fumarate
a-Ketoglutarate
FADH,.\ 6 \
~NADH+CO'
fNADH + CO, Sucdnyl-CoA
Succinate
5
GTP
Enzymes 1. Citrate synthase 2. Aconitase 3. Isocitrate dehydrogenase 4. a-Ketoglutarate dehydrogenase 5. Succinyl-CoA thiokinase 6. Succinate dehydrogenase 7. Fumarase 8. Malate dehydrogenase
Figure 1-3-6. The citric acid cycle.
Mnemonic Citric acid is Krebs starting substrate for mitochondrial oxidation: citrate, cis-aconitate, isocitrate, a-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, oxaloacetate
The entry of acetyl-CoA into the cycle involves condensation with oxaloacetate to produce citrate, a C6 tricarboxylic acid. After isomerization to isocitrate, two sequential oxidative decarboxylation reactions occur, resulting in the formation of succinyl-CoA. Each of these reactions releases CO 2 and transfers electrons to NADH. Succinyl-CoA contains a thioester linkage that has sufficient energy to drive the synthesis of a high-energy phosphate bond. Thus, in the next reaction, hydrolysis of succinyl-CoA is coupled with the synthesis of GTP from GDP and Pi. GTP is energetically equivalent to ATP, and the two molecules are interconvertible. The remaining reactions complete the cycle by regenerating oxaloacetate. They include two oxidation reactions: the oxidation of succinate to fumarate, producing FADH2 ; and the oxidation of malate to oxaloacetate, producing NADH. All of the enzymes of the cycle are located in the mitochondrial matrix except for succinate dehydrogenase, which is embedded in the inner mitochondrial membrane. The localization of these enzymes provides for efficient transfer of electrons from the carriers, NADH and FADH2 , to the electron transport chain.
e. Key
enzymes. The four dehydrogenases (isocitrate, a-ketoglutarate, succinate, and malate) all catalyze oxidation reactions. In the isocitrate and a-ketoglutarate dehydrogenase reactions, decarboxylation also occurs. Isocitrate dehydrogenase catalyzes one of the
30
meClical
Bioenergetics
rate-limiting steps in the cycle. This enzyme is allosterically inhibited by ATP and NADH and is activated by ADP. Secondary sites of regulation are a-ketoglutarate dehydrogenase and citrate synthase, which are also inhibited by ATP and NADH. These three enzymes catalyze reactions that are essentially irreversible. Although pyruvate dehydrogenase is not an enzyme in the citric acid cycle, it oxidizes pyruvate to acetyl-CoA, thereby allowing carbohydrate to be oxydized to COz. D. Anaplerotic ("filling-up") reactions. When intermediates of the cycle are removed for synthetic purposes, they must be replenished in order to ensure that acetyl-CoA can continue to be oxidized. The most important reaction for replenishing the cycle with intermediates is the conversion of pyruvate to oxaloacetate. This reaction, catalyzed by pyruvate carboxylase, requires biotin, ATP, and bicarbonate as substrates, and it has an absolute requirement for acetyl-CoA as an allosteric activator (Figure 1-3-7). The accumulation of acetyl-CoA, due to insufficient levels of oxaloacetate, activates the synthesis of this intermediate.
In a Nutshell Enzyme
Inhibitors Activators
Pyruvate ATP dehydrogenase Acetyl-CoA NADH
ADP CoASH NAD+ Ca 2+ Insulin
Citrate synthase
ATP
Iso citrate ATP dehydrogenase NADH
ADP
a-Ketoglutarate Succinyl-CoA dehydrogenase NADH ATP
biotin pyruvate + HC03 - + ATP - - - - - - - - - - - - - - . . . Oxaloacetate + ADP + Pi pyruvate carboxylase
0 acetyl-CoA Figure 1-3-7. The pyruvate carboxylase reaction.
ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION The final step in the aerobic oxidation of metabolic fuels is achieved by the electron transport chain (ETC). The ETC accepts electrons from NADH and FADH z and passes them, through a series of carriers, to molecular oxygen, forming water. The energy released at specific steps in the ETC is used to synthesize ATP via a process called oxidative phosphorylation. All of the enzymes and cofactors required for electron transport and oxidative phosphorylation are localized in the inner mitochondrial membrane. A. The electron transport chain 1. Components. The ETC is composed of a specific sequence of enzymes and their coenzymes, including NAD+ and FAD-linked dehydrogenases, iron-sulfur proteins (FeS), coenzyme Q, and several cytochromes (Figure 1-3-8). The components of the chain can be separated into four protein-lipid complexes (I, II, III, and IV) and two mobile components (CoQ and cyt c) that move freely in the lipid bilayer.
meCtical
31
Biochemistry
Succinate
Complex II FAD FeS
Complex III
Complex I NADH
~
Complex IV ~
FMN
~
FeS
cytc
~
cyt a
t
~cyt
a3
~~
fatty acyl-CoA
a-glycerol-P
Figure 1-3-8. Components of the electron transport chain.
2. Sources ofNADH and FADH. The electrons in NADH arise mainly from the mitochondrial oxidations and to a lesser extent from cytosolic oxidations. Almost all of the FADH arises from mitochondrial oxidations.
a. Mitochondrial oxidations. Several substrates, including isocitrate, a-ketoglutarate, malate, pyruvate, and glutamate, are oxidized in mitochondria with the formation of NADH. The electrons in FADH2 come from the oxidation of succinate, fatty acylCoA, and a-glycerol phosphate. The electrons from NADH and FADH2 enter at different positions along the electron transport chain. Electrons from NADH enter at complex I, whereas electrons from several FAD-linked dehydrogenases enter at CoQ. Note that complex II is succinate dehydrogenase, the same enzyme utilized in the citric acid cycle. b. Shuttles for getting cytoplasmic NADH electrons into mitochondria. Although most oxidations occur in the mitochondria, there are a few oxidative reactions in the cytosol that produce NADH. The mitochondrial membrane, however, is impermeable to NADH; thus, the transfer of electrons from cytosolic NADH into the mitochondria for oxidation by the electron transport chain requires special shuttle systems. Two types of shuttles are found in cells: the malate shuttle and the a-glycerol phosphate shuttle (Figure 1-3-9). In these shuttles, malate and a-glycerol phosphate act as carriers of electrons across the inner mitochondrial membrane. In the malate shuttle, electrons originating in the cytosol are incorporated into mitochondrial NADH, whereas in the a-glycerol phosphate shuttle, cytosolic electrons are incorporated into mitochondrial FADH 2.
32
meClical
Bioenergetics
Inner mitochondrial membrane
Cytosol
Malate Shuttle
NADH
X
NAD+
X
Mitochondrial matrix
Aspartate
Aspartate
t
t
Oxaloacetate
Oxaloacetate
Malate
Malate
X
NAD+
a-Glycerol-P Shuttle NADH NAD+
NADH
Dihydroxyacetone-P
Dihydroxyacetone-P
a-Glycerol-P
a-Glycerol-P
X FAD
Figure 1-3-9. Shuttles for getting cytosolic NADH electrons into the mitochondria.
3. Energetics. A simplified version of the electron transport chain is shown in Figure 1-3-10. The carriers in the electron transport chain are arranged so that the reduction potential progressively increases from negative to positive values. Thus, as electrons move from one carrier to the next in the chain, the ~EO > 0 and the ~GO < 0, ensuring that electrons flow spontaneously to oxygen. FADH2
NADH
~
---J"~ CoQ
-0.32
+0.10
\.."--'y~
__~1
..
cyt b
..
+0.07
\
cyt c
+0.25
Tm
I
+0.42 volt
+0.18
-19.3 kcal/mol
-9.4 kcal/mol
..
..
cyt a
..
cyt a3
+0.55
+0.29
\
1/202 +0.82 volt
I
T +0.53 volt
-24.4 kcal/mol
Figure 1-3-10. Energy released by electron flow through the ETC.
B. Oxidative phosphorylation. The energy required for synthesis of ATP is 7.3 kcal/mol. It is clear from Figure 1-3-10 that transfer of electrons from 1 mol ofNADH through complexes I, III, and IV releases sufficient energy to drive the synthesis of ATP. Thus, it is at these three sites in the electron transport chain that energy can be harnessed for oxidative phosphorylation. Note that electrons from FADH 2 bypass complex I and flow through only two of these energy transduction sites.
I KAPLAlf medlea
33
Biochemistry
In a Nutshell P/O ratio NADH FADH2
3 2
DNP (2A-dinitrophenol, an uncoupler that dissipates the W gradient) has a P/O ratio of o.
1. PIO ratio. By definition, the PIO ratio is the number of ATP molecules produced per 0 atom reduced. Thus, substrates that are oxidized with the generation of NADH (malate, isocitrate, a-ketoglutarate) support the synthesis ofthree molecules of ATP and have PIO ratios of 3. Substrates that are oxidized with the production of FADH2 (succinate, aglycerol phosphate) have PIO ratios of 2. 2. Efficiency. The calculations shown below indicate that the oxidation ofNADH by the ETC releases a total of 53 kcal of energy. LlE' LlG'
= E' oxygen -
E' NADH
= -nFL\E' = -(2)
= +0.82 -
(-0.32)
= 1.14 volts
(23 kcallvolt) (1.14 volt) = -53 kcal
The synthesis of ATP requires 7.3 kcal. Thus, in theory, the oxidation of 1 mole of NADH should release sufficient energy to drive the synthesis of 7 moles of ATP. However, because only 3 moles of ATP are synthesized per mole of NADH oxidized, the efficiency of oxidative phosphorylation is approximately only 40%. The remainder of the energy is released as heat. 3. Mechanism of energy transduction. The chemiosmotic hypothesis is the most widely accepted theory for how electron transport is coupled with ATP synthesis (Figure 1-3-11). This theory is based on the observation that when electrons are flowing through the ETC, complexes I, III, and IV are pumping protons out of the matrix, creating a proton gradient across the inner mitochondrial membrane. Complex II does not pump protons. The chemiosmotic hypothesis asserts that the protomotive force associated with the proton gradient drives the synthesis of ATP. The movement of protons down the gradient as they re-enter the matrix releases energy that is available for ATP synthesis. ATP synthase, also known as complex V, is associated with the inner mitochondrial membrane in close proximity to the electron transport chain (Figure 1-3-11).
Clinical Correlate LHON (Leber hereditary optic neuropathy) is a disease that affects the central nervous system, resulting in the loss of bilateral vision secondary to insufficient ATP to support neuronal activity. The disease has been shown to arise from point mutations in regions of mitochondrial DNA that code for proteins in complex I and complex III of the electron transport chain. Mutations in either of these genes impairs ATP production by oxidative phosphorylation. Patients frequently present with lactic acidosis arising from an inhibition of pyruvate dehydrogenase secondary to NADH accumulation.
34
KAPLAN"dme leaI
ADP+ Pi
Matrix
NADH
l e-
ATP
FADH2
!
e-
Inner mitochondrial membrane
Cytosol
Figure 1-3-11. Structure of mitochondrial ATP synthase.
ATP synthase consists of two types of subunits. The Fo subunits span the membrane, creating a proton channel that allows protons to move back into the matrix. The FI subunit protrudes into the matrix and, in the presence of a proton gradient, catalyzes the condensation of ADP and Pi to form ATP.
Bioenergetics
4. Inhibitors of oxidative phosphorylation. A large number of compounds inhibit oxidative phosphorylation. The sites at which selected inhibitors exert their effects are shown in Figure 1-3-12. Inhibitors of oxidative phosphorylation always decrease ATP production. They are organized into three major classes based on their effects. a. Inhibitors of electron transport bind to specific components of the electron transport chain, blocking electron transfer. This blockage results in a decreased proton gradient and decreased ATP synthesis. For example, cyanide binds to cyt a3 and blocks the transfer of electrons from complex IV to oxygen; antimycin binds to complex III and blocks the transfer of electrons from cyt b to cyt c; rotenone and barbiturates bind to complex I and inhibit the transfer of electrons to CoQ.
NADH
..
------~~
Rotenone, Amytal
cyt b
---
~
cyt c
..
-----I~ cyt a
Antimycin A
~
o
Cyanide
Figure 1-3-12. Inhibitors of electron transport.
b. Inhibitors of ATP synthase bind directly to the enzyme. For example, oligomycin binds to the Fo subunit and prevents re-entry of protons into the mitochondrial matrix. The increased proton gradient across the inner mitochondrial membrane eventually leads to diminished electron transport. c. Uncouplers of oxidation and phosphorylation make the membrane permeable and
abolish the proton gradient. Electron transport is increased by uncouplers, but ATP synthesis cannot occur because the energy cannot be harnessed. The energy resulting from electron transport is dissipated as heat. Examples of uncouplers are dinitrophenol and the protein thermogenin, which is found in the mitochondrial membrane of brown fat. Brown fat surrounds the major blood vessels of the neonate, where it functions to keep the blood warm.
Clinical Correlate Cyanide binds to the heme Fe3+ of cytochrome a3 in the ETC, blocking the transfer of electrons to oxygen and, consequently, the synthesis of ATP. Inhibition of mitochondrial respiration can lead to coma and death unless diagnosed and treated early.
COORDINATE REGULATION OF THE CITRIC ACID CYCLE AND OXIDATIVE PHOSPHORYLATION The rates of oxidative phosphorylation and the citric acid cycle are closely coordinated and are dependent mainly upon the availability of O 2 and ADP. If O 2 is limited, the rate of oxidative phosphorylation decreases, and the concentrations of NADH and FADH2 increase. The accumulation of NADH, in turn, inhibits the citric acid cycle. The coordinated regulation of these pathways is known as "respiratory control." In the presence of adequate 02> the rate of oxidative-phosphorylation is dependent upon the availability of ADP. The concentrations of ADP and ATP are reciprocally related; an accumulation of ADP is accompanied by a decrease in ATP and the amount of energy available to the cell. Therefore, ADP accumulation signals the need for ATP synthesis. ADP allosterically activates isocitrate dehydrogenase, thereby increasing the rate of the citric acid cycle and the production of NADH and FADH 2. The elevated levels of these reduced coenzymes, in turn, increases the rate of electron transport and ATP synthesis.
KAPLAdlf • me leaI
35
Biochemistry
CONGENITAL DEFECTS IN OXIDATIVE PHOSPHORYLATION Mitochondrial DNA contains genes coding for ATP synthase and for some of the proteins in each of the complexes of the electron transport chain. Defects in several of these components have been found. Infants and children present with various combinations of seizures, hypotonia, failure to thrive, and delay of developmental milestones. Focal deficits include abnormalities of eye movements and stroke-like symptoms. Identified entities include mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). All of the defects arising from mutations in mitochondrial DNA are inherited maternally.
36
KAPLllfilme leaI
Carbohydrates
Carbohydrates participate in many metabolic pathways and serve as structural components of cells and tissues. In human biochemistry, there are three major classes of carbohydrates: monosaccharides, disaccharides, and polysaccharides. The most important monosaccharide in human metabolism is glucose. All cells and tissues require the use of some glucose for energy. The brain relies almost entirely on glucose for its energy, and red blood cells derive all of their energy from glucose. Following a highcarbohydrate meal, glycogenesis in liver and skeletal muscle provides a pathway for storage of excess glucose. Conversely, the mobilization of glucose from glycogen is achieved via glycogenolysis. Gluconeogenesis is the pathway for de novo synthesis of glucose from amino acids, glycerol, propionate, and lactate. This chapter emphasizes the role that these pathways play in homeostasis, the tissues where they occur, the key enzymes involved, and the mechanisms for regulation. Numerous clinical examples are provided that stress the importance of carbohydrates in both health and disease.
CLASSIFICATION Carbohydrates are chains of carbon atoms with attached hydrogen and hydroxyl groups. The length of the carbon chain may vary, and carbohydrates can generally be represented by the formula Cn(H 2 0)n, where n is a minimum of three carbons. This formula emphasizes the idea that this class of molecules are "hydrates of carbon:' In some specialized monosaccharides, one of the hydroxyl groups may be replaced by another chemical group, such as a hydrogen atom (deoxyribose in DNA), an amino group (glucosamine in proteoglycans), or phosphate groups (most intermediates of carbohydrate metabolism pathways). A. Monosaccharides. The simplest carbohydrates are the monosaccharides. Most monosaccharides in human metabolism are trioses (C 3 ), pentoses (Cs ), and hexoses (C 6 ). Monosaccharides containing an aldehyde are known as aldoses, and those containing a keto group are called ketoses. The most important monosaccharide is D-glucose, a hexose and an aldose whose structure is shown in Figure 1-4-1.
meClical
37
Biochemistry
---I."
Aldehyde group (Anomeric carbon)
---I."
Penultimate carbon
D-Glucose Figure 1-4-1. Structure of o-glucose.
1. Nomenclature and definitions. The numbering system for monosaccharides always starts with the carbon atom nearest the carbonyl group (::::C=O). a. Anomeric carbon. The carbonyl carbon is defined as the anomeric carbon. This carbon participates in internal ring structures and glycosidic bonds between monosaccharides (discussed below). b. Penultimate carbon. The penultimate carbon is the next-to-the-last carbon in the chain. For hexoses, it is C-5; for pentoses it is C-4. c. D and L isomers. The "D" designation describes the configuration around the penulti-
mate carbon. If the hydroxyl group is on the right, it is a D-sugar; if it is on the left, it is an L-sugar. Almost all important monosaccharides in human biochemistry have the D configuration. d. Epimers. Two monosaccharides are epimers if they differ in the configuration around a single carbon atom. For example, galactose is a 4-epimer of glucose. Thus, knowing the structure of glucose allows you to automatically recognize the structure of galactose. Enzymes that catalyze the interconversions of these compounds are epimerases. e. Aldose-ketose isomers. Glucose, an aldose, and fructose, a ketose, differ only in the position of the carbonyl group. In glucose, the carbonyl group is at C-1, whereas in fructose, it is at C-2. 2. Cyclic structure of monosaccharides. The straight chain structure of monosaccharides exists in equilibrium with a ring structure, resulting from the reaction of the hydroxyl of the penultimate carbon with the anomeric carbon. The cyclic structure of glucose is a sixmember ring (a pyranose) that can be drawn in one of two ways (Figure 1-4-2).
or
Figure 1-4-2. Cyclic structures for l3-o-glucose.
38
meClical ~~~~~~~~~-
~--
------~---
~
Carbohydrates
In the structure on the right, the ring projects from the plane of the paper and the hydroxyl and hydrogen groups project up and down from the carbons in the ring. By convention, hydroxyl groups on the right point down, and those on the left point up. When a ring structure is formed, the anomeric carbon can exist in two configurations, (J, and ~. In (J,-Dglucose, the hydroxyl group on C-l points down, whereas in ~-D-glucose it points up. The predominant form of glucose in the body is ~-D-glucose. D-fructose, shown in Figure 1-4-3, is an important hexose that forms a five-member ring (a furanose).
6
----J.~ ~:J;OH '~6H OH a-O-Fruclose
Figure 1-4-3. Structure of fructose.
3. Derivatives of glucose. Glucose can be modified to give three important compounds found in metabolism (Figure 1-4-4). a. D-gluconic acid is formed by the oxidation of the aldehyde at C-l, producing an "aldonic" acid. The phosphorylated form of gluconic acid is an important intermediate in the hexose monophosphate shunt. b. D-glucuronic acid is formed by oxidation of the alcohol at C-6, producing a "uronic" acid. Ascorbic acid (vitamin C) is synthesized from glucuronic acid. The activated carrier of glucuronic acid (UDP-glucuronic acid) is used in the synthesis of proteoglycans. c. D-glucosamine is formed by the substitution of an amino group for the hydroxyl group of C-2, resulting in glucosamine. Glucosamine and galactosamine are important structural components of glycoproteins and proteoglycans.
CHO
COOH
I
I
H-C-OH
H-C-OH
I
I
HO-C-H
HO-C-H
I
I
H-C-OH
H-C-OH
I
I
H-C-OH
H-C-OH
I
I
COOH
CHpH
O-Gluconic Acid
O-Glucuronic Acid
In a Nutshell • D-gluconic acid ~ hexose monophosphate shunt • D-glu(uronic acid proteoglycans
~
• D-glucosamine ~ glycoproteins, proteoglycans, and glycoilpids
CHO
I I
H-C-NH 2
HO-C-H
I
H-C-OH
I
H-C-OH
I
CH2 0H
O-Glucosamine
Figure 1-4-4. Derivatives of D-glucose. B. Disaccharides are formed when two monosaccharides are connected by a glycosidic linkage.
The anomeric carbon of one monosaccharide is usually linked to a hydroxyl group on the
,mattical
39
Biochemistry
Clinical Correlate Primary lactose intolerance is caused by a hereditary deficiency of lactase, most commonly found in persons of Asian and African descent. Secondary lactose intolerance can be precipitated at any age by gastrointestinal disturbances such as celiac sprue, colitis, or viral-induced damage to intestinal mucosa. Common symptoms of lactose intolerance include vomiting, bloating, explosive and watery diarrhea, cramps, and dehydration. The symptoms can be attributed to bacterial fermentation of lactose to a mixture of CH 4, H2, and small organic acids. The acids are osmotically active and result in the movement of water into the intestinal lumen. Diagnosis is based on a positive hydrogen breath test following an oral lactose load. Treatment is by dietary restriction of milk and milk products or by lactase pills, which can be added to the diet.
Note Heparan is a proteoglycan found in the extracellular matrix and is associated with the cell surface of skin and fibroblasts; heparin is a proteoglycan found mainly in mast cell granules and functions primarily as an anticoagulant.
40
meClical
second monosaccharide. The glycosidic bond is designated as a or 13, depending on the configuration of the anomeric carbon in the linkage. The two most important disaccharides in human biochemistry are lactose and sucrose (Figure 1-4-5).
Galactose
Glucose Lactose
Glucose
Fructose Sucrose
Figure 1-4-5. Structures of lactose and sucrose.
1. Lactose, also known as milk sugar, is a disaccharide of galactose linked through its
anomeric carbon to the 4-hydroxyl group of glucose. Thus, the monosaccharides are joined by a 13-1,4 glycosidic linkage. Lactose is hydrolyzed to glucose and galactose by lactase, an enzyme found in the brush border membrane of the small intestine. 2. Sucrose, or table sugar, is a disaccharide of glucose and fructose linked together through their anomeric carbon atoms in an a-l,2 linkage. Sucrose is hydrolyzed to glucose and fructose in the small intestine by the enzyme sucrase. C. Oligo saccharides are arbitrarily defined as having between 2 and 10 monosaccharides
linked by glycosidic bonds. They are found in mucoproteins and glycolipids. D. Polysaccharides are arbitrarily defined as having more than 10 monosaccharide units. They
serve as structural components of cells and the extracellular matrix, as storage forms for monosaccharides, and as dietary fiber. The most common polysaccharides are starch, glycogen, cellulose, and proteoglycans. 1. Starch and glycogen are both storage forms for glucose. Starch, the major plant polysac-
charide, is composed of amylose and amylopectin. Amylose is a long, unbranched chain of glucose units linked by a-l,4 bonds. Amylopectin has a similar structure, but it also contains branches, where the monosaccharide at the branch point is linked to a monosaccharide in the straight chain by an a-l,6 glycosidic bond. Glycogen, the major animal polysaccharide, is structurally similar to amylopectin, except it is more highly branched. 2. Cellulose is a linear plant polysaccharide composed of glucose units linked together by 131,4 glycosidic bonds. Cellulose is not digested by humans because there is no intestinal enzyme for hydrolyzing glucose units linked by 13-1,4 glycosidic bonds. Therefore, cellulose acts as a dietary fiber, providing "roughage" in the diet. 3. Proteoglycans, also known as mucopolysaccharides, are major structural components of the extracellular matrix. Examples are hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate. These compounds are linear carbohydrate polymers with more than one kind of monosaccharide, linked covalently to a protein core. The carbohydrate portions of proteoglycans are known as glycosaminoglycans (GAGs). GAGs contain repeating disaccharides, usually a hexosamine (glucosamine or galactosamine) and a uronic acid (either glucuronic acid or iduronic acid). The polymers are usually sulfated, and the hexosamines are acylated. The exception is hyaluronic acid; it is not linked to a protein core, it is not sulfated, and it is the only member of this class of compounds found in bacterial sources. The proteoglycans are highly asymmetrical and have a high density of negative charge, allowing them to absorb large quantities of water and form
Carbohydrates
viscous solutions. These physical properties account for their ability to serve as excellent shock absorbers and lubricants. a. Mucopolysaccharidoses are a group of diseases that result from the inability of lysosomes to degrade proteoglycans because of a deficiency of a number of lysosomal hydrolases. The enzyme deficiency leads to excessive storage and urinary excretion of partially degraded mucopolysaccharides. Musculoskeletal deformities and mental retardation are seen in affected individuals. Table 1-4-1 lists the enzyme defect, the urinary metabolite, and the signs and symptoms of the various mucopolysaccharidoses. The enzymatic defects in the mucopolysaccharidoses are inherited in an autosomal recessive manner, except for Hunter disease, which is X-linked recessive.
Mnemonic A Hunter will aim for the X: Hunter disease is the only mucopolysaccharidosis that is X-linked recessive.
Table 1-4-1. Mucopolysaccharidoses. Urinary Metabolites'
Signs and Symptomst
a-L-iduronidase Iduronosulfate sulfatase 4 variants, incl. heparan N-sulfatase (sulfamidase) N-acetylglucosaminidase N-acetylgalactosamine-6sulfatase a-L-iduronidase N-acetylgalactosamine-4sulfatase
HS,DS HS,DS HS
C, MS, CNS, CV MS,CNS MS,CNS
KS, C-6-S
C,MS,CV
HS,DS DS
C,MS,CV C,MS,CV
~-glucuronidase
HS,DS
C(+/-), MS, CNS( +/-), CV
Type
Enzyme Defect
III
(Hurler) (Hunter) (Sanfilippo)
IV
(Morquio)
V VI
(Scheie) (MaroteauxLamy) (Sly)
II
VII
*HS = heparan sulfate; DS = dermatan sulfate; KS = keratan sulfate; C-6-S = chondroitin-6-sulfate. corneal clouding; MS = musculoskeletal abnormalities (dwarfism); CNS = neurologic deterioration; CV = cardiovascular abnormalities.
Clinical Correlate
tc =
4. Glycoproteins and mucoproteins also contain carbohydrate covalently linked to protein, but there are no repeating disaccharides, and the carbohydrate portion usually contains fewer than 20 monosaccharides. Glycoproteins are found in connective tissue (collagen), in plasma (plasma proteins and some peptide hormones), on cell surfaces as antigens (i.e., blood groups), and as components of mucus. There are three types of linkages between protein and carbohydrate. The amino acid side chains involved in the protein-carbohydrate linkage are: a. Asparagine (N-linkage) is found in plasma and cell surface proteins. b. Serine (O-1inkage) is found in mucous and connective tissue proteins. c. 5-Hydroxylysine (O-linkage) is found in collagen.
Glycoprotein synthesis starts in the endoplasmic reticulum and is completed in the Golgi. One of the last steps in glycoprotein synthesis is putting "zip codes" on proteins that are targeted for a particular destination. For example, glycoproteins that are targeted for lysosomes have mannose-6-phosphate in their carbohydrate chains. Receptors on lysosomes recognize this signal and vesicles containing these proteins pinch off and fuse with lysosomes.
I-cell disease is characterized by coarse facial features, musculoskeletal deformities, and recurrent respiratory problems; death usually occurs within the first decade. The distinguishing feature of I-cell disease is a deficiency of mUltiple lysosomal enzymes and the presence of high concentrations of these enzymes in the plasma. The enzymes are being synthesized but are being sent to the wrong destination due to a deficiency in N-acetylglucosaminylphosphotransferase, resulting in the absence of mannose-6-phosphate.
meClical
41
Biochemistry
GLUCOSE ENTRY AND TRAPPING IN CELLS Circulating glucose supplies all cells with a source of energy. The entry of glucose into cells is mediated by a group of carrier proteins (GLUT proteins) that span the plasma membrane. Net transport across the membrane is ensured by coupling glucose transport with phosphorylation, a process that keeps the intracellular glucose concentration low and continues to shift the equilibrium toward glucose uptake by cells (Figure 1-4-6). The membrane is highly impermeable to the phosphorylated compounds. Therefore, phosphorylation renders glucose transport irreversible.
Hexokinase (all tissues)
[Glucose]
[Glucose]
mM
J..LM
--------il..~
Glu-6-P
Glucokinase (liver)
GLUT Carrier protein (tissue specific)
Figure 1-4-6. Glucose transport and trapping in cells.
A. Glucose carriers (GLUT proteins). A family of homologous proteins, known as GLUT proteins, are responsible for transporting glucose into the cell. These proteins differ in tissue specificity, their affinity for glucose, and the maximum rate at which they can transport glucose across the plasma membrane. 1. Skeletal muscle and adipose tissue respond to insulin by increasing their uptake of glucose. These tissues contain GLUT-4, a glucose carrier with spare copies in the Golgi membrane. Stimulation by insulin results in translocation of spare carriers into the plasma membrane. 2. Liver has a high density of GLUT-2, a transporter that has a low affinity (high Km) for glucose and is not saturated by the increased concentration of glucose in the portal circulation following a high-carbohydrate meal.
Mnemonic Hexokinase places a hex on glucose, with lKm and lv max. Hexokinase is inhibited by i[G6P]. Glucokinase is a glutton for glucose, with iKm and ivmax ; it does not seem to be affected by the amount of glucose-6-phosphate.
3. The brush-border membrane of intestinal and kidney cells contains S-GLUT, a carrier that requires sodium for glucose transport.
4. Brain and red blood cells are rich in GLUT-I. This transporter has a high affinity (low Km) for glucose and is normally saturated, ensuring a constant supply of glucose to these tissues. B. Glucose phosphorylation and trapping. The phosphorylation of glucose in most cells is catalyzed by hexokinase. Liver contains glucokinase, an isozyme that has a much lower affinity (higher Km) for glucose and is never saturated under physiologic conditions. Therefore, the properties of glucokinase, in concert with GLUT-2, allow the liver to reduce the concentration of glucose in the portal circulation following a meal.
GLYCOLYSIS Glycolysis, also known as the Embden-Myerhof pathway, is the central pathway of glucose metabolism. It occurs in the cytosol of all cells. Glycolysis is defined as the pathway that converts glucose to pyruvate. For each mol of glucose converted to pyruvate, 2 mols of ATP are consumed and 4 mols are generated, with a net production of 2 mols of ATP per mol of glucose (discussed below). In the presence of well oxygenated mitochondria, pyruvate can be completely oxidized to CO 2 and H 20, resulting in a total of 36-38 mols ATP per mol of glucose. Under anaerobic conditions, however, pyruvate is converted to lactate. The formation of lactate provides a mechanism
42
KAPLA~. I me....ca
Carbohydrates
for regenerating NAD+ from NADH, a condition that is essential for glycolysis to continue. Anaerobic glycolysis plays an important role in ATP production under conditions where O2 is limited, e.g., in exercising skeletal muscle or in cells that lack mitochondria, such as red blood cells. A. Functions. Glycolysis generates ATP and provides intermediates that can be used in other pathways. For example, both glucose-6-phosphate and pyruvate act as branch points in metabolism because they are substrates for enzymes in other pathways. B. Pathway and enzymes. The pathway of glycolysis is shown in Figure 1-4-7. It can be divided
into two stages. The first stage requires the expenditure of ATP, whereas the second stage results in the net production of ATP.
*2(3-Phosphoglycerate)
a-D-glucose
A~+1 Glucokinase Hexokinase
COOH I H-C-OH
Mg ADP
I Glucose-6-Phosphate
1
1
~I Phosphoglucoisomerase
CH -o-® 11 Phosphoglycerate ~ Mutase
I
Fructose-6-®
M;;+1 ADP
1
*2(2-Phosphoglycerate)
H-1~:~
Phosphofructokinase- 1 1
I
CH20H
~
Fructose-1 ,6-bisphosphate
1:
HjJ
Glyceraldehyde-3-®
Dihydroxyacetone- ®
H-?=O H-C-OH
9H2- 0- ® C=O
I
CH2-0-®
R
NAD+
~
NADH + H+
Enolase 1
1
[AfdOfase]
I C~OH
~ *2(2-Phosphoglycerate)
Triosephosphate Isomerase
IGlyceraldehyde-3P-Dehydrogenase I
*2( Phosphoenolpyruvate) COOH I c-cr® II CH 2
2ADP~
I
I
MgH pyruvate Kinase . . 2ATP *2(Enolpyruvate) 00H
9
C-OH II CH 2
*2(1,3)-Bisphosphoglycerate
tSpontaneous
O=?-O-® H-C-OH
2(Pyruvate)
I
2A~;~~ ::hogl~emte K'oose I 1
*2ATP
2(3-Phosphoglycerate) - - - - - - - - - - - - '
EH NADH+~ NAD
+
.-'L-a-,ct-at;-e----, Dehydrogenase
*2 (Lactate) COOH I
HO-C-H I
*2 moles per mole glucose
CH3
Figure 1-4-7. Glycolysis.
meClical
43
Biochemistry
1. Stage 1 of glycolysis. Glucose is converted to fructose-1,6-P 2 through three sequential reac-
tions. ATP is required for the addition of phosphate groups to both ends of the monosaccharide. The two key enzymes in stage 1 are hexokinase and phosphofructokinase-1 (PFK-1). a. Hexokinase (or glucokinase in liver) uses ATP to convert glucose to glucose-6phosphate. In the next step, glucose-6-phosphate is isomerized to fructose-6-phosphate. b. PFK-l uses ATP to add phosphate to the C-1 of fructose-6-phosphate, with the formation of fructose-1,6-bisphosphate. This reaction is the rate-limiting step in glycolysis. PFK-1 is therefore the primary site of regulation in glycolysis. c. Phosphofructokinase-l and phosphofructokinase-2 both use fructose-6-phosphate as substrate. However, these enzymes have distinctly different functions. The product of the PFK-1 reaction is fructose-1 ,6-P 2, an intermediate in glycolysis. The product of the PFK-2 reaction is fructose-2,6-P 2 • The only known function offructose-2,6-P 2 is to act as an allosteric effector that activates glycolysis and inhibits the opposing pathway of gluconeogenesis. The relationship between PFK-1 and PFK-2 is shown in Figure 1-4-8.
GLUCOSE ~-------
!i
Fructose-6-phosphate
Pi Fructose-1,6-bisphosphatase
f
\ : Fructose-2,6-bisphosphate
..... . . . . .
/
',,+, . . . . . .v_/ /
!i
Fructose-1,6-bisphosphate
---- ----------
' - ....... (activator)
.........
/ /
PYRUVATE ..-/
/
/
e
(inhibitor)
/ /"
Figure 1-4-8. Relationship between PFK-1 and PFK-2.
2. Stage 2 of glycolysis. The function of the second stage is to produce ATP. It begins with the cleavage of fructose-1,6-bisphosphate by aldolase into two phosphorylated trioses (dihydroxyacetone phosphate and glyceraldehyde-3-phosphate). These two compounds are interconvertible through the action of triosephosphate isomerase. This reaction allows both trioses to proceed by a common pathway. Thus, beginning from this step in glycolysis, each mol of glucose can be considered to produce two mols of glyceraldehyde-3-phosphate and all subsequent intermediates. The remaining steps in the pathway are concerned with generating intermediates having high-energy phosphate groups that can be transferred to ADP with the formation of ATP. Two intermediates, 1,3-bisphosphoglycerate and phosphoenolpyruvate, have enough energy to drive the synthesis of ATP. There are three important enzymes in stage 2 of glycolysis: glyceraldehyde-3-phosphate dehydrogenase (G-3-P DH), 3-phosphoglycerate kinase, and pyruvate kinase. Under anaerobic conditions, lactate dehydrogenase is an also important enzyme in glycolysis. a. Glyceraldehyde-3-phosphate dehydrogenase catalyzes a reversible reaction that occurs in two steps. First, the aldehyde group is oxidized to a carboxylic acid, with NAD+ being reduced to NADH. (Note that this is the only oxidation reaction in glycolysis.) Second,
44
KAPLA~. I meulca
---------------
Carbohydrates
inorganic phosphate is covalently linked to the carboxyl group, forming 1,3bisphosphoglycerate. The phosphate bond created in this reaction is a high-energy bond, with a L1GO of -ll.S kcal/mol (only 7.3 kcal/mol are needed to convert ADP to ATP). b. 3-Phosphoglycerate kinase transfers the high-energy phosphate group from 1, 3-bisphosphoglycerate to ADP, producing ATP and 3-phosphoglycerate. Thus, 3-phosphoglycerate kinase is one of the ATP-producing enzymes in glycolysis. At this point, the energy consumed in stage 1 has been replaced. In the next two reactions, the phosphate group of 3-phosphoglycerate is rearranged to form another high-energy bond. Phosphoglycerate mutase moves the phosphate group from carbon-3 to carbon2, forming 2-phosphoglycerate. Enolase then dehydrates 2-phosphoglycerate to form phosphoenolpyruvate, a high-energy compound with a L1GO of -14.S kcal/mol.
In a Nutshell Intermediate Enzyme
Product
1,3-bisphospho- Phosphoglycerate glycerate kinase
ATP
Phosphoenolpyruvate
Pyruvate ATP kinase (irreversible)
Glyceraldehyde- G-3P 3-phosphate dehydrogenase
NADH
c. Pyruvate kinase catalyzes the last reaction in glycolysis. The phosphate group from phosphoenolpyruvate is transferred to ADP with the formation of ATP and pyruvate. Thus, the pathway results in a net of mols of ATP per mol of glucose. This reaction is irreversible, and pyruvate kinase is a secondary site for regulation of glycolysis.
d. Lactate dehydrogenase participates in glycolysis only under anaerobic conditions. Lactate dehydrogenase reduces pyruvate to lactate in a reaction that uses NADH and regenerates NAD+. In cells with well oxygenated mitochondria, lactate does not form in significant amounts. However, when oxygenation is poor, as in heavily exercising muscle, shock, or cardiopulmonary arrest, both the citric acid cycle and oxidative phosphorylation become relatively inactive, and most of the cellular ATP is generated by glycolysis. Because glycolysis requires NAD+ (step "a" above), the formation of lactate serves as a mechanism for regenerating NAD+ so that glycolysis can continue. In states of prolonged anoxia, large amounts oflactate accumulate. The lactate is transported to the liver where it can be used to resynthesize glucose. C. Regulation of glycolysis. There are three steps in glycolysis that are regulated: The primary
site of regulation is PFK-1, the enzyme that catalyzes the slowest step in the pathway. Secondary sites of regulation are hexokinase/glucokinase and pyruvate kinase. The regulatory properties of these enzymes are summarized in Table 1-4-2. PFK-1 is inhibited by ATP and citrate, molecules that indicate a high-energy status. When AMP accumulates, signaling the need for ATP, PFK-1 is activated. Pyruvate kinase is also inhibited by molecules that indicate a high-energy state (ATP and acetyl-CoA). When fructose-1,6-bisphosphate accumulates, it feeds forward and activates pyruvate kinase. In liver, the most important activator of glycolysis is fructose-2,6-bisphosphate, a compound whose concentration is increased by insulin and decreased by glucagon.
Table 1-4-2. Regulation of glycolysis. Enzyme
Mode of Regulation
Effect
Hexokinase
Allosteric
Inhibition: glucose-6-P*
Glucokinase t
Enzyme synthesis
Induced by insulin, not inhibited by glucose-6-P
PFK-1
Allosteric
Activation: AMP, fructose-2,6-P 2 t Inhibition: ATP, citrate
Pyruvate kinase
Allosteric
Activation: fructose-1 ,6-P2 Inhibition: ATP, acetyl-CoA, alanine t
Covalent t
Inhibited by phosphorylation t
Clinical Correlate Hemolytic anemia is caused by excessive destruction of red blood cells. A deficiency in pyruvate kinase is a common enzyme deficiency that results in premature lysis of the red blood cell. The red blood cell has no mitochondria and is totally dependent on glycolysis for ATP. A deficiency in pyruvate kinase, therefore, markedly decreases the ability to synthesize ATP. Maintenance of the biconcave shape of the red blood cell is dependent on ATP. Loss of shape signals uptake and turnover by the spleen. In addition, decreased ion pumping by Na+/K+ ATPase results in loss of ion balance and causes osmotic fragility, leading to swelling and lysis.
'Accumulates when ATP is high. t Effects are liver specific.
meCtical
45
Biochemistry
D. Fate(s) of pyruvate. Pyruvate, the end product of glycolysis, plays a central role in metabolism, as shown in Figure 1-4-9. Pyruvate can be reversibly converted to lactate and to alanine. Both of these reactions occur in the cytosol. Lactate is generated under anaerobic conditions by lactate dehydrogenase. Alanine is formed mainly in skeletal muscle, where it serves as a vehicle for transferring amino groups from muscle to liver, where they are then incorporated into urea (discussed in Chapter 6 of this section). Pyruvate also undergoes two irreversible reactions, both occurring in the mitochondria. It can be carboxylated to oxalo-acetate, which can replenish TCA cycle intermediates or be used for gluconeogenesis. The oxidative decarboxylation of pyruvate results in acetyl-CoA, which can be used as a building block for fatty acids, or it can be oxidized to C02 and H 20 by the TCA cycle and oxidative phosphorylation to generate ATP.
Pyruvate
2
Lactate
4
Alanine
3
Acetyl-CoA
Oxaloacetate
/~
/~ Fatty acids
TeA cycle
Gluconeogenesis
Enzymes: 1. Lactate dehydrogenase; 2. transaminase; 3. pyruvate carboxylase; 4. pyruvate dehydrogenase
Figure 1-4-9. Metabolic fates of pyruvate.
1. Pyruvate dehydrogenase (PDR). PDH is a multienzyme complex (Figure 1-4-10) that
converts pyruvate to acetyl-CoA by oxidative decarboxylation. This reaction is irreversible, and there is no enzyme in humans that will reverse the reaction. The overall reaction catalyzed by PDH is: Pyruvate + CoA + NAD+
46
KAPLAlfdme leaI
-7
Acetyl-CoA + CO 2 + NADH
The enzyme complex is located in the mitochondria and consists of three distinct enzyme activities (decarboxylase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase). Five coenzymes are required (thiamine pyrophosphate, lipoic acid, Coenzyme A, FAD, and NAD+). The reaction occurs in several steps as shown in Figure 14-10. In the first step, the decarboxylase (E 1) and its cofactor, thiamine pyrophosphate (TPP), release CO 2. The C2 fragment that remains is oxidized by lipoic acid, transferred to CoA, and finally released as acetyl-CoA. These reactions are catalyzed by dihydrolipoyl transacetylase (E z). The last step, catalyzed dihydrolipoyl dehydrogenase (E 3 ), requires FAD and NAD+ to regenerate the initial oxidized form of lipoic acid. The cofactors TPP, lipoic acid, and FAD never leave the enzyme complex.
carbohydrates
CHTCH-TPP
I OH
Figure 1-4-10. The pyruvate dehydrogenase complex.
2. Regulation of pyruvate dehydrogenase. The activity of PDH is dependent on the energy state of the cell as reflected by the levels of acetyl-CoA, ATP, and NADH. It is activated by ADP, CoA, and NAD+. Acetyl-CoA and NADH are powerful inhibitors of pyruvate dehydrogenase activity. As shown in Figure 1-4-11, PDH undergoes phosphorylation/dephosphorylation. A specific protein kinase and protein phosphatase are associated with the multienzyme complex. The kinase is activated by acetyl-CoA and NADH. The phosphatase is activated by insulin. The regulation of pyruvate dehydrogenase is important to fuel conservation. For example, the oxidation of carbohydrate, fat, and proteins all produce acetyl-CoA and NADH (as seen in Figure 1-3-1 in the Bioenergetics chapter). Thus, when fatty acids are being oxidized, carbohydrate (pyruvate) and protein are conserved.
NADH ~~ Acetyl-CoA
.lG> Kinase
Insulin
~ Phosphatase
Figure 1-4-11. Regulation of PDH activity.
KAPLA~.
I
meulC8
47
Biochemistry
3. Homology of pyruvate dehydrogenase with other enzymes. Two other multienzyme complexes in human metabolism have similar structures to pyruvate dehydrogenase. These are a-ketoglutarate dehydrogenase (in the TCA cycle) and branched-chain keto acid dehydrogenase (in amino acid catabolism). All three of these multienzyme complexes catalyze the oxidative decarboxylation of a-ketoacids. They all have E1> E2> and E3 enzyme activities that require the same five coenzymes.
Clinical Correlate Lactic acidosis arising from a deficiency in pyruvate dehydrogenase (PDH) may be an acquired or an inherited condition. The inherited form frequently presents in the neonatal period with vomiting, hypotonia, neurologic deficits, and persistent acidosis. The accumulation of pyruvate behind the metabolic block pushes the equilibrium of the reversible lactate dehydrogenase and alanine transaminase reactions, thus accumulating lactate and alanine. Acquired PDH deficiency is frequently seen in chronic alcoholics who have a thiamine deficiency. The elevated fat content of the diet given as treatment results in an excess of acetyl-eoA, which is converted to ketones. The ketones can be used as fuel by the brain, thereby bypassing the PDH reaction.
E. Energetics of glycolysis. The amount of ATP derived from glucose oxidation is dependent on the availability of oxygen. 1. Anaerobic glycolysis. When oxygen is limited, glycolysis can be summarized by the equation given below. For each mol of glucose consumed, 2 mols oflactate and 2 mols of ATP are produced. There is no net accumulation of NADH. Glucose + 2 ADP + 2 Pi ~ 2 lactate + 2 ATP 2. Aerobic glycolysis. When oxygen is plentiful, glycolysis can be summarized as follows: Glucose + 2 NAD+ + 2 ADP + 2 Pi ~ 2 pyruvate + 2 ATP + 2 NADH For each mol of glucose consumed, 2 mols each of pyruvate, ATP, and NADH are produced. The NADH can undergo oxidative phosphorylation, producing either 2 or 3 mols of ATP, depending on how electrons in the cytosolic NADH are transported into the mitochondria. Use of the malate shuttle results in 3 mols of ATP per NADH, whereas use of the a-glycerol-phosphate shuttle results in 2 mols of ATP per NADH. Additionally, each mol of pyruvate can be transported into the mitochondria and oxidized to CO 2 and H 2 0 by the TCA cycle and oxidative phosphorylation, resulting in 15 mols of high-energy phosphate (ATP or GTP) per mol of pyruvate. The enzymes involved in generating ATP (or GTP) from glucose are summarized in Table 1-4-3.
F. Poisons of glycolysis. Many compounds shut down the glycolytic pathway by inhibiting one or more of the enzymes. 1. 2-Deoxyglucose is converted by hexokinase to 2-deoxyglucose-6-phosphate. This compound is not a substrate for phosphoglucoisomerase. It accumulates and inhibits hexokinase, preventing the phosphorylation of glucose to glucose-6-phosphate. 2. Iodoacetate and other reagents that react with sulfhydryl groups inactivate glyceraldehyde3-phosphate dehydrogenase, which has an essential sulfhydryl group in its active site. 3. Fluoride inhibits enolase by complexing with 2-phosphoglycerate and Mg2+, making the substrate unavailable. 4. Arsenate inhibits ATP production by acting as a substrate for glyceraldehyde-3-phosphate dehydrogenase, producing a compound that breaks down spontaneously to 3-phosphoglycerate and arsenate. Thus, no ATP is formed in the reaction.
48
meclical
Carbohydrates
Table 1-4-3. Energetics of aerobic oxidation of glucose. Pathway
Enzyme
Product
Method of Energy Generation
1. Glyceraldehyde-3-P dehydrogenase
2 NADH
Oxidative phosphorylation
2 Phosphoglycerate kinase
2 ATP
Substrate-level phosphorylation
2
3. Pyruvate kinase
2 ATP
Substrate-level phosphorylation
2
ATP/Glucose
(Cytosol) Glycolysis 4-6*
ATP produced/glucose ATP consumed/glucose (hexokinase and phosphofructokinase)
8-10*
Net ATP produced/glucose
6-8*
(Mitochondria) PDH & TCA Cycle 1. Pyruvate dehydrogenase
-2
2 NADH
Oxidative phosphorylation
6
2. Isocitrate dehydrogenase
2 NADH
Oxidative phosphorylation
6
3. a-Ketoglutarate dehydrogenase
2 NADH
Oxidative phosphorylation
6
4. Succinate thiokinase
2 GTP
Substrate-level phosphorylation
2
5. Succinate dehydrogenase
2 FADH2
Oxidative phosphorylation
4
6. Malate dehydrogenase
2 NADH
Oxidative phosphorylation
6
Net ATP produced/glucose
30
Total ATP per glucose (aerobic oxidation) = 36-38* Total ATP per glucose (anaerobic oxidation) = 2 (Reactions 2 and 3 of glycolysis) *Transfer of electrons in cystolic NADH via malate shuttle produces mitochondrial NADH and 3 ATP, whereas transfer via a-glycerol-P shuttle produces FADH2 and 2 ATP.
GLUCONEOGENESIS Gluconeogenesis is the pathway for de novo synthesis of glucose from C 3 and C 4 precursors. This pathway is distinct from glycogenolysis, which gives rise to preformed glucose. Gluconeogenesis occurs mainly in the liver and kidney, with a small amount occurring in the epithelium of the small intestine. The pathway requires both mitochondrial and cytosolic enzymes. A. Function. The role of gluconeogenesis in homeostasis is to maintain proper blood glucose levels and to provide glucose for the body. The brain, central nervous system, and red blood cells are dependent on glucose for all or most of their energy. When fasting persists for more than 12 to 24 hours, liver glycogen stores are exhausted and gluconeogenesis provides glucose for these tissues. The primary precursors for de novo glucose synthesis are lactate (from KAPLAlf I meillea
49
Biochemistry
red blood cells and anaerobic muscle), glycerol (from triacylglycerol degradation in adipocytes), and amino acids (from protein degradation in skeletal muscle).
Clinical Correlate
B. Pathway and key enzymes. Gluconeogenesis uses the seven enzymes in the glycolytic pathway that catalyze reversible reactions. Additionally, there are four enzymes unique to gluconeogenesis that are required to bypass the three irreversible reactions in glycolysis (Figure 1-4-12).
A deficiency in any of the four key enzymes used in gluconeogenesis will result in hypoglycemia.
GLUCONEOGENESIS
GLYCOLYSIS
E
GK
Pyruvate Kinase
PFK-1
f \G6P~F6P f \F-1,6-P2~)
DHAP
Glu
"---/
G6Pase
G3P~ 3PG~
"---/
2PG~
Pyruvate
PEP
/
\
FBPase-1
Pyruvate Carboxylase
PEPCK
\
I
Oxaloacetate Figure 1-4-12. Gluconeogenesis and glycolysis.
Each of the four enzymes unique to gluconeogenesis also catalyzes an irreversible reaction, but in the opposite direction from the corresponding enzyme in glycolysis. To bypass the pyruvate kinase reaction, two enzymes are required: pyruvate carboxylase and phosphoenolpyruvate carboxykinase (PEPCK). 1. Pyruvate carboxylase, an enzyme located in the mitochondria, catalyzes the carboxylation
of pyruvate to oxaloacetate, the first step in gluconeogenesis (Figure I-4-13). Acetyl-CoA is an allosteric activator that must be present for the enzyme to function. Biotin serves as a carrier of the "activated carboxyl" group derived from bicarbonate. ATP hydrolysis to ADP and Pi provides the energy needed to generate the "activated carboxyl" group.
COOH
I + C=O I CH 3
Pyruvate
Pyruvate Carboxylase
COOH
I
C=O
I
CH 2
I
COOH Oxaloacetate
Figure 1-4-13. The pyruvate carboxylase reaction.
2. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the second step in gluconeogenesis (Figure 1-4-14). PEPCK is a cytosolic enzyme that phosphorylates and decarboxylates oxaloacetate to form phosphoenolpyruvate. The phosphate group is derived from GTP, which is energetically equivalent to ATP. Thus, to bypass the irreversible pyruvate
50
meClical
Carbohydrates
kinase reaction, two enzymes are required and the equivalent of 2 mols of ATP are consumed in converting pyruvate to phosphoenolpyruvate.
COOH
I
C=O
I
CH2
I
Phosphoenolpyruvate Carboxykinase
f"\
GTP
COOH
•
I C-O-® + CO 2
II
CH 2
GOP
COOH Oxaloacetate
Phosphoenolpyruvate Figure 1-4-14. The PEPCK reaction.
3. Fructose-I,6-bisphosphatase (FBPase-l) catalyzes the hydrolysis offructose-l,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. This reaction bypasses the irreversible step in glycolysis catalyzed by PFK-1.
FBPase-l Fructose-l,6-bisphosphate + H 2 0
------~ ..~
fructose-6-phosphate + Pi
FBPase-l is a cytosolic enzyme that is activated by ATP and citrate and inhibited by AMP and fructose-2,6-bisphosphate. These same compounds alter the activity of the opposing enzyme, PFK-l, but in the opposite direction (see Table 1-4-2). Thus, glycolysis is inhibited when gluconeogenesis is occurring, and vice versa. 4. Glucose-6-phosphatase (G6Pase) catalyzes the last step in gluconeogenesis by removing the phosphate from glucose-6-phosphate and releasing free glucose. G6Pase Glucose-6-phosphate + H 2 0
------~ ..~
glucose + Pi
Clinical Correlate A deficiency in glucose-6phosphatase results in von Gierke disease, which is characterized by low serum glucose and high serum lactate. The elevated lactate results from both a compensatory increase in liver glycolysis and a decreased ability to use lactate as a substrate for gluconeogenesis.
This reaction bypasses the irreversible hexokinase step in glycolysis. G6Pase is associated with the endoplasmic reticulum and is found only in liver, kidney, and intestinal epithelium. The absence of G6Pase in skeletal muscle accounts for the fact that muscle glycogen cannot serve as a source of blood glucose. C. Overall reaction of gluconeogenesis. The conversion of pyruvate to glucose via gluconeogenesis is shown below. For every mol of glucose synthesized, six ATP equivalents are required. Four ATP equivalents are required to convert pyruvate to phosphoenolpyruvate, and two are required to reverse the 3-phosphoglycerate kinase step in glycolysis. The NADH is used to reverse the step catalyzed by glyceraldehyde-3-phosphate dehydrogenase.
2 pyruvate + 4 ATP + 2 GTP + 2 NADH
--7
glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+
The ATP required for gluconeogenesis is derived from the oxidation of fatty acids. It should be noted that the reaction may begin with lactate rather than pyruvate because the lactate dehydrogenase reaction is reversible. KAPLA~.
meulCa
I
51
Biochemistry
I Blood I ~
Glucose
ILiver I
Glucose-6-phosphate
t t ..
. .. ...
Pyruvate
I Muscle I
Glycogen
Glycogen
Lactate
Lactate
. .. ollIE
, Glucose-6-phosphate
tt ... Pyruvate
~
I Blood I Lactate
""'-
Figure 1-4-15. The Cori cycle.
D. Precursors for gluconeogenesis. Any compound that can be converted to an intermediate in glycolysis or the citric acid cycle can serve as a precursor for gluconeogenesis. 1. Lactate. The function of the Cori cycle is to conserve glucose by recycling lactate
formed from anaerobic glycolysis in red blood cells and skeletal muscle. Lactate is released from these tissues into the blood and is taken up by the liver. It is then reconverted to glucose via gluconeogenesis to provide more glucose for energy. The recycling process is illustrated in Figure 1-4-15. 2. Alanine. Skeletal muscle releases large amounts of alanine formed by the transamination of pyruvate. The alanine is taken up by the liver and is reconverted to pyruvate, which is used for glucose synthesis. This recycling is known as the alanine cycle. The alanine cycle provides a means of conserving carbohydrate. Note that neither the alanine cycle nor the Cori cycle result in the net synthesis of glucose. 3. Glucogenic amino acids. All of the common amino acids except lysine and leucine are glucogenic, i.e., they can be degraded to intermediates in either glycolysis or the TCA cycle and thus can serve as precursors for glucose synthesis. The nitrogen arising from these amino acids is disposed of as urea. Lysine and leucine are strictly ketogenic and cannot be converted to glucose. 4. Glycerol. The hydrolysis of triacylglycerols in adipose tissue produces glycerol that is
In a Nutshell
released into the blood. The liver takes up glycerol and converts it to glycerol-3 phosphate, which can be oxidized to dihydroxyacetone phosphate, a gluconeogenic intermediate.
Enzyme
Activators Inhibitors
Pyruvate carboxylase
Acetyl-CoA
Phosphoeno~
ADP ADP
pyruvate carboxykinase Fructose-l,6ATP bisphosphatase Citrate Glucagon Epinephrine
AMP Fructose-2,6bisphosphate
Glucose-6phosphatase
Glucose
52
meCtical
5. Odd-numbered fatty acids. The oxidation of odd-numbered fatty acids produces one molecule of propionyl-CoA from the ffi-end. Propionyl-CoA can be converted to succinylCoA, an intermediate in the TCA cycle, and then to glucose. In contrast, degradation of even-numbered fatty acids produces only acetyl-CoA, which is not a precursor of glucose. 6. Fructose. Fructose (from dietary sucrose) can be phosphorylated to fructose-I-phosphate and cleaved to glyceraldehyde and dihydroxyacetone phosphate (DHAP), a glucogenic intermediate. Glyceraldehyde can be converted to glucogenic intermediates either by reduction to glycerol or by phosphorylation to glyceraldehyde-3-phosphate. 7. Galactose. Galactose can be phosphorylated by the liver to galactose-I-phosphate and then converted to glucose-6-phosphate by a series of reactions discussed below.
Carbohydrates
E. Regulation of gluconeogenesis. Gluconeogenesis is tightly regulated in order to maintain blood glucose at a normal level. Regulation is accomplished by three methods: substrate availability, enzymatic control, and hormonal control. l. Substrate availability. Under conditions of fasting, the primary substrates for gluconeo-
genesis are amino acids derived from protein degradation. The oxidation of fatty acids provides the energy required. All of these processes are coordinated by hormonal control. 2. Enzymatic control. The enzymes that are regulated are unique to gluconeogenesis. The opposing enzymes of glycolysis and gluconeogenesis at the irreversible steps in these pathways are regulated reciprocially so that when one is active the other is inactive. This prevents futile cycling of substrate by ensuring that glycolysis and gluconeogenesis are not occurring at the same time. a. Pyruvate carboxylase is activated by acetyl-CoA. The opposing enzyme in glycolysis, pyruvate kinase, is inhibited by acetyl-CoA. b. Fructose-l,6-bisphosphatase is activated by ATP and citrate and is inhibited by AMP and fructose-2,6-bisphosphate. The opposing enzyme in glycolysis, PFK-l, is inhi-bited by ATP and citrate and is activated by AMP and fructose-2,6-bisphosphate. c. Glucose-6-phosphatase and the opposing enzyme glucokinase are both regulated by
substrate availability. Both enzymes have high Km values and are not saturated under the conditions in the cell. Therefore, as glucose-6-phosphate and glucose concentrations increase, the activities of glucose-6-phosphatase and glucokinase increase, respectively. 3. Hormonal control. The major hormones involved in regulating blood glucose levels are glucagon and insulin, which have opposing effects. Glucagon promotes glucose synthesis and release into the blood, and insulin promotes glucose uptake and storage. Both of these effects are mediated by intracellular concentrations of cAMP. Cellular cAMP levels increase in response to glucagon and decrease in response to insulin. cAMP activates protein kinase-A. The active form of the protein kinase phosphorylates a subset of enzymes and alters their activities. Under conditions of low blood glucose, the secretion of glucagon initiates several responses that lead to restoring blood glucose levels. Responses to elevated glucagon include: a. Inhibition of pyruvate kinase. Phosphorylation of this enzyme leads to its inactivation, thereby decreasing the amount of glucose consumption by glycolysis. b. Decreased concentration of fructose-2,6-bisphosphate. The degradation of fructose2,6-bisphosphate is stimulated by glucagon. Therefore, its inhibitory effect on FBPase1 and its stimulatory effect on PFK-l are both relieved, resulting in increased gluconeogenesis and decreased glycolysis. c. Increased synthesis of key enzymes. The synthesis of PEPCK, FBPase-l, and glucose-
6-phosphatase are increased by glucagon. This is a slower response, taking hours rather that minutes or seconds to occur. Synthesis of these enzymes is also induced by glucocorticoids and catecholamines. d. Increased protein degradation. During gluconeogenesis, proteolysis in skeletal muscle is increased, thus providing an increased supply of amino acids for glucose synthesis. e. Increased lipolysis. The release of fatty acids from adipose triglyceride is stimulated by any hormone that increases intracellular cAMP. Fatty acids are taken up by the liver and oxidized to supply the energy required for gluconeogenesis.
In a Nutshell Increased glucagon ---+ increased cAMP ---+ increased protein kinase-A activity ---+ phosphorylation ---+ decreased pyruvate kinase activity ---+ increased gluconeogenesis
In a Nutshell Increased insulin ---+ decreased cAMP ---+ increased protein phosphatase activity ---+ dephosphorylation ---+ increased F-2, 6-P2 ---+ increased glycolysis
Clinical Correlate Ethanol-induced hypoglycemia is due to the oxidation of ethanol by the liver, leading to the accumulation of excessive NADH and a deficit of NAD+. This prevents the oxidation of lactate to pyruvate, a-glycerol-P to dihydroxyacetone phosphate, and malate to oxaloacetate. Gluconeogenesis therefore fails due to inadequate prescursors, resulting in hypoglycemia.
KAPLAlf _ I meillea
53
Biochemistry
GLYCOGENESIS AND GLYCOGENOLYSIS Glycogen is a highly branched polymer containing glucose molecules linked by a-1,4 glycosidic bonds, with a-1,6 glycosidic bonds between the two glucose molecules at the branch points. Branching increases the solubility of the molecule and facilitates breakdown of glycogen. At the core of the glycogen particle is the protein glycogenin, which serves as the initial primer in the synthesis of glycogen. Glycogenin is not only the primer for the first glucose molecule; it is also the catalyst for the synthesis of the first eight glucose residues of the glycogen molecule. The synthesis and degradation of glycogen occur mainly in the liver and skeletal muscle. The liver may store up to 10% of its wet weight as glycogen. All of the synthesis and degradation occurs at the ends of the branch points. A. Functions. Glycogen is the storage form for glucose in animal tissues. The function of glycogen in skeletal muscle differs from that in the liver.
Mnemonic The well-"red" person is slow, but complete: red muscle fibers are slow, but completely oxidize fuel (TCA cycle).
1. Skeletal muscle degrades glycogen and rapidly metabolizes glucose via glycolysis. This process generates ATP required for muscle contraction. Thus, the function of glycogen in muscle is to provide energy for contraction. In white ("fast") muscle fibers, glucose is released from glycogen and metabolized by glycolysis, with lactate being the major end product. In red ("slow") muscle fibers, pyruvate is completely oxidized by the TeA cycle and oxidative phosphorylation. 2. Liver uses glycogen mainly to regulate blood glucose levels. In response to hypoglycemia, liver glycogen is degraded, and glucose is released into the blood. However, liver glycogen stores become depleted after approximately 12 hours of fasting. In response to hyperglycemia, glucose is removed from the blood and stored as liver glycogen. The pathways of glycogenesis and glycogenolysis are shown in Figure 1-4-16.
y
UDP
Glycogen
UDP-glu
PFl
Glu-1-P
Gll.p
()
Glucose (blood) Figure 1-4-16. Overview of glycogen metabolism.
B. Key enzymes in glycogenolysis. Glycogen degradation occurs at the ends of the branches, where glucose-1-P units are sequentially released by glycogen phosphorylase, the most
54
KAPLAlfdme leaI
Carbohydrates
important enzyme in glycogenolysis. Total degradation requires an additional "debranching" enzyme system. 1. Glycogen phosphorylase cleaves a-I,4 glycosidic bonds by the addition of inorganic
phosphate, a process known as "phosphorolysis': This reaction is the rate-limiting step in glycogenolysis and is regulated both allosterically and hormonally. The glycogen phosphorylase reaction is shown below, where n is the number of glucose units in glycogen. glycogen phosphorylase (Glucose)n
------------.~
P'~
(Glucose)n_l + Glucose-I-P
1
Glycogen phosphorylase will remove glucose units until it gets to within 4-5 glucose units of a branch point, when it no longer binds efficiently to the partially degraded glycogen. 2. Debranching enzymes. The complete degradation of glycogen requires two additional enzymes that make up the "debranching system" (Figure 1-4-17). a[1--t4] chain
Ja·1,6-glucosidase +
o Figure 1-4-17. The glycogen debranching enzymes.
a. a-[1,4] --7 a-[1,4] glucan transferase removes three or four glucose units from a branch point and transfers them to the end of another chain. In this reaction, one a-I,4 bond is being cleaved, and another is being formed. The elongated chain now becomes a substrate for glycogen phosphorylase. b. a-I,6 glucosidase removes the single glucose unit remaining at the branch point and releases it as free glucose.
In a Nutshell
C. Key enzymes in glycogen synthesis. As shown in Figure 1-4-16, the synthesis of glycogen
begins with glucose phosphorylation to glucose-6-phosphate. This reaction is catalyzed by glucokinase in the liver and by hexokinase in other tissues. Glucose-6-phosphate is rapidly and reversibly converted to glucose-I-phosphate by phosphoglucomutase. Before glucose can be added to the glycogen polymer, it must be "activated" or energized. This is achieved by the formation ofUDP-glucose, which serves as the donor of glucose to a glycogen primer. Most monosaccharides are activated by reacting with UTP to form a UDP-sugar. 1. Formation of UDP-glucose. The phosphate group of glucose-I-phosphate reacts with
UTP to form UDP-glucose and pyrophosphate (PPi). Thus, one of the phosphate groups in UDP-glucose is derived from glucose-I-phosphate and the other from UTP, as shown
Enzyme
Activator Inhibitor
Muscle AMP ATP glycogen Ca2+ Glucose-Gphosphorylase Epinephrine phosphate Insulin Glucagon Glucose Liver glycogen Epinephrine Insulin phosphorylase Glycogen synthase
Glucose Insulin
Glucagon Epinephrine
KAPLA~. I meulca
55
Biochemistry
Clinical Correlate Glycogenesis is coupled to the influx of K+ in the cells. Insulin and glucose are therefore given to treat hyperkalemia (high serum K+), inducing glycogenesis and causing an influx of K+ into the cells.
below. The reaction is catalyzed by UDP-glucose pyrophosphorylase. The equilibrium of this reaction is "pulled" toward UDP-glucose formation by the hydrolysis of pyrophosphate to inorganic phosphate. UDP-glucose pyrophosphorylase Glu-1-P + Uridine-P-P-P
..
UDP-Glu + PP j
The linkage between glucose and UDP is a high-energy bond, making it a suitable donor in many biosynthetic reactions. 2. Elongation of glycogen chains. To be active, glycogen synthase requires an existing glycogen chain serving as a primer. If the chain has been totally degraded, then glycogenin, the protein found at the core of the glycogen particle, serves as a primer for glycogen synthesis and accepts the initial glucose residue. Glycogen synthase catalyzes the transfer of glucose from UDP-glucose to the end of a chain. The linkage created is an o,-1,4-glycosidic bond. This is the rate-limiting step in glycogen synthesis and is therefore the site of regulation. glycogen synthase .. (Glucose)n+l + UDP (Glucose)n + UDP-glucose
3. Branching enzyme. After approximately 10 glucose units have been added, a branch point is created by 0,-[1,4] 0,-[1,6] glucan transferase. Forming a branch point involves breaking an 0,-1,4 linkage and creating an 0,-1,6 linkage. The two shorter chains that are produced can now be elongated by glycogen synthase. There are always at least four glucose molecules between branch points. D. Coordinate regulation of glycogenesis and glycogenolysis. Glycogen phosphorylase and glycogen synthase are regulated in a reciprocal manner, such that when one of these enzymes is active, the other is inactive. The primary mode of regulation for both enzymes is hormonal and is mediated by phosphorylation/dephosphorylation. In addition, both glycogen synthase and phosphorylase activities can be affected allosterically.
56
meClical
------~~~-
-~~~
Epinephrine
Glucagon (liver cell)
ADENYLATE CYCLASE
..
"
0
& liver cells)
- . ---= ,R ADENYLATE '
ATP
GLYCOGEN PHOSPHORYLASE KINASE
cAMP-DEPENDENT PROTEIN KINASE
I
CYCI,ASE active
cAMP-
Phosphodiesterase active
Glucose
0(
0(
Glucose i-Phosphate
t
GLYCOGEN PHOSPHORYLASE
a active
Glycogen
5Ie CD; A,"'l
gO
~
.....
*Reactions catalyzed by PHOSPHOPROTEIN PHOSPHATASE Figure 1-4-18. Cascade for activating glycogenolysis.
*
..go ~
{
i
Biochemistry
1. Glycogen phosphorylase. A comprehensive diagram describing the regulation of this
In a Nutshell Hormone (glucagon and epinephrine)
~
G-protein
~
ieAMP
~
Activates PKA
I
Phosphorylates glycogen synthase
~
\
Phosp horylates phosphorylase kinase
~
Inactive Phosphorylates (no glycogenesis) glycogen phosphorylase
~
Active (iglycogenolysis)
enzyme is shown in Figure 1-4-18. Phosphorylation of a specific serine side chain activates the enzyme, resulting in glycogen degradation. The activation of phosphorylase is initiated by the binding of glucagon to liver cell receptors or by the binding of epinephrine to muscle receptors. Both of these hormones increase the intracellular synthesis of cAMP, resulting in the activation of a cAMP-dependent protein kinase (protein kinase A). A single molecule of hormone can generate many molecules of cAMP; each molecule of cAMPdependent protein kinase can phosphorylate (and activate) many molecules of phosphorylase kinase; each molecule of phosphorylase kinase can phosphorylate (and activate) many molecules of glycogen phosphorylase; and each molecule of glycogen phosphorylase can release many molecules of glucose-I-phosphate from glycogen. This "cascade" system amplifies the response initiated by the binding of a few hormone molecules by generating millions of molecules of glucose-I-phosphate. It is noteworthy that phosphorylase kinase can also be activated directly (without phosphorylation) by binding calcium. This is particularly important in skeletal muscle, where the coupling of excitation with contraction is mediated by calcium. Thus, the same regulatory molecule that activates contraction also activates glycogenolysis, which provides the energy for contraction. The active (phospho) form of glycogen phosphorylase can be allosterically inhibited by glucose and ATP, whereas the inactive (dephospho) form can be activated allosterically by AMP. Thus, although covalent modification of these enzymes is the primary mode of regulation, the activity of the phospho- and dephospho- forms can be fine-tuned by the accumulation of intracellular metabolites that act as allosteric effectors. 2. Glycogen synthase. The same signals that activate glycogen phosphorylase inactivate glycogen synthase. Glucagon and epinephrine (via cAMP-dependent protein kinase) stimulate the phosphorylation and inactivation of glycogen synthase. The coordinate regulation of these enzymes prevents futile cycling of substrate that would exist if both enzymes were active at the same time. The inactive (phosphorylated) form of glycogen synthase in muscle can be allosterically activated by glucose-6-phosphate. Many texts refer to glycogen synthase-1 and glycogen synthase-D. The D form is the inactive (phospho) enzyme, and the 1 form is the active (dephospho) form. E. Glycogen storage diseases. A number of enzyme deficiencies have been identified that result in the accumulation of an abnormal type or quantity of glycogen in tissues. Table 1-4-4 lists the major types of glycogen storage disease, the deficient enzyme, and the cardinal clinical feature of each type.
58
me&ical
Carbohydrates
Table 1-4-4. Glycogen storage diseases. Type
Deficient Enzyme
Cardinal Clinical Feature
von Gierke Pompe
Glucose-6-phosphatase a-I, 4-glucosidase
III
Cori
Glycogen debranching
IV
Andersen (amylopectinosis)
Branching enzyme
Severe hypoglycemia Cardiomegaly, muscle weakness, death by 2 years Mild hypoglycemia, liver enlargement Infantile hypotonia, cirrhosis, death by 2 years
McArdle
Muscle glycogen phosphorylase
Hers disease
Hepatic glycogen phosphorylase Muscle phosphofructokinase Hepatic phosphorylase kinase
I II
V VI
VII VIII
Muscle cramps and weakness on exercise Hypoglycemia, cirrhosis Muscle cramps No neuromuscular symptoms, hypoglycemia
It is clear from Table I -4-4 that the clinical consequences of an enzyme deficiency depend on
which enzyme is missing. For example, in type I deficiency, the absence of glucose-6phosphatase, impairs the ability of the liver to release free glucose by glycogenolysis, resulting in severe hypoglycemia. Other consequences include increased uric acid levels due to increased pentose phosphates, increased purine synthesis, and increased purine degradation; acidosis due to the accumulation of lactate; elevated serum lipids due to fat degradation in adipose; and growth retardation due to accelerated protein degradation. In type III deficiency, the absence of debranching enzyme only partially impairs glycogenolysis, resulting in mild hypoglycemia. Glycogen structure is normal in all of the types except III and Iv.
HEXOSE MONOPHOSPHATE SHUNT This pathway, also known as the pentose phosphate pathway, provides an alternative route for the oxidation of glucose. The products of the pathway are CO 2 , pentose phosphates, and NADPH. The shunt branches off of glycolysis at glucose-6-phosphate and re-enters at fructose6-phosphate (Figure 1-4-19). The hexose monophosphate (HMP) shunt is present in the cytosol of all cells. Unlike glycolysis, this pathway neither consumes nor produces ATP. A. Functions. This pathway supplies the cell with NADPH and pentoses. 1. NADPH. Almost all of the NADPH required in reductive biosynthetic processes, such as
the synthesis of cholesterol, fatty acids, and steroid hormones, comes from the hexose monophosphate shunt. Tissues that synthesize large amounts of these compounds have high levels of the NADPH-producing enzymes in the pathway. Red blood cells require large amounts of NADPH to maintain the reduced form of glutathione. Reduced glutathione helps prevent hemolysis by neutralizing the effects of strong oxidizing agents such as superoxide and hydrogen peroxide. Neutrophils, macrophages, monocytes, and other phagocytosing cells require large quantities of NADPH to generate superoxide. These cells use superoxide as a part of the "cidal" process for killing bacteria they engulf. NADPH oxidase reduces molecular oxygen to superoxide in these cells. A deficiency in either NADPH or NADPH oxidase can result in chronic infection.
Clinical Correlate Chronic granulomatous disease is caused by a defect in the NADPH oxidase complex, resulting in neutrophils that do not produce superoxide. Affected individuals therefore present with an inability to kill invading microbes that are engulfed.
meClical
59
Biochemistry
2. Pentoses. The synthesis of nucleotides and some coenzymes (NAD+, NADP+, FAD, CoA) requires ribose-5-phosphate, which is supplied by the HMP shunt. B. Pathway and key enzymes. As shown in Figure 1-4-19, the HMP shunt branches off glycolysis at glucose-6-phosphate. The two pathways equilibrate by having fructose-6-phosphate as a common intermediate. After the early steps in the pathway, which lead to NADPH and ribose-5-phosphate synthesis, a large number of intermediates (shown in brackets) allow excess pentoses to be reconverted to fructose-6-phosphate. The numbers at each reaction refer to the enzyme used. The reactions in the HMP shunt can be divided in two phasesthe oxidative and non oxidative phases. 1. The oxidative phase produces 2 mols of NADPH per glucose oxidized. This phase consists of three reactions starting with glucose-6-phosphate and resulting in ribulose-5phosphate. All of these reactions are essentially irreversible. In the first reaction, the carbon-1 of glucose-6-phosphate is oxidized to a cyclic acid (lactone), with the simultaneous production of NADPH (Figure 1-4-20). In the next reaction, the lactone of 6-phosphogluconate is hydrolyzed to the straight chain form by lactonase. In the last step of the oxidative phase, 6-phosphogluconate is oxidized and decarboxylated, with the formation ofNADPH, CO 2 , and ribulose-5-phosphate. Glucose
~
Glucose-6-P
I
Fructose-6-P
NADPH
NADPH
H2 0
~•
/ . 6-phosphoglucono-o-lactone
6-P-gluconate
2
CO 2
~
/
Ribulose-5-P
3 4
.....If-----~.~
Glyceraldehyde-3-P Erythrose-4-P Xylulose-5-P Fructose-6-P Sedoheptulose-7-P
5,6,7
.....E-----------___ • Ribose-5-P
1
1
Pyruvate
Nucleotide Synthesis
Figure 1-4-19. The hexose monophosphate shunt. (1. Glucose-6-phosphate dehydrogenase; 2. lactonase; 3. 6-phosphogluconate dehydrogenase; 4. pentose-P-isomerase; 5. pentose-P-epimerase; 6. transketolase; 7. transaldolase)
COOH
CH2-00~
~ OH
HO
OH
I
CH2 0@
NADPH
~
~ OH
0
0
H-C-OH
NADPH
I
~
HO-C-H
I
H-C-OH
HO
I
OH
H-C-OH
I
CH2 0@ Glucose-6-@
6-Phosphoglucono-1)-lactone
6-P-Gluconate
Ribulose-5-P
Figure 1-4-20. The oxidative phase of the hexose mono phosphate shunt.
60
meClical
Carbohydrates
2. The nonoxidative phase. All of these reactions are reversible. Ribulose-5-phosphate is isomerized to ribose-5-phosphate, the pentose required for nucleotide synthesis. The remaining reactions involve the transfer of Cz- and Crunits from one sugar to another. Intermediates having 3, 4, 5, 6, and 7 carbons are involved. The key enzymes in the transfer reactions are transketolase and transaldolase. Transketolase transfers C2 -units, using thiamine pyrophosphate as a Cz-carrier between donor and acceptor. Transal-dolase transfers C3 units. The major function of the non oxidative phase is to provide a pathway for recycling excess pentoses. This is particularly important in tissues that require larger amounts of NADPH than pentoses. The recycling is achieved by the formation of fructose-6-phosphate, a common intermediate in the HMP shunt and glycolysis. Fructose-6-phosphate can be isomerized back to glucose-6-phosphate and reused. For every 6 mols of glucose-6-phosphate used in the HMP shunt, 6 mols of CO 2 are produced and 5 mols of fructose-6-phosphate can be returned to glycolysis. C. Regulation of the HMP shunt. The rate-limiting step in the pathway is the initial reaction cat-
alyzed by glucose-6-phosphate dehydrogenase. The amount of this enzyme present in liver and adipose increases when the diet contains large amounts of carbohydrate, a condition that leads to fatty acid synthesis and an increased requirement for NADPH. Glucose-6-phosphate dehydrogenase is allosterically activated by NADP+ and inhibited by NADPH and palmitoyl-CoA.
FRUCTOSE METABOLISM Fructose is second to glucose as a dietary source of carbohydrates. It is mostly derived from the hydrolysis of sucrose in the brush border of the small intestine. The primary site of fructose metabolism is the liver. As the portal blood enters the liver, fructose is very efficiently removed and metabolized by the liver. The liver has three enzymes (fructokinase, aldolase B, and glyceraldehyde kinase) that convert fructose into triose phosphates, intermediates in glycolysis. Because these reactions bypass the rate-limiting step in glycolysis, fructose is metabolized to pyruvate and acetyl-CoA much more rapidly than is glucose. The enzymes of glycolysis, gluconeogenesis, and glycogenesis also allow dietary fructose to be converted to blood glucose or glycogen. The very rapid phosphorylation of fructose by fructokinase may result in a transient decrease in intracellular phosphate concentrations and a diminished ability to synthesize ATP. A. Pathway and key enzymes of fructose metabolism. The metabolism of fructose starts with the conversion to fructose-I-phosphate by fructokinase (Figure 1-4-21). Fructose-I-phosphate is split into two C3 fragments by aldolase B, producing dihydroxyacetone phosphate and glyceraldehyde. Glyceraldehyde can be phosphorylated to glyceraldehyde-3-phosphate by triose kinase. Alternatively, glyceraldehyde can be reduced to glycerol and used for gluconeogenesis or for triacylglycerol synthesis. Both dihydroxyacetone phosphate and glyceraldehyde-3phosphate are intermediates in glycolysis. They can be further degraded by glycolysis, or they be condensed to fructose-I,6-bisphosphate and used for gluconeogenesis or glycogenesis.
Note Transketolase is used to diagnose thiamine deficiency by measuring erythrocyte transketolase activity upon addition of thiamine pyrophosphate.
Clinical Correlate Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive disorder causing t NADPH production -7 a hemolytic anemia. Microscopic examination of erythrocytes reveals the presence of "Heinz bodies," small, dark inclusion bodies of precipitated hemoglobin. Oxidizing agents such as sulfonamides, aspirin, and antimalarial and antituberculous drugs t the amount of superoxide and hydrogen peroxide in the blood. The lack of NADPH -7 a lack of reduced glutathione, which is needed to neutralize the toxic effects of these substances. These agents can therefore aggravate the hemolytic anemia.
In a Nutshell Hexose Monophosphate Shunt • Produces ribose-5phosphate for nucleotide synthesis • Reactions occur in cytoplasm • Produces NADPH for use in anabolic processes (e.g., fatty acid biosynthesis) • Does not produce or consume ATP
meClical
61
Biochemistry
Dihydroxyacetone-P Fructokinase Fructose
)I
Aldolase-8
Fructose-1-P
-----
. ~
C ::> M
Figure 11-4-4. Structure of a typical gene transcribed by RNA polymerase II.
a. TATA box (Hogness box). This sequence of base pairs, which is important in the initiation of transcription, is found in all eukaryotes. It is located approximately 25 base pairs upstream (-25) from the start point and is almost identical to the Pribnow box found in bacterial systems. TFIID, the critical transcription factor for RNA polymerase II, binds here. b. CAAT box. This sequence is usually found between positions -75 and -80, but it can KAPLAN"dme leaI
139
Molecular Biology
function at distances that vary considerably from the start point. Binding of transcription factors at this site may influence the formation of initiation complexes at other sites.
In a Nutshell RNA polymerase II initiation requires: • Specific transcription factors bound to enhancer sequences • General transcription factors bound to promotor sequences • Association of RNA polymerase II with transcription factors to form initiation complex
3. Regulatory regions. Sequences that either increase or decrease the rate at which transcription is initiated by RNA polymerase II exist at various places in the gene. Although they are usually located upstream from the start site, they may also be internal to the gene or downstream from the gene. Enhancer sequences bind transcription factors that increase the rate of transcription, whereas silencer sequences bind factors that decrease the rate of transcription. 4. Exon and introns. The segment(s) of the gene that are maintained in the mature mRNA and code for protein are known as exollS. Introns are the portions of the primary RNA transcript that are removed by splicing together the exons (Figure 11-4-5). The splicing usually occurs at a consensus sequence (GU ... AG) found at the boundaries between introns and exons (discussed in more detail below).
prima7rahnn:c~; Mature mRNA
5'
--l 5'
Exon 1
---1
H
Exon 1
Exon 2
H
Exon 3
H
I Exon 2 I Exon 3 I Exon
Exon 4
r--
3'
4}-- 3'
Figure 11-4-5. hnRNA and mature mRNA.
In a Nutshell RNA polymerase III initiation requires: • Transcription factors bound at intemal promotor sequence and at start site • Association of RNA polymerase III with transcription factors at start site to form initiation complex
Note In eukaryotes, because transcription and translation are not coupled, the primary transcript (hnRNA) is extensively modified in the nucleus, yielding mature mRNA that is then transported to the cytoplasm for translation into protein.
140
ineClical
D. Genes coding for tRNA and 5S rRNA. These genes are transcribed in the nucleoplasm by RNA polymerase III. The promoters for both the 5S rRNA and the tRNA genes are internal and are found downstream from the start site of transcription. l. 55 rRNA genes. The genes for this small rRNA are tandemly repeated in a single cluster
located far away from the genes for tRNA. These are the only rRNA genes that are transcribed outside the nucleolus. 2. tRNA genes. The genes for tRNA are clustered and are transcribed as larger precursor RNA molecules, which are processed by endonucleases. Internal promoters are found in two regions downstream from the start site. tRNA is subject to a number of posttranscriptional modifications, including modification of specific bases and the addition of the sequence -CCA to the 3' -OH of the tRNA.
PROCESSING OF EUKARYOTIC RNA The heterogeneous nuclear RNAs (hnRNAs), synthesized by RNA polymerase II, leave the nucleus as messenger RNAs (mRNAs). The processing of the hnRNA starts while transcription is still occurring and involves covalent modification of both the 5' and 3' ends, followed by cutting and splicing to eliminate the intervening sequences that separate the coding regions. A. 5' Capping. The 5' end of the RNA is "capped" shortly after the initiation of RNA synthesis. This process involves the addition of an "inverted" methylated guanosine molecule to the first nucleotide in the RNA transcript. The 7-methyl-guanosine is linked through a 5'-5' triphosphate linkage. The 5' cap plays an important role in the initiation of protein synthesis and protecting the mRNA chain from degradation. B. Polyadenylation of the 3'-OH end. Mature mRNA molecules have a poly-A tail that is
between 20 and 250 nucleotides long. The tail is added to hnRNA by the enzyme poly-A polymerase. A consensus sequence near the end of the gene provides a signal that initiates
Transcription
polyadenylation. Polyadenylation is believed to increase the stability of hmRNA. Not all mRNA molecules are polyadenylated: histone mRNAs, for example, have no poly-A tails. C. RNA splicing. hnRNA contains coding sequences (exons) that are separated from one another by intervening sequences (intrans). In the conversion ofhnRNA to mature mRNA, the introns are removed and the exons are spliced together. This process is illustrated in Figure 11-4-6. During the excision of introns, the 5' cap and the poly-A tail are not removed. The base sequence at the beginning of an intron is GU ... and at the end of an intron is ... AG. This consensus sequence defines the sites at which cutting and splicing occur. The intron is excised as a loop (lariat) of RNA that is degraded. After excision, ligation of the exons occurs. These reactions occur in the nucleus. STEP 1.
-0 Q)
'til
-0
co
!:;c
Ui c
~'rn
!:;c
Q)
Ui c
'til
co
co
Co
Q) l!)lo..
t
~'rn Q) C')lo..
Intron 1
Exon 1
Exon 2 AAUAAA
5' Cap AGGUAAGU
t
t
AGG
t
["'il
AAAn
HnRNA
t
2
STEP 2.
'(jj
+ «'co >'c Exon 1
0-0 a.. co
a..'(jj
~.... AAUAAA
.......""'"""'........ AG
« .Q b:g
DOl
Exon 2
5' Cap
+ c
[ii,1
AAAn
Formation of lariat
G
STEP 3. Exon 1
Exon 2
====;.;.1 AAUAAA leII AAAn
5'Cap
G
AG STEP 4.
Excision of lariat
Exon 1 Exon 2
5' Cap
........··.··, ••. ·\.. 1 AAUAAA
II
(mRNA) AAAn
Ligation of exons
AGG
plus
Lariat (degraded in nucleus) Figure 11-4-6. Processing of hnRNA. KAPLAtf _ I med lea
141
Protein Synthesis
Translation is the process by which the base sequence in mRNA is decoded into an amino acid sequence. All three types of RNA play different and essential roles in translation. The genetic code is defined as the relationship between the sequence of bases in DNA (or its RNA transcript) and the sequence of amino acids in proteins. mRNA is the template for protein synthesis and acts as a "working copy" of the gene in which the code words for each amino acid (codons) have been transcribed from DNA to mRNA. The tRNA has a three-base anticodon that hydrogen bonds with the complementary codon in mRNA thus aligning amino acids in the appropriate sequence prior to peptide bond formation. Ribosomes are complexes of protein and rRNA that serve as the molecular machines, coordinating the interactions between mRNA, tRNA enzymes, and protein factors required for protein synthesis. This chapter reviews the structure and function of the three types of RNA, the genetic code, the structure and function of ribosomes, and the events involved in protein synthesis. Commonly used antibiotics that inhibit protein synthesis are also reviewed.
ROLE OF RNA IN PROTEIN SYNTHESIS Each of the three major types of RNA plays an essential role in protein synthesis. A. Messenger RNA (mRNA) acts as the "working copy" of the gene coding for a protein. The mRNA carries the information from the genome in the nucleus to the cytosol where protein synthesis occurs. Messenger RNA is synthesized as an hnRNA precursor and is processed to mature mRNA in the nucleus. The mature mRNAs are capped at their 5' end with 7-methylguanosine attached through a triphosphate linkage to the first nucleotide in the mRNA. Most mRNAs also contain a poly-A tail attached to the 3' end. Messenger RNA constitutes approximately 5% of the total cellular RNA and has a shorter half-life than other types of RNA. The length of the mRNA is related to the size of the gene. The key structural features of mRNA are shown in Figure 11-5-1.
KAPLAN"iIme leaI
143
Molecular Biology
5' Untranslated region
Coding region or open reading frame
Poly-A+ signal
Poly-A+ tail
UGA 5' Cap - - AUG - - - - - - UAA - - - AAUAAA--- AAAn 3' UAG Start codon Stop codon (Methionine)
t
t
3' Untranslated region
Figure 11-5-1. Structure of eukaryotic mRNA.
B. Ribosomal RNA (rRNA). Ribosomal RNA is the most abundant form of RNA, comprising approximately 80% of the cellular RNA. Ribosomes, the machines for synthesizing protein, are complexes containing protein and rRNA. In prokaryotic systems, there are three forms of rRNA: 23S, 16S, and 5S rRNA, which vary in length from 120 to 3,700 nucleotides. Eukaryotic rRNA has four forms of rRNA: 28S, 18S, 5.8S, and 5S. In eukaryotic systems, all of the forms of rRNA except 5S rRNA are synthesized in the nucleolus. The precise function of rRNA is unclear, but it is necessary for the organization of a functional ribosome.
Note The 5' end of an RNA that has been cut out of a larger precursor RNA is a monophosphate; the 5' end of a precursor or unprocessed RNA is a triphosphate.
C. Transfer RNAs (tRNAs) are the adaptor molecules in protein synthesis. They have a threebase region (anticodon region) that recognizes and hydrogen bonds to the complementary codon in mRNA, which specifies a particular amino acid. The tRNAs react with amino acids at their 3' ends. Transfer RNAs are small, containing approximately 80 nucleotides. The key structural features of tRNA are shown in Figure 11-5-2 and are described briefly below. 3' Amino acid OH - - attachment site A
C C
Phosphorylated 5' terminus - - 5' P
A G···· C
nvC loop
"" ______________"Extra arm" (variable)
Figure 11-5-2. Structure of tRNA.
144
mectical ---
._---_.----- - - - - - -
Protein Synthesis
1. The 5' end of tRNA is a monophosphate rather than a triphosphate, and the base is usually G. 2. The 3' end of tRNA has the sequence CCA. The "activated amino acid" is covalently attached to the 3' OH of the terminal adenosine. The bond linking the amino acid to tRNA is a high-energy bond that provides energy for peptide bond formation, an endergonic reaction.
Note The CCA amino acid attachment site of tRNA is encoded in prokaryotes but is added posttranscriptionally in eukaryotes.
3. Unique structural features of tRNA include three loops and an "extra arm" created by internal hydrogen bond formation. The anticodon loop contains three bases that are antiparallel and complementary to the bases in the codon of mRNA. 4. A high degree of secondary structure is found in tRNA due to the bending back of the molecule on itself with the formation of internal base pairs that stabilize the secondary structure. About half of the nucleotides in tRNA are base paired to form double helices. 5. A large number of unusual bases are found in tRNA that are not found in other types of RNA. These are believed to be important in maintaining the characteristic secondary structure. Inosine, pseudouridine, dihydrouridine, ribothymidine, and methylated guanosine are found exclusively in tRNA.
THE GENETIC CODE The genetic code is the relationship between the sequence of bases in DNA (or its working RNA transcript) and the sequence of amino acids in proteins.The genetic code is read in triplets. The code words are almost (but not absolutely) universal. Human mitochondria have a few differences in codons. A. A codon is a group of three nitrogenous bases (base triplet) that represents an amino acid or signals the initiation or termination of protein synthesis. The codons in mRNA are antiparallel and complementary to the anticodons (recognition sites) found in tRNA. The codons in mRNA are shown in Figure II-5-3. There are 64 possible combinations of the four bases in mRNA, and thus the genetic code contains 64 codons. B. Amino acid codons. There are 61 triplets that specify 20 amino acids. (The remaining three codons are "stop" codons.) Because more than one codon can specify the same amino acid, the genetic code is said to be redundant or degenerate. Different co dons for the same amino acid usually differ in the third base. C. Stop codons. There are only three codons that do not specify an amino acid. These codons (UAA, UAG, UGA) act as termination signals during protein synthesis.
D. Start codon. AUG (which codes for methionine) and sometimes GUG are signals for the initiation of translation. In prokaryotic systems, polypeptide chains start with a modified amino acid, formylmethionine (fMet). Prokaryotes have a specific tRNA that carries fMet and recognizes the initiating AUG codon in mRNA. In eukaryotic systems, the AUG closest to the 5' end of the mRNA is the start signal for protein synthesis. Eukaryotes also have a specific initiating tRNA that carries fMet.
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8econd base U U
C
UCUj
UUU} UUC Phe UCC UCA UUA} UUG Leu UCG
CUUj CUC
C CUA CUG
Ser
CCUj CCC Leu
CCA CCG
Pro
ACUj
AUU} lie AUC ACC A AUA ACA AUG fJ~1 ACG
Val
GCA GCG
G
UAU} UAC Tyr UAA Stop UAG Stop
UGU} UGC Cys UGA Stop UGG Trp
U C A G
CAU}H' CAC IS
CGUj CGC
U C A G
CGA CAA} Gin CAG CGG
Arg
Thr
U AAU} AGU} AAC Asn AGC Ser C A AGA} ~}LYS AGG Arg G
Ala
GAU} GAC Asp GGC GGA GAA} GAG Glu GGG
GUUj GCUj GCC
GUC G GUA GUG
A
GGUj Gly
U C A G
Figure 11-5-3. The genetic code.
PROKARYOTIC AND EUKARYOTIC RIBOSOME STRUCTURE The structure and function of ribosomes are very similar in prokaryotes and eukaryotes. A. Common features. E. coli and eukaryotic ribosomes share the following features. 1. Function. Ribosomes are the sites at which protein synthesis occurs.
2. Components. Protein and rRNA are the building blocks for ribosomes. Each of the forms of rRNA and most of the proteins are present in only one copy per ribosome.
Note In general. the small subunit is necessary for initiating protein synthesis; the large subunit catalyzes the elongation steps.
3. Subunits. As shown in Figure 11-5-4, ribosomes have two subunits, one large and one small. (Note that the Svedberg units [S] are not additive.)
8mall subunit
Prokaryotes: 308: 168 rRNA + 20 proteins --- Eukaryotes: 408: 188 rRNA + 30 proteins Prokaryotes: 508: 238 and 58 rRNA + 34 proteins ---Eukaryotes: 608: 288,5.88, and 58 rRNA + 40 proteins
Ribosome
Prokaryote ribosome: 308 + 508 = 708 Eukaryote ribosome: 408 + 608 = 808 Figure 11-5-4. Composition of ribosomes.
B. Differences in prokaryotic and eukaryotic ribosomes. As seen in Figure 11-5-4, eukaryotic ribosomes are larger than prokaryotic ribosomes. In prokaryotes, the 70S ribosome is made up of a large subunit (50S) and a small subunit (30S), whereas eukaryotes have an 80S ribosome containing a large subunit (60S) and a small subunit (40S). Eukaryotic ribosomes contain four forms of rRNA, whereas prokaryotes have only three forms. Other minor differences are found in the number of proteins found in both the large and small subunits of eukaryotic and prokaryotic systems.
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meCtical
---~~~~~-
Protein Synthesis
C. Eukaryotic ribosome assembly. In eukaryotes, the large ribosomal subunit (60S) is assembled in the nucleolus, whereas the small subunit (40S) is assembled in the nucleus. The ribosomal proteins are synthesized in the cytoplasm and are transported into the nucleus, where they combine with the appropriate rRNA species to form the large and small ribosomal subunits. After assembly, the ribosome moves through the nuclear pores into the cytoplasm by an unknown mechanism. Because prokaryotes have no defined nucleus, all of these processes occur in the cytoplasm.
In a Nutshell
ACTIVATION OF AMINO ACIDS AND ATTACHMENT TO tRNA The formation of peptide bonds between amino acids is an endergonic process and therefore requires a source of energy. The energy is derived from ATP and is transferred to the bond that links the amino acid to the 3'-OH group of the tRNA molecule. Enzymes that "activate" the carboxyl group of amino acids are known as aminoacyl-tRNA synthetases. These enzymes are highly specific for both the amino acid and the tRNA. There is a specific enzyme for each amino acid. As shown in Figure 11-5-5, the attachment of the amino acid to the tRNA occurs in a twostep process. R I
Step I
I
0
II
NH2-CH-C-AMP+tRNA R I
Sum
I
0
II
ATP+NH 2 -CH-COOH - - - - - NH2-CH-C-AMP+PPj R
Step 2
R
Each tRNA must be "charged" with the correct amino acid by a separate tRNA synthetase.
R I
0
II
- NH 2-CH-C-tRNA+AMP R I
0
II
NH 2-CH-COOH+ATP+tRNA - - NH 2-CH-C-tRNA+AMP+PPj
Figure 11-5-5. Formation of aminoacyl-tRNA.
The AGO for this reaction is close to zero, but in the cell, the overall reaction is highly exergonic and essentially irreversible because the high-energy bond in the pyrophosphate product is rapidly hydrolyzed by pyrophosphatases.
STEPS INVOLVED IN TRANSLATION The process of translating the genetic code found in mRNA into a specific sequence of amino acids in a protein involves three steps: initiation, elongation, and termination. Protein synthesis occurs in the direction of amino terminal to carboxy terminal, and the mRNA is read from the 5' end to the 3' end. A. Initiation of protein synthesis. Initiation of protein synthesis requires special tRNA molecules and the formation of an initiation complex. 1. Initiating tRNA molecules. The codon AUG in mRNA usually signals the beginning of protein synthesis. AUG is the codon for methionine. a. Prokaryotes. The initial methionine residue has a formyl group attached to it (fMet). The initiating tRNA is known as tRNAfMet. The tRNA that carries methionine to any other position is known as tRNAMet. b. Eukaryotes. The initiating methionine has no formyl group. The initiating tRNA is known as tRNAiMet, whereas the tRNA involved in elongation of the protein is known simply as tRNAMet. KAPLAN" . ._
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2. Formation of initiation complex. The assembly of the 70S initiation complex found in prokaryotes is a stepwise process involving the formation of an intermediate 30S complex.
In a Nutshell In prokaryotes, the initiating AUG is defined by its position near a Shine-Oalgarno sequence. Therefore, prokaryotic mRNAs may contain multiple genes ("polycistronic"). Because each gene in the mRNA begins with a Shine-Oalgarno + AUG sequence, each will be independently translated by ribosomes. In eukaryotes, the ribosomal small subunit (with tRNAMet + e1F-2 + GTP bound) must begin each time at the 5' cap, then scan the mRNA for the first AUG to initiate, where it is joined by the large subunit. Eukaryotic mRNAs are typically monocistronic (one gene per mRNA).
a. Assembly of the 30S complex. Formation of the 30S complex requires mRNA, the 30S ribosomal subunit, three initiation factors (IF-I, IF-2, and IF-3), GTP, and tRNAtMet. The mRNA contains the start codon (AUG or GUG) that recognizes tRNAtMet. There is a special sequence of bases upstream from the AUG codon called the ShineDalgarno sequence. This sequence of bases binds with the 16S rRNA in the 30S ribosomal subunit. The binding of the mRNA to the 30S ribosomal subunit requires the transient help of IF-3. After mRNA binds to the 30S ribosomal subunit, IF-3 dissociates. The complex between mRNA and the 30S ribosomal subunit then binds IF-2, GTP, and tRNAtMet to produce an active 30S initiation complex. This reaction requires IF-I. b. Assembly of the 70S initiation complex occurs by the interaction of the 50S ribosomal subunit with the 30S complex. The formation of the 70S complex results in the hydrolysis of GTP to GDP and Pi and the dissociation of both IF-l and IF-2. The structure of the 70S initiation complex is shown in Figure 11-5-6. The association of the ribosomal subunits creates two important sites for protein synthesis within the ribosome, the A site and the P site. (1) The aminoacyl site (A site) binds the incoming tRNA molecule carrying an activated amino acid. (2) The peptidyl site (P site) is the site on the ribosome at which tRNAtMet initially binds. After formation of the first peptide bond, the P site is occupied by the growing peptide chain. The tRNAtMet recognizes two sites: the P site on the ribosome and the start site (AUG) on the mRNA. B. Elongation of the protein. This process is a three-step cycle that is illustrated in Figure II-5-6.
Each step is described below.
----,TT"'"""Trl~,.,.---
----,TT"'"""Trl.,..,.,.---3'
3'
Delivery of activated tRNAArg
P site
•
A site
P site
A site
70S Initiation complex Peptide bond formation by peptidyl transferase
-rl...--
mRNA 5' --r..,-".c"....
P site
..
tI
----,TT"'"""Trl.,..,.,.--- 3' Translocation
r
Empty A site
r
UAC
P site
Uncharged tRNA
Figure 11-5-6. Elongation phase of protein synthesis in bacteria.
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mellical
A site
Protein Synthesis
1. Binding of aminoacyl-tRNA to the A site. The A site will always contain the next amino acid to be added to the peptide chain. The specific aminoacyl-tRNA that comes into the A site is determined by the mRNA codon that is positioned above the A site. Thus, in Figure II-5-6, tRNAArg is delivered to the A site because CGC codes for Arg (see Figure 11-5-3). The delivery of the aminoacyl-tRNA to the A site requires elongation factor EF-Tu and the hydrolysis of GTP to GDP and Pi. GDP remains associated with EF-Tu until it is displaced by another elongation factor, EF-Ts. The new Tu-Ts complex is dissociated by the binding of a second GTP to give GTP-Tu complex.
2. Peptide bond formation. Formation of a peptide bond between the amino acid (or peptide) in the P site and the amino acid in the A site is catalyzed by peptidyl transferase, which is an integral part of the 50S ribosomal subunit. This reaction results in the release of the amino acid from the tRNA in the P site. The resulting peptide is bound to the tRNA in the A site. 3. Translocation. In order for elongation to continue, translocation has to occur. This involves three movements. First, the uncharged tRNA leaves the P site. Next, the peptidyl tRNA moves from the A site to the P site. Finally, the mRNA moves a distance of three nucleotides to bring a new codon into a position above the empty A site. The movement of mRNA requires an elongation factor known as translocase (EF-G). Hydrolysis of GTP is required to release EF-G from the ribosome. At some point during elongation of the peptide chain, the formyl group is removed from the initial methionine residue. C. Termination of protein synthesis. When anyone of the three stop codons is encountered,
elongation is terminated. Cells do not have tRNAs with anticodons that are complementary to stop codons. Release of the peptide from the ribosome requires a protein known as a release factor (RF) that binds to the stop codon. Hydrolysis of the peptidyl-tRNA bond requires an enzyme, peptidyl-tRNA hydrolase, and GTP, which is hydrolyzed to GDP and Pi' The polypeptide leaves the ribosome, the ribosome dissociates into its 30S and 50S subunits, and the mRNA is released.
Note During peptide bond formation, the amino group of the amino acid bound to the A site tRNA is bound to the a-carboxy group of the amino acid bound to the Psite tRNA.
In a Nutshell Elongation is catalyzed by the large subunit: • Charged tRNA binds to the A site (requires GTP hydrolysis) • Peptide bond formation • Translocation (requires GTP hydrolysis)
INHIBITORS OF PROTEIN SYNTHESIS Several antibiotics recognize differences between prokaryotic and eukaryotic protein synthesis and can be used to inhibit one independent of the other. Additionally, some bacterial toxins inhibit protein synthesis in animal cells. Table II -5-1 lists several inhibitors of protein synthesis.
Table II-5-l. Inhibitors of protein synthesis. Inhibitor
Prokaryote or Eukaryote Step/Site of Action
Tetracycline
Prokaryotes
Streptomycin Erythromycin Chloramphenicol Puromycin
Prokaryotes Prokaryotes Prokaryotes Both
Cycloheximide Sparsomycin
Eukaryotes Eukaryotes
Ricin Eukaryotes Diphtheria toxin Eukaryotes Pseudomonas toxin Eukaryotes
Initiation; prevents binding of aminoacyl-tRNA to ribosome Initiation; causes misreading of code Translocation Ribosomal peptidyl transferase Elongation; binds to A site and prematurely terminates chain growthl Ribosomal peptidyl transferase . Initiation; inhibits formation of initiation complex Initiation; binds to 60S subunit Translocation; inactivation ofEF-2 Translocation; inactivation ofEF-2
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Regulation of Ciene Expression
Regulation of gene expression is an essential feature in maintaining the functional integrity of a cell. Regulation of a gene may occur in a variety of ways; some are positive, whereas others are negative. Regulation of gene expression in prokaryotes almost always involves either initiation or termination of transcription. In eUkaryotes, transcription is more complicated than in prokaryotes, and, therefore, there are more possible sites for regulation. In both prokaryotes and eukaryotes, transcriptional regulation is usually achieved by the interaction of proteins with specific sequences in the DNA, resulting in either an increase or a decrease in the rate of transcription. All of the cells in eukaryotic organisms contain the same genome, yet different types of cells contain unique complements of mRNAs and proteins. Therefore, mechanisms for specifying cell-specific transcription exists in eukaryotes but not in prokaryotes. In this chapter, the basic mechanisms for regulating transcription in both prokaryotes and eukaryotes are summarized. A few selected examples of gene regulation are discussed that illustrate different mechanisms of transcriptional control.
REGULATION OF TRANSCRIPTION IN PROKARYOTES The regulation of gene expression in prokaryotes is usually achieved by regulating the rate at which transcription is initiated. The examples that will be considered are the positive and negative control of the lactose (lac) operon in E. coli. A. Operon concept. In bacteria, a set of structural genes and a regulatory region constitute an operon. The structural genes code for a group of proteins required for a particular metabolic function. The regulatory region is upstream (to the 5' side) of the structural genes. The structural genes in an operon are coordinately regulated, i.e., the expression of all the structural genes is controlled by the same regulatory region in the DNA. B. Description of the lac operon. The arrangement of the structural and regulatory genes in the lac operon of E. coli is shown in Figure 11-6-1. The regulatory gene (i) codes for a repressor that can interact with the operator sequence (0). The operator sequence is situated adjacent to the three structural genes (Z, Y, and A) that code for three enzymes concerned with lactose metabolism (B-galactosidase, permease, and transacetylase). The expression of these three genes is regulated by the operator sequence. RNA polymerase binds at the promoter site (P).
1. Negative regulation of the lac operon. In the absence of lactose, the repressor protein binds to the operator sequence and blocks the movement of RNA polymerase along the template, thus inhibiting transcription (Figure II -6-1A). When lactose is present, it induces transcription by the following mechanism: Lactose is converted to 1,6-allolactose, a molecule that binds to the repressor and changes its conformation so that it no longer binds to the operator region. Thus, the repressor is released and the three structural genes are transcribed as a single mRNA that codes for all three proteins (Figure II-6-1B).
Note Each structural gene in the polycistronic lac mRNA has a Shine-Dalgarno sequence to define its initiating AUG.
Note The lac repressor is made constitutively, i.e., whether or not lactose is present.
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Messenger RNA coding for more than one protein is known as a polycistronic mRNA and is found only in prokaryotic systems. In this model of negative regulation of an operon, a protein acts as the repressor and lactose (or l,6-allolactose) acts as the inducer of transcription.
In a Nutshell Full transcription of the lac operon requires: • Absence of the repressor (negative regulator) • Presence of the activator (positive regulator)
2. Positive regulation of the lac operon. The function of ~-galactosidase in lactose metabolism is to cleave lactose to glucose and galactose. The galactose is ultimately converted to glucose by other enzymes. Therefore, if the bacteria has both glucose and lactose in the growth medium, there is no reason to activate the lac operon. When glucose is low, the lac operon is activated, and the bacteria begin to use lactose to generate glucose. The effects of glucose are mediated by cAMP. When glucose is low, cAMP concentration is high and vice versa. When cAMP increases, it binds to a protein known as CAP (catabolite activator protein). The CAP-cAMP complex activates transcription of the lac operon by binding to the promoter region and allowing RNA polymerase to initiate transcription. Thus, the CAP-cAMP complex is a positive regulator of the lac operon.
A. In the absence of lactose Regulatory region
Control region
Structural genes jrj----------------------------~
p
01
z
y
A
! Repressor mRNA
t
Repressor binds to 0 and structural genes cannot be transcribed
Repressor
B. In the presence of lactose Regulatory region
Control region
p
! t
Structural genes
jrj-----------------------------,
o
z
y
A
!
.-
Repressor mRNA
Beta-galactosidase, permease, transacetylase polycistronic mRNA
Irll
Repressor-Inducer complex
Repressor + Inducer
The repressor cannot bind to 0 because it is bound to the inducer and so structural genes are transcribed.
Figure 11-6-1. Negative control of the lac operon.
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Regulation of Gene Expression
PACKAGING OF EUKARYOTIC DNA The DNA molecule in eukaryotes is associated with histones. The DNA-histone complex is known as chromatin and is organized into discrete units of higher structure in the eukaryotic chromosome. A. Eukaryotic chromosomes contain two specialized structures that are required for replication and segregation during mitosis. 1. Telomeres are G-rich, block-like structures located at the end of eukaryotic chromosome
arms. They are essential for the completion of DNA replication. 2. Centromeres are the chromosomal sites where the two daughter chromatids attach during mitosis. Centromeres are essential for aligning the DNA molecule on the mitotic spindle during mitosis. They are required for segregation and stability of the mitotic apparatus. The centromeres attach to the spindle fibers and are responsible for movement during both mitosis and meiosis.
Note Telomeres protect the ends of the linear DNA molecules from being degraded by exonucleases and are needed to complete replication.
B. Chromatin composition. Chromatin is composed oflinear double-stranded DNA and five types of histones (HI, H2A, H2B, H3, and H4). Histones have a high density of positive charge that interacts with negatively charged phosphate groups of DNA. Histones contain a large number of side chains that are modified by methylation, acetylation, or phosphorylation after the protein is translated. C. Chromatin organization. There are various levels of organization that chromatin undergoes
during packaging.
Note
1. Nudeosomes. Electron micrographs of chromosomes show long fibers that are 10 nm
Highly transcribed regions of DNA contain fewer nucleosomes than less transcribed regions.
in diameter and contain discrete bead-like structures equally spaced along the lO-nm fiber. The bead-like particles are DNA-histone complexes known as nucleosomes, which are connected to one another by linear regions of DNA. The core of the nucleosome contains approximately 140 base pairs of DNA wrapped twice around eight histones (two each of H2A, H2B, H3, and H4). Approximately 160 base pairs of linear DNA connect adjacent nucleosomes. Nucleosomes constitute the first level of packaging of DNA. 2. Solenoid fibers. Histone HI binds to the linear DNA between nucleosomes. Chromatin that contains HI forms solenoid-like fibers, with six nucleosomes per turn of the helix. The solenoid fibers constitute the second level of packaging of eukaryotic DNA. A still higher level of organization occurs when the solenoid fibers are organized into loops of greater than 20 kilobases. D. Chromatin and gene regulation. Chromatin can be organized into two classes based on their genetic activity. 1. Heterochromatin contains DNA that is genetically inactive and highly condensed. The
regions of DNA that condense in early prophase are known as heterochromatin. Most of these regions are located near the centromere, and, although they are not transcribed, they playa role in pairing and segregation of homologous chromosomes. Heterochromatin is found in the 30-nm solenoid fibers folded into looped domains. 2. Euchromatin makes up most of the genome, and about 10% of euchromatin contains genes that are actively transcribed. This active euchromatin is made up of lO-nm fibers of nucleosomes connected to one another by linear DNA.
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In a Nutshell
REGULATION OF EUKARYOTIC GENE EXPRESSION
The major eukaryotic gene regulation mechanisms occur in the nucleus, including:
Eukaryotic gene expression is much more complex than that of prokaryotes and can occur at several levels. Although regulation of mRNA transcription initiation appears to be the most common type of gene regulation in eukaryotes, several other levels of control also exist. Alternative processing of mRNA primary transcripts into different mature mRNAs provides an important mechanism for cell-type specific gene expression. Within the cytoplasm, both the stability of the mRNA and the initiation of translation can be regulated. Other examples are also mentioned.
• Regulation of transcription initiation • Alternative mRNA processing • DNA modifications or rearrangements
Note Regulatory proteins (transcription factors) exert their effects in trans, i.e., they can diffuse through the nucleoplasm to affect any and all genes that contain their recognition sequences (enhancers, silencers).
A. Initiation of eukaryotic mRNA transcription 1. Description. As with prokaryotes, the rate of mRNA transcription is regulated by the interaction of regulatory proteins that bind to DNA sequences near the promoter and modulate the rate at which the initiation complex can form on the promoter. In eukaryotes, general transcription factors (including TFIID and TFIIB) must bind to the promoter to allow RNA polymerase II to bind and form the initiation complex. Specific transcription factors bind to regulatory DNA sequences, often called enhancer or silencer sequences, and modulate the formation of the initiation complex, thus regulating the rate of transcription. 2. Transcription factors. Transcription factors are proteins that bind to regulatory DNA sequences and to other transcription factors or to RNA polymerase II in order to regulate initiation of transcription. Typically, transcription factors contain at least two recognizable domains, a DNA-binding domain and an activation domain. a. The DNA-binding domain binds to a specific sequence in the promoter, enhancer, or silencer. Several types of DNA-binding domain motifs have been characterized and have been used to define certain families of transcription factors. Families based on DNA-binding domains include "zinc fingers" (seen on steroid hormone receptors), and "homeodomain" (seen on some transcription factors involved in development). b. The activation domain allows the transcription factor to bind to another transcription factor or to RNA polymerase II to stabilize the formation of the initiation complex. These are less well characterized. c. Some transcription factors must dimerize in order to form functional DNA-binding domains and functional activation domains. These include the "helix-loop-helix" family and "leucine zipper" family. Within each family, partners may form heterodimers or homodimers, creating dimeric combinations with a variety of DNA sequence specificity and a variety of activating or inhibiting abilities. Because different cells can express different partners within each family, this greatly increases the possible range of gene regulation. The genes that code for fos and jun, two transcription factors important in regulating cell proliferation, are members of the "leucine zipper" family. When expressed abnormally (or overexpressed), these two genes can contribute to transformation of the cell; therefore, fos and jun are both proto-oncogenes.
Note DNA sequences (promoters, enhancers, silencers) exert their effects in cis, i.e., they can affect only the gene to which they are attached.
3. Enhancer and silencer sequences. These are DNA sequences to which the specific transcription factors bind to regulate transcription. Enhancer sequences contain binding sites for specific transcription factors that increase transcription. Enhancers are functional both upstream and downstream of the promoter and in either orientation. Silencers contain binding sites for specific transcription factors that inhibit transcription. B. Regulation by specific transcription factors. Each gene contains a variety of enhancer and
silencer sequences in its regulatory region. Each enhancer or silencer sequence may be recognized by a different combination of specific transcription factors. The exact combination
154
iile&ical
Regulation of Gene Expression
of specific transcription factors available (and active) in a particular cell at a particular time determines which genes will be transcribed at what rates. 1. Regulating transcription factor function. Because specific transcription factors are pro-
teins, their expression can be cell-type specific. Additionally, hormones can regulate the activity of specific transcription factors. 2. Examples
Note
a. Thyroid receptor (T3R) is localized in the nucleus. Besides its DNA-binding and activation domains, it contains a thyroid-binding domain at its C-terminus. This receptor is found in many tissues, but it is expressed in very high levels in the anterior pituitary, where it increases transcription of the growth hormone gene. The effects of thyroid hormone on the anterior pituitary are shown in Figure 11-6-2. Thyroid hormone activates growth hormone transcription. T3 (or T 4) enters the cell and moves into the nucleus, where it binds to its receptor. The T3R complex undergoes a conformational change that allows it to bind to specific enhancer sequences located upstream from the growth hormone gene. In the nucleus, the rate of transcription is regulated by RNA polymerase II, resulting in increased mRNA for growth hormone. Translation of mRNA in the cytosol results in increased growth hormone synthesis. The steroid hormones exert their effects by a similar mechanism. The DNA-binding domain of thyroid hormones and steroid hormones contains "zinc fingers;' a structural motif that binds to DNA.
_....,t""-_AAAn HnRNA
Steroid and thyroid hormone receptors are hormoneactivated specific transcription factors that allow these hormones to change gene expression.
•
Processing
1
Growth 5'Cap - - - AAAn hormone mRNA Transport to cytoplasm
Cytoplasm
Growth 5'Cap --:---- AAAn hormone mRNA Translation •
•
!
Growth hormones
Secreted growth hormone
Figure 11-6-2. Activation of growth hormone transcription by thyroid hormone. KAPLA~. I meulca
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b. CREB is a specific transcription factor found in many cell types that is active only when cAMP levels rise in the cell. The cAMP-dependent protein kinase phosphorylates CREB, enabling it to bind to its enhancer sequence in various genes and activate their transcription. In hepatocytes, CREB affects the transcription of some of the key enzymes in gluconeogenesis. c. AP-l is a family of transcription factors. Members include the fos-jun complex consisting of one molecule of fos bound to one molecule of jun. Fos and jun are proteins encoded by proto-oncogenes; mutations in these genes have been associated with a wide variety of tumors. A range of cellular stimuli, including growth factors, phorbol esters, and neurotransmitters, regulate the activity of the fos-jun complex. The dimerization of these proteins is highly specific and occurs through a domain on the proteins known as the "leucine zipper." This domain consists of repeating leucine residues occurring at every seven amino acids in a region of a-helical structure.
Note
C. Alternative mRNA processing
Alternative mRNA processing allows different proteins to be synthesized from the same gene and pre-mRNA.
1. mRNA processing. Eukaryotic mRNA primary transcripts are extensively processed before the mature mRNA is transported into the cytoplasm. On each mRNA the 5' end is capped, the 3' end is cleaved to allow addition of polyadenosine, and the introns are spliced out. The presence of the sequence AAUAAA in the mRNA signals a 3' cleavage/polyadenosine addition site, and splice donor (GU) and splice acceptor (AG) sequences define splicing patterns.
2. Cell-type specific gene regulation. Alternative use of such signals allows the formation of different mRNAs from transcripts of the same gene. The alternative mRNAs are translated into different proteins. This is often seen in the production of different proteins in different cell types produced from the same gene. 3. Example. The calcitonin gene contains exons for both calcitonin, a hormone that regulates blood calcium and phosphate levels, and CGRP, a protein that regulates neural receptor expression. In thyroid C cells (stromal cells located between the follicles in the thyroid gland), this primary gene transcript is cleaved in front of the CGRP exon and spliced to form an mRNA containing the calcitonin exon. In neural cells, a cleavage signal after the CGRP exon is used and the calcitonin exon is spliced out, leaving an mRNA containing the CGRP exon. D. Regulation of initiation of translation
Note Regulation of gene expression can also occur in the cytoplasm via: • Regulation of translation initiation • Regulation of mRNA stability
1. Initiation of eukaryotic translation requires the small ribosomal subunit, charged initiator tRNA (tRNAiMet), GTP, and several initiation factors. The two important initiation factors are eIF-2 and eIF-4.
a. eIF-2, GTP, and tRNAiMet form a complex and bind to the small (40S) ribosomal subunit, forming a pre-initiation complex. b. The cap-binding protein subunit of eIF-4 recognizes the 7-methyl-guanosine "cap" at the 5' end of eukaryotic mRNA and allows the pre-initiation complex to bind. c. The pre-initiation complex scans down the mRNA until it encounters the first AUG in the appropriate sequence; GTP is hydrolyzed to GTP, the large ribosomal subunit joins the complex, and the initiation factors leave. Translation continues much as in prokaryotes. 2. Some viruses, such as polio virus, ensure preferential translation of viral mRNAs by cleaving the eIF-4 cap-binding subunit but providing a cap-independent alternative recognition site for the pre-initiation complex (called a "ribosomal landing pad").
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Regulation of Gene Expression
3. Initiation of translation can also be regulated by proteins that bind to sequences on the mRNA. a. Ferritin is a cytoplasmic Fe2+-binding protein expressed in hepatocytes only when cytoplasmic Fe2+ levels are high. Ferritin sequesters the Fe2+, preventing it from being toxic to the cell. b. Ferritin mRNA contains three "IRE" sequences between the 5' cap and the first AUG. The hepatocyte also makes an IRE-binding protein that binds to the IRE sequence and prevents the pre-initiation complex from scanning from the cap to the AUG; this inhibits the translation of ferritin.
Note Certain sequences found near the 3' end of some mRNAs decrease mRNA stability. Regulatory proteins can reversibly mask these destabilizing sequences, increasing protein synthesis.
c. As Fe z+ levels rise in the cell, iron binds the IRE-binding protein and changes its con-
formation. The Fe2+ -IRE-binding protein complex cannot bind to the IRE, so ferritin is translated. E. The level of packing of the DNA can also affect transcription. 1. Condensed DNA is not accessible to transcription factors or RNA polymerases and so is not transcribed. The level of packing of the DNA can be regulated by the frequency of methylation of cytosines found in the sequence 5' CG 3'. Such methylation inhibits the binding of specific transcription factors to their enhancer sequences and promotes higher levels of packing of the DNA. Different methylation patterns are found in different cells; the regulation of these patterns is not clear. 2. Transient modification of histones, such as acetylation or binding of ubiquitin (which may target them for degradation by proteases), is associated with unpackaging the DNA before transcription. F. Regulation of 5S rRNA gene (Xenopus). Regulation of rRNA transcription is much less common than the regulation of mRNA transcription but does occur in Xenopus oocytes. The
5S rRNA genes are tandemly repeated and organized in clusters. These genes have an internal promoter and are transcribed by RNA polymerase III. 1. Xenopus has two types of gene clusters for 5S rRNA: One gene cluster code for the somat-
ic-type 5S rRNA and the other cluster codes for the oocyte 5S rRNA. Both types of genes are present in all cells. Although the gene types differ from each other at only six positions in the 120 base-pair coding region, they are transcribed at very different rates. The somatic gene population constitutes 2% of 5S rRNA genes and accounts for more than 95% of the 5S rRNA synthesized in somatic cells. In the oocyte, almost all of the 5S rRNA is transcribed from the oocyte gene cluster. 2. The differential transcription of the two 5S rRNA genes can be accounted for by different interactions between the promoter and a transcription factor, TFIIIA. TFIIIA binds to the internal promoter, signaling RNA polymerase to initiate transcription. TFIIIA has a greater affinity for somatic 5S rRNA genes than for the oocyte 5S rRNA genes. In fact, the binding of TFIIIA to oocyte 5S rRNA genes is prevented in regions of the chromosome that contain histone HI. Early in oogenesis, there are very high levels of TFIIIA (101Z molecules/oocyte), and under these conditions all internal promoters of the 5S rRNA genes (both somatic and oocyte) bind to TFIIIA, activating transcription. As the 5S rRNA gene is expressed and the level of 5S rRNA increases in the nucleus, the 5S rRNA begins to bind TFIIIA and deplete the transcription factor, thus decreasing transcription. Therefore, the 5S rRNA regulates its own synthesis.
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Genetic Engineering
Genetic engineering, also known as recombinant DNA technology, provides a means of analyzing and altering genes and proteins. Additionally, this technology can provide a source of specific proteins in almost unlimited quantities and has many applications in clinical medicine. Genetic engineering depends on having an array of tools for analyzing genes and proteins, including enzymes that can cut, join, and replicate genes. This chapter reviews the procedures and related techniques of genetic engineering and indicates how these procedures can be applied to clinical medicine.
TOOLS USED IN RECOMBINANT DNA TECHNOLOGY A. Enzymes that modify nucleic acids. Many enzymes that alter the structure of nucleic acids are used in genetic engineering. 1. Nucleases constitute a family of enzymes that hydrolyze phospho diester bonds. They are classified either as ribonucleases or deoxyribonucleases, depending on whether they hydrolyze RNA or DNA. Both ribonucleases and deoxyribonucleases can be further classified as endonucleases (if they cleave internal phosphodiester bonds) or as exonucleases (if they cleave terminal phosphodiester bonds, either at the 5' or 3' end of the chain). 2. Restriction endonucleases are enzymes that recognize specific double-stranded sequences in DNA and cleave the DNA at or near the recognition or "restriction" site. These enzymes are powerful tools in recombinant DNA technology, allowing specific genes to be excised from the genome. Many restriction endonucleases have been isolated from bacterial sources. Isochizomers are restriction endonucleases from two different sources that recognize the same DNA sequence. Most restriction nucleases recognize sites consisting of four to eight base pairs. The sequences in the two strands of the recognition site are palindromes, having the same 5' to 3' sequence in both strands. Two examples of restriction endonucleases are shown in Figure 11-7-1.
!
t
5'-GGCG-3' 3'-CCGG-5'
t
!
feoRI
5'-G + 3'-CTTAA-5'
!
Haelll
5'-MTTC-3' G-5'
The location of restriction sites along a DNA helix depends upon its nucleotide sequence: • Use of a restriction enzyme on mUltiple samples of the same sequence will always generate the same pattern of fragment sizes.
!
5'-GMTTC-3' 3'-CTTMG-5'
Note
5'-GG-3' + 5'-CC-3' 3'-CC-5' 3'-GG-5'
Figure 11-7-1. Examples of restriction endonucleases.
• A mutation or difference in the DNA sequence can change the presence or absence of a restriction site, thereby changing the sizes of the resulting fragments. KAPLA~.
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a. EcoRI recognizes a specific six-base-pair sequence in double-stranded DNA and cleaves one site in each strand, as indicated by the arrows in Figure II-7-1, producing staggered cuts that leave short single-stranded tails at the two ends of each fragment. Ends of this type are known as sticky ends because each tail can form complementary base pairs with the tail of any other fragment produced by EeoR!. b. HaeIII recognizes a specific four-base-pair sequence in double-stranded DNA and cleaves both strands at positions opposite one another, producing blunt ends with no unpaired tails. 3. Reverse transcriptase is an enzyme found in retroviruses whose genome is composed of RNA rather than DNA. This enzyme is an RNA-dependent DNA polymerase that can synthesize a single strand of DNA using a complementary RNA strand as a template. It is used in recombinant DNA technology to construct synthetic DNA molecules known as cDNA (complementary DNA) by copying the corresponding mRNA. 4. DNA and RNA polymerases are used to synthesize a particular DNA or RNA, respectively. Unlike RNA polymerase, DNA polymerase requires a primer (a 3' -OH group at the end of an oligonucleotide chain) to initiate synthesis. 5. DNA ligase forms a phosphodiester bond between two DNA fragments. This is an essential step in creating recombinant DNA molecules. 6. SI nuclease acts exclusively on single-stranded polynucleotides or single-stranded regions of double-stranded polynucleotides. Double-stranded regions are resistant to its action. 7. Terminal deoxynucleotidyl transferase catalyzes the addition of deoxynucleotides to the 3'-OH end of the DNA molecule. It does not require a template (Figure II-7-2). Most restriction endonucleases produce complementary "sticky" ends. However, DNA fragments that have "blunt" ends can be joined by using terminal deoxynucleotide transferase to add complementary homopolymer tails. Thus, two molecules can be joined if poly( dA) tails are put on one molecule and poly( dT) tails are put on a second molecule.
5 ' - - - - - - - - 3' 3' 5'
tI
Terminal deoxynucleotidyl transferase dTTPP
5'-------TTTT3' 3'TTTT 5'
In a Nutshell
Figure 11-7-2. Terminal deoxynucleotide transferase reaction.
To radioactively label the 5' end of a nucleic acid:
8. Alkaline phosphatase removes the S'-phosphate group from either single-stranded or double-stranded DNA or RNA to give a 5' -OH group.
• Remove existing 5' phosphate with alkaline phosphatase.
9. Polynucleotide kinase catalyzes the transfer of the terminal phosphate group of ATP to a 5' -OH group in either DNA or RNA. The physiologic function of this enzyme is unknown. However, it is useful in radiolabeling nucleic acids, a procedure used in determining the base sequence of polynucleotides. Because naturally occurring nucleic acids usually contain 5' phosphate rather than 5' OH, labeling with polynucleotides can be accomplished only after the S'-phosphate group is removed with alkaline phosphatase.
• Use radioactive ATP and poly-nucleotide kinase to replace it with radioactive phosphate. 160
meClical
10. Eco RI methylase catalyzes the methylation of the adenine (A) residue in the EeoR! recognition sequence, thereby protecting the DNA sequence from cleavage by BeoR!.
Genetic Engineering
B. Oligonucleotides. Short DNA molecules, usually 16 to 40 base pairs, can be chemically synthesized from nucleotide building blocks. Two specific uses of these oligonucleotides are discussed below. 1. Hybridization screening probes. Radiolabeled oligonucleotides can be used in
hybridization experiments to screen cells for specific DNA clones. Oligonucleotides with base sequences complementary to different parts of the gene of interest are synthesized. Colonies of cells that hybridize with two different oligonucleotide probes are considered to be strong candidates for containing the desired gene. 2. Detection of specific mutations in genes. The knowledge of either the protein sequence or the DNA sequence of a particular gene allows synthesis of specific oligonucleotides that will hybridize with specific regions in the gene. A mismatch of a single base can result in the formation of unstable hybrids. The shorter the oligonucleotide, the more sensitive the probe to base changes in the gene.
TECHNIQUES AND APPLICATIONS IN MOLECULAR BIOLOGY A. Gel electrophoresis is very useful in recombinant DNA technology for separating fragments resulting from cleaving DNA with restriction endonucleases. Electrophoresis permits the separation of DNA (or RNA) molecules by measuring the rate at which they move in an electric field. DNA molecules have a negative charge because of the phosphate groups. In an electric field, these molecules move toward the positive pole at a rate that is dependent upon their size. Smaller DNA molecules move more rapidly than do larger molecules. 1. Types of gels. Two types of gels are typically used for separating DNA.
a. Polyacrylamide gels are used to separate small single-stranded DNA fragments ranging from about 10 to 500 nucleotides.
In a Nutshell Probes (in a single-stranded form) will hybridize with any homologous sequence in the (denatured) genomic DNA. • Long probes can be made from known genes or cDNAs. Minor sequence differences, such as allelic differences, or sometimes even the difference between a mouse and a human version of a gene, will not prevent hybrid formation. • Short oligonucleotide probes are more sensitive to differences in nucleotide sequence.
b. Agarose gels are used to separate double-stranded DNA fragments ranging from approximately 300 to 20,000 base pairs in length. 2. Applications a. Separation of DNA on the basis of size. In the experiment shown in Figure II -7 -3, a 5-kilobase (kb) DNA molecule was digested with two different restriction endonucleases (RE). Digestion with RE-A results in two fragments of the same size (2.5 kb), whereas digestion with RE-B results in three fragments of different sizes (2.5 kb, 1.5 kb, and 1 kb). After digestion, the fragments are separated by electrophoresis on an agarose gel. Molecular weight markers (MW) of different sizes ranging from 0.7 kb to 5 kb and a sample of undigested DNA (5 kb) are also submitted to electrophoresis. The position of DNA fragments in the gel is detected by staining the gel with ethidium bromide, a dye that binds to DNA and can be visualized as a fluorescent band when the gel is illuminated with UV light. Note that the pattern of electrophoresis of the DNA fragments differs between RE-A and RE-B. With RE-A, only one band is detected because the enzyme cuts the 5-kb DNA into two pieces of equal size.
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7
(5 kb)
Sample DNA
Res'dct;on Enzyme
,,\es.,;cllon Enzyme B
~(2.5kb)
~(2.5kb)
~(2.5kb)
~
(1.5kb) (1 kb)
2 DNA fragments
3 DNA fragments
MW UC RE-A RE-B kb 5
- pole
2.5 2
Electrical field
0.7
+ pole
UC = uncut DNA
Figure 11-7-3. Restriction enzyme digestion and agarose gel electrophoresis.
b. Separation of DNA molecules on the basis of shape. Gel electrophoresis can distinguish between different configurations of a DNA molecule. Supercoiled DNA is more compact and migrates faster than open circular DNA and linear DNA of the same mass.
B. Hybridization. In molecular biology, hybridization is defined as the complementary pairing of an RNA and a DNA strand, or of two different DNA strands, to form a stable doublestranded molecule. 1. Definitions and principles of hybridization are illustrated in Figure II -7 -4. The breaking of hydrogen bonds between two complementary nucleotide strands is defined as denaturation. Conditions promoting denaturation are high temperatures or treatment of DNA with high salt, alkali, urea, or formamide. The temperature at which the double-stranded DNA molecule denatures into single-stranded DNA is defined as the melting temperature (Tm). In addition to solution conditions of ionic strength, pH, etc., the Tm is dependent upon the relative number of A-T (or A-U) and G-C base pairs in a given DNA or DNA-RNA hybrid. Because G-C base pairs have three hydrogen bonds, they are more stable than the A-T and A-U base pairs with only two hydrogen bonds. Thus, the higher the number of G-C base pairs in a given DNA molecule, the higher the Tm. The Tmis also dependent upon the number of mismatched base pairs: The greater the number of mismatches, the less stable the hybrid and the lower the Tm. Renaturation or annealing between two separated complementary strands occurs upon the cooling of the heatdenatured DNA or by the removal of agents that promote denaturation.
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Genetic Engineering
~ ~ ~
Double-stranded DNA
~aturat;o" ~ ~
~ ~ ~ ~
Initial base pairing
Single-stranded DNA
Figure 11-7-4. Denaturation and renaturation.
2. Applications. The principles of hybridization are used in numerous techniques, including Southern blotting, Northern blotting, and in situ hybridization. a. Southern blotting is used to detect DNA fragments in an agarose gel by hybridization with a specific radiolabeled oligonucleotide probe. The technique is illustrated in Figure II-7-5 and involves the following steps:
A.
B.
* Isolate genomic DNA
* Restriction
C.
* Agarose gel
electrophoresis
digestion
kb MW 27
Long DNA fragments
DNA fragments 0.5 Genomic DNA
Short DNA fragments
DNA fragments Agarose gel
D.
E.
* Denature gel in alkali * Blot-transfer onto
* Hybridize with
F.
* Autoradiography
radioactive probe * Wash blot
nitrocellulose * Bake nitrocellulose
Singlestranded -+-----+- DNA fragments ...
Nitrocellulose filter
Washed nitrocellulose filter
Figure 11-7-5. Southern blotting.
-
Photographic film
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Note A Southern blot of two samples of DNA exposed to the same restriction enzyme and probe may reveal different fragments (in size and/or number). Such a difference is due to a sequence difference that has affected the position of a restriction site and is called a restriction fragment length polymorphism (RFLP).
In a Nutshell Northern blots are often used to detect which cell types express mRNA from a particular gene.
Bridge to Physiology In situ hybridization can detect the chromosomal location of a gene, or it can detect the cells in an intact tissue that express that gene.
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(1) Cleavage of genomic DNA by treatment with a specific endonuclease. The fragments are separated according to size by agarose gel electrophoresis. Because the sample used in the figure was genomic DNA, thousands of fragments were produced. When submitted to electrophoresis, the digested DNA appears as a continuous smear of fragments from the top to the bottom of the gel. (2) Denaturation of the DNA fragments is achieved by treatment with alkali. The gel is then placed on a buffer-saturated filter paper, covered with a sheet of nitrocellulose, and overlaid with dry filter paper. (3) Immobilization on nitrocellulose. The filter paper draws buffer out of the gel. The buffer carries single-stranded DNA with it, and when the DNA comes in contact with the nitrocellulose filter, it binds to the nitrocellulose and is immobilized. (4) Detection of a specific DNA fragment by hybridization. The filter is dried and incubated with a solution containing a radioactive DNA probe that is complementary to the sequence of the DNA of interest. The radioactive probe and the DNA on the filter are allowed to renature under conditions that promote hybridization. Under these conditions, only the DNA that is complementary to the radioactive oligonucleotide probe will be recognized by the probe. The filter is washed and dried, and the region of hybridization is visualized by autoradiography. Southern blotting is a very sensitive method and can detect DNA sequences present in a single copy per genome. b. Northern blotting is a technique similar to Southern blotting, but it is used to determine the size and abundance of mRNA for a specific gene in a sample of total cellular RNA. Figure II -7 -6 uses the total RNA from liver to test for the mRNA expression for albumin. The total RNA includes rRNA, tRNA, and mRNA. Because mRNA contains poly-A tails, it can be isolated from the other types of RNA by hybridizing it to an oligo d(T) column. The mRNA is removed from the column by heat denaturation and is run on a denaturing agarose gel containing formamide. The mRNA is then transferred onto nitrocellulose filter paper. The filter is baked and hybridized with a singlestranded radioactive probe. The filter is then washed, and the position of the mRNA for albumin is identified by autoradiography. By use of marker RNA molecules of known size, the size of the specific mRNA can be determined. c. In situ hybridization can be used to detect the distribution of a specific mRNA in tissue sections. The radioactive nucleic acid probe is incubated directly with the tissue. After hybridization, it is possible to determine exactly which population of cells in the tissue expresses the specific mRNA of interest. This technique can also be used to detect the position of a specific DNA sequence on a chromosome. C. Cloning vectors. Cloning vectors are DNA molecules that have inserts of foreign DNA incorporated into their genome. There are two important features of cloning vectors: They possess sites at which the foreign DNA can be inserted without disrupting its function, and they replicate in the host cell independently of the host cell chromosome. Two of the most commonly used cloning vectors are plasmids, which are routinely used for cloning short DNA fragments (fewer than 5-10 kilobases), and bacteriophages, which are used for cloning larger DNA fragments (up to 20 kilobases).
Genetic Engineering
A.
* Prepare liver total RNA
B.
* Isolate poly-A+ RNA
using oligo d(T) column • mRNA with poly· (A) tail
IIIIIII
C.
* Agarose-formaldehyde
gel electrophoresis rRNA poly A+
An
IIIIIII An • rRNA (288, 188)
I II III
288
-
188
-
• tRNA
+
D.
+ Agarose-formaldehyde gel
rRNA poly A+
* Blot-transfer onto
nitrocellulose * Bake nitrocellulose * Hybridize with
radioactive probe (albumin cDNA or oligonucleotide) * Wash blot * Autoradiography
288 -
c::=I
188 -
c::=I
-
....~- Albumin mRNA
Photographic film Figure 11-7-6. Northern blotting.
1. Plasmids
a. Plasmids are small, circular, double-stranded DNA molecules (2-10 kilobases) that replicate in bacteria. Multiple copies of plasmid DNA can be found in bacterial cells. The important features of a plasmid cloning vector are shown in Figure 11-7-7. They contain an origin of replication (ori) that permits autonomous replication, one or more genes that confer specific antibiotic resistance, and sites for restriction endonucleases that can be used for inserting specific DNA fragments. b. Polylinker DNA, containing many overlapping sites for restriction endonucleases, can also be inserted into the plasmid (Figure 11-7-8). The advantage of adding polylinker DNA is that it can provide unique sites that can be used for inserting other foreign pieces of DNA. In principle, there is no limit on the size of foreign DNA that can be inserted into the plasmid, but larger molecules do not multiply as well as smaller ones.
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Molecular Biology
Pvull
~ .....
Q)
I:: Q)
III
0>
(')
'S.
Q)
EcoRI
I::
ell
U5
Pvull
s·
u
'iii ~
§
'u
!
!
'6.
(!l
CD (/) iii'
Neal
iii ~
(') (!l
E
«
EcoRI
Bgnl
San Figure 11-7-7. Important features of a plasmid cloning vector.
BamHI
I
Hind/II
I
I
I
S'GGATCCCGGGAAGCTI3' 3'CCTAGGGCCCAAGCTIS'
I
Smal
I
Figure 11-7-8. Polylinker DNA.
Note Transformation has two definitions. It can refer to the introduction of exogenous DNA into a bacterium, or it can refer to the process by which an animal cell becomes a cancer cell.
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c. The cloning of a DNA fragment into a plasmid involves cleaving the plasmid with a restriction endonuclease (such as EeoR!) that results in sticky ends. If the DNA fragment to be cloned also has EeoR! sticky ends, it can be joined to the plasmid by DNA ligase.
d. The plasmid, containing foreign DNA, is introduced into the host bacterium by a process called transformation. Bacterial membranes are made permeable to plasmids by treatment with CaCho The efficiency of transformation is low (0.001 %). Transformed bacteria can be selected by growing them on a medium containing antibiotic. Only the bacteria that contain plasmids with the antibiotic resistance gene will grow under these conditions.
--------~--~----
Genetic Engineering
2. Bacteriophages (phages) a. Phages are bacterial viruses with a relatively large and linear DNA molecule (40-50 kilobases) enclosed in a shell of protein. The phage genome codes for the coat proteins and a number of other specific viral proteins. The structure of the protein coat imposes a physical limitation on the size of the DNA that can be enclosed in the virus (about 50 kilobases). Consequently, there is a limit to the size of foreign DNA that can be inserted into this type of cloning vector. Genetic engineering has tried to overcome this problem by replacing parts of the viral DNA that do not contain essential viral proteins with foreign DNA. b. Lambda phages that replicate in E. coli are most commonly used as cloning vectors. They have a genome of approximately 50 kilobases. However, they can be reduced to 30 kilobases by elimination of nonessential genes, thus providing room for insertion of up to 20 kilobases of foreign DNA. If the recombinant DNA is too small, the phage is typically not viable. Infection of E. coli is initiated by adsorption of lambda onto the surface of E. coli. The phage DNA enters the bacterium and converts it into a "phagesynthesizing factory" that eventually lyses and releases approximately 106 phages that can infect other bacteria. Not all phages are lytic; however, only lytic phages are used as cloning vectors.
Note Plasmid or bacteriophage vectors can be used to express human genes in bacteria; this is a source of human insulin used to treat diabetics.
D. Host cell for cloning vectors. Cloning vectors are usually transformed into a prokaryotic host cell, with E. coli usually being the host of choice. E. coli grows quickly, with a dividing time of approximately 20 minutes. This makes E. coli an ideal choice for a cloning vector. The growth curve of E. coli has three distinct phases. In the lag phase, the bacteria are adjusting to their environment and are activating their replication machinery. In the log phase, exponential growth is occurring. With each cell division per generation, the number of bacteria is doubled. Transformations are performed at this stage of growth. In the stationary phase, the nutrients in the medium become scarce, and the replication machinery slows down.
CONSTRUCTION OF DNA LIBRARIES The purpose of cloning is to isolate a particular gene in large quantities. The most common approach to this problem is to construct a set of recombinant DNA clones from a source that contains the gene or DNA sequence of interest. A collection of clones containing all of the sequences found in the original source is known as a DNA library. There are two approaches to constructing a library. One approach starts with the total population of mRNA of a given cell type, synthesizes complementary DNA (cDNA) copies of the mRNA, and inserts the cDNA into cloning vectors, thereby producing a cDNA library. The other approach is to start with digested genomic DNA and insert the fragments into bacteriophage cloning vectors, producing a genomic library. A. cDNA libraries. A cDNA library is a collection of recombinant vectors that together contain a complete copy of all of the messenger RNAs in a particular cell. There are five steps involved in constructing a cDNA library: isolation of the total mRNA population from a cell, synthesis of cDNA using mRNA as a template, introducing the cDNA library into a cloning vector, transforming the cDNA library into E. coli, and screening the cDNA library for specific cDNA clones. The chief advantage to a cDNA library versus a genomic library is that the cDNA clones do not have introns or other noncoding sequences found in genomic DNA.
In a Nutshell A cDNA library represents the genes expressed in a particular cell type; a genomic library contains all of the genes present in the DNA.
1. Preparation of mRNA. The cell or tissue type used as a source of mRNA must contain the
specific mRNA of interest. For example, reticulocytes are a good source for mRNA that codes for (1,- and l3-globin proteins. Once a cell type has been identified, the total RNA population (rRNA, mRNA, and tRNA) is isolated from the cells. mRNA usually constitutes 800 MW molecules • Adhesins and/or evasins-Function: an adhesin is a gene product promoting attachment and colonization. An evasin is a gene product promoting evasion of host defenses. • Lipoprotein: - Function-covalently anchors outer membrane onto PDG; also loosely linked to LPS • Fimbriae or pili: - Structure-filament of pilin, helical arrangement, addition at base Sex pili: - Genetic exchange (plasmiddetermined) - Only see a few per cell (3-4) Common pili or fimbriae: - Adhere to host cells - Several 100 per cell - Almost all Gram-negatives have them; some Gram-positives as well Can also act as evasins or aggresins
mallical --~-----
Bacteriology Overview
BACTERIAL GROWTH How rapidly an infection can progress before host defenses respond can determine the severity of disease. In a closed system, growth is dependent upon the availability of nutrients, the external environment (e.g., temperature), and the growth rate of the specific species. Figure V-2-1 depicts a typical bacterial growth curve.
c d
b
a FigureV-2-1. Growth curve in a closed system. a =lag phase; b =exponential or log phase; c =stationary phase; d =phase of decline (death phase).
A. Bacterial growth in a dosed system 1. The lag phase is a period of no growth when the organisms are adapting to a new envi-
ronment. 2. The exponential or log phase describes the steady state of growth, typically at the organism's fastest rate. It continues until the nutrients are depleted or toxic waste products accumulate. Many antibiotics, especially those that target cell wall synthesis, are maximally effective during this phase. 3. The stationary phase occurs when nutrients are exhausted or toxins accumulate. Cell number is stable (cell loss = cell formation). Different outcomes can occur depending on the species of bacteria. Some bacteria stop growing, but remain viable for long periods of time. Some organisms cannot maintain a viable, nongrowing state. When they reach the stationary phase, they immediatedly start to die. Other organisms, if conditions do not improve, start forming spores. 4. The phase of decline is observed when the death rate increases, due to cell starvation or sensitivity to toxins.
SURVIVAL IN OXYGEN Survival in oxygen is an important parameter used to classify bacteria. All bacteria produce the superoxide ion (° 2-) in the presence of oxygen. Three enzymes are important to detoxify this ion. Superoxide dismutase converts the superoxide ion to hydrogen peroxide. Catalase or peroxidase then metabolizes the hydrogen peroxide to water and oxygen. Obligate anaerobes (strict anaerobes) lack these enzymes or have such low levels that an oxygen environment is toxic for them. Examples of obligate anaerobes are Clostridium and Bacteroides. Facultative organisms grow with or without oxygen. Whether they ferment or respire is an independent issue.
In a Nutshell The ABCs of Anaerobes
• Actinomyces • Bacteroides • Clostridium
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ENERGY PRODUCTION Energy production requires a source of carbon. Some bacteria are very versatile, requiring only a few essential nutrients. Many pathogenic bacteria have become so host adapted that they have lost much of their metabolic machinery. They have many additional requirements and are termed fastidious bacteria. A. Siderophores are iron (Fe 3+)-chelating compounds that are essential for growth of many pathogenic bacteria. The siderophore is a low-molecular weight material such as a catechol that is excreted from the cell. This molecule binds iron and is then bound to a cell wall or outer membrane protein and transported into the cytosol of the cell, where it is utilized in energy metabolism. B. Mechanisms of energy production 1. Fermentation is the anaerobic degradation of glucose to obtain ATP.
a. It is much less efficient than respiration for generating energy. It can be used by both obligate anaerobes and facultative organisms. End products of fermentation differ depending upon the organism and are often useful in identification. b. Most obligate anaerobes and all Streptococcus species use fermentation. Streptococcus species can only ferment because they cannot make cytochromes or catalase. However, they do have superoxide dismutase and peroxidase, so they can survive in an oxygen environment. 2. Respiration completely oxidizes organic fuels and requires an electron transport chain to drive the synthesis of ATP. a. Respiration produces almost 20 times as much ATP as fermentation. It requires a terminal electron acceptor. The usual electron acceptor is oxygen; however, alternate electron acceptors, such as nitrate and fumarate, are used by some organisms. b. Given a choice, bacteria will opt for respiration over fermentation. However, bacteria differ in their intrinsic ability to use fermentation or respiration. (1) Strict or obligate aerobes respire only and must use oxygen as a terminal electron
acceptor. Mycobacterium tuberculosis is a good example of an obligate aerobe. (2) Some bacteria ferment only. (3) The majority of bacteria use the most versatile strategy, fermentation or respiration, depending upon the conditions. Figure V-2-2 summarizes this information.
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Baderiology Overview
....-_ _ _ _ _---,NO Obligate Can the organism aerobes; grow without Facultative oxygen? \
Obligate aerobe -respires only (Mycobacterium tuberculosis) Ferments only (Streptococcus; no cytochromes = no catalase)
'---------'YE~ Facultative
How does the organism get ATP?
(E. coli, Strep species, Pseudomonas aeruginosa )
YES
Obligate anaerobes The enzymes for processing the superoxide ion are absent or minimal. Need superoxide dismutase, catalase, and peroxidases to detoxify the superoxide ion.
~
Ferments or respires with oxygen as terminal electron acceptor (E. coli)
Ferments only ( Clostridium species)
How does the organism get ATP? Ferments and possesses a primitive respiratory chain (uses something other than oxygen as a terminal electron acceptor, e.g., fumarate in Bacteroides fragilis)
Figure V-2-2. Bacterial survival in oxygen and methods of ATP production.
SPORULATION A. A spore is a dormant structure capable of surviving prolonged periods of unfavorable envi-
ronmental forces. Spores are capable of re-establishing the vegetative stage of growth when environmental factors become more favorable. Spores are resistant to radiation, drying, and disinfectants. Thermal resistance to denaturation is due to the high content of calcium and dipicolinic acid in the core. Spore formation is observed in Bacillus and Clostridium species.
B. Initiation of sporulation is related to the guanosine triphosphate pooL Regulation is by means of negative feedback. In addition, the availability of carbon and nitrogen sources are important to sporulation activity. C. Germination and outgrowth occurs when environmental and nutritional factors allow for
renewed cell growth. 1. Vegetative growth is triggered by the exposure to stimulants such as glucose, nucleic acids, and amino acids. 2. Activation of autolysin results in autolysis of the cortex. The synthesis of protein and structural components follows. The spore core membrane develops into the cell wall.
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GENETIC TRANSFER Genetic transfer refers to three principal mechanisms that result in the movement of genetic material into a host organism. A. Transformation is the uptake and integration of naked DNA from the environment. 1. Once inside the cell, homologous recombination with the chromosome of the recipient
must occur for the transformation to be successful. 2. Transformation can be induced in the laboratory with salt and heat shock. This technique is used to make cells take up plasmids carrying genes of interest (competency). 3. There are natural transformers among both the Gram-positive and Gram-negative bacteria. The medically important natural tranformers are: Streptococcus species, Haemophilus species, Neisseria gonorrhea, and Helicobacter pylori.
B. Transduction is the phage-mediated transfer of bacterial DNA. There are two kinds of transduction: generalized transduction and specialized transduction.
Note
1. In generalized transduction, bacterial DNA is mistakenly packaged into an empty phage
The gene for diphtheria toxin is carried on a lysogenic bacteriophage.
head. This is a very low frequency event, but any gene can be transferred. Once inside the recipient cell, homologous recombination must occur for the transduction of information to be successful. 2. During specialized transduction, a lysogenic bacteriophage that is integrated into the bacterial chromsome excises itself, accidentally taking some chromosomal DNA. When the phage replicates, any bacterial gene that it has picked up is also replicated. These genes will be carried into cells that the progeny viruses infect. Specialized transduction occurs at a higher frequency that generalized transduction. C. Conjugation is the direct transfer of bacterial DNA between organisms. It requires cell-to-
In a Nutshell • Transformation ~ Uptake and integration of naked DNA fragments • Transduction ~ Phagemediated exchange of information • Conjugation ~ Direct bacteria to bacteria transfer of DNA (bacterial sex)
cell contact. It is the most important mechanism for widespread transfer of genetic information between bacteria. 1. Most conjugation is plasmid-mediated. A plasmid is an extrachromosomal piece of cir-
cular DNA that can replicate itself. It often carries genes such as those that encode resistance to antibiotics, and virulence factors such as enterotoxins or adhesins. Plasmids vary in size, copy number per cell, and host range. 2. If a plasmid can exist only within a single species, it is called a narrow-host-rangeplasmid. If it can transfer between different genera of organisms, it is called a broad-host-range plasmid. 3. All plasmids can replicate themselves within the appropriate host, but not all plasmids can transfer themselves. a. A conjugative plasmid codes for the genes involved in transfer between cells. b. A nonconjugative, or mobilizable, plasmid requires the help of a conjugative plasmid to transmit itself to another bacteria. D. Insertion sequences are small (1,000 bp) pieces of DNA that code for the enzyme transpo sase, which allows them to jump into and out of DNA. 1. A transposon consists of two insertion sequences flanking another gene or set of genes.
2. Transposons and insertion sequences can insert into target DNA without significant homology at the site of insertion.
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--------
- - -
Bacteriology Overview
3. This ability to move between chromosomes, plasmids, or bacteriophages is an efficient mechanism for moving genes through a bacterial population. In fact, transposons are frequently associated with the formation of multiple-drug resistance plasmids.
DIFFUSION ACROSS BACTERIAL CELL MEMBRANES Clearly, bacteria must transport a number of macromolecules across the cell wall for survival. Larger ions and macromolecules diffuse slowly unless transported across the membrane by one of three mechanisms: facilitated diffusion, active transport, or group translocation. A. Facilitated diffusion Substrates that cross the membrane without the use of energy. Substrates bind to carrier proteins in the membrane whose affinity is equal both outside and inside the cell, allowing transport in both directions. Glycerol is transported into E. coli by this method and ATP may be brought into Rickettsia by this method. B. Active transport
Similar to facilitated diffusion except that energy is required. This allows substrates to be transported against a concentration gradient. Sugars, amino acids, and ions are transported by this mechanism. This mechanism utilizes ATP-binding cassette transport proteins. C. Group translocation
This mechanism does not expend energy to transport, but instead modifies the substrate to be transported (i.e., via phosphorylation of the substrate). Mannitol, mannose, and glucose are examples of substrates transported by this mechanism.
KAPLAlf I medlea 265
Bacteriology: Gram-Positive Cocci There are two medically important genera of Gram-positive cocci-staphylococci and streptococci. Both are nonmotile and do not form spores. Staphylococci are catalase positive, whereas streptococci are catalase negative.
STAPHYLOCOCCUS A. Genus characteristics and classification 1. Staphylococci are Gram-positive cocci that divide perpendicular to the last plane of divi-
sion, forming clumps or clusters. Depending on the age of the culture, they can be observed singly, in pairs, in short chains, or in grape-like clusters. 2. They are hardy organisms because they are relatively resistant to heat and drying. 3. Metabolically, the staphylococci are facultative aerobes and possess both superoxide dismutase and catalase. 4. Clinically, the most important distinction is between S. aureus and all other species that are nonpathogenic members of the normal flora. The coagulase test is a simple way to differentiate S. au reus from the coagulase-negative staphylococci. 5. Although there are six species of coagulase-negative staphylococci, the most numerous species on the skin is Staphylococcus epidermidis. The other coagulase-negative species of clinical relevance is S. saprophyticus. B. Staphylococcus aureus is a common infectious agent of humans and tends to cause localized or toxin-mediated disease. transiently colonizes the nasopharynx, skin, and vagina of up to 30% of the population.
1. Staphylococcus aureus
2. Table V-3-1 summarizes the conditions commonly caused by S. aureus.
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Microbiology
Table V-3-1. Common conditions caused by Staphylococcus aureus. Direct infection Skin: Folliculitis, furuncles, carbuncles, abscesses, cellulitis, wound infection Deep infection: Osteomyelitis (often post-trauma and/or surgery) Systemic infections secondary to above Osteomyelitis, endocarditis, lung abscesses, pneumonia Toxin-mediated disease Food poisoning, scalded skin syndrome, bullous impetigo, toxic shock syndrome
3. Staphylococcus aureus does not produce a single factor that is necessary for virulence. The best host defense against infections are PMNs. No protective immune response is raised, so one can get infections again and again. Table V-3-2Iists the multiple virulence factors of s. aureus. 4. Treatment. The drug of choice is a penicillinase-resistant penicillin (e.g., methicillin, nafcillin, or oxacillin) or a first generation cephalosporin. Methicillin-resistant s. aureus (MRSA) requires therapy with vancomycin.
Note Unfortunately, there are now vancomycin-resistant MRSA.
C. Staphylococcus epidermidis is most commonly a nosocomial pathogen. In contrast to aureus, it is coagulase negative.
s.
1. The major virulence factor it produces is a viscous exopolysaccharide biofilm (slime).
2. When foreign bodies like IVs, catheters, and prosthetic valves are inserted into the host, Staphylococcus epidermidis can grow on their surface, embedded in the biofilm. This biofilm makes it difficult for the immune system to destroy the organism. 3. Clinical manifestations are typically related to instrumentation and procedures and include endocarditis and urinary tract infections. 4. Treatment is with vancomycin. D. Staphylococcus saprophyticus
1. Coagulase negative
2. Differentiated from S. epidermidis by its resistance to novobiocin. 3. Causes urinary tract infections in newly sexually active women. 4. Treatment is with penicillin. Table V-3-2. Major virulence factors of Staphylococcus aureus. I. Binding proteins and capsules 1. Protein A: • Binds IgG by the Fc portion-IgG can't react with the Fc receptor on phagocytes 2. Fibronectin, fibrinogen, vitronectin, and collagen type II binding proteins: • Localization and adherence through binding to body proteins 3. Capsule: • Carbohydrate • Prevents phagocytosis but most adults have opsonizing antibodies
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meclical
(Continued)
Bacteriology: Gram-Positive Cocci
Table V-3-2. Major virulence factors of Staphylococcus aureus (cont'd). II. Enzymes 1. Coagulase: Exists in a bound form and a free form • Stimulates the conversion of fibrinogen to fibrin by binding coagulase-reacting factor (CRF), a derivative of prothrombin. This complex becomes active and initiates fibrin polymerization. • Coated with fibrin, staph are resistant to phagocytosis; the fibrin deposition in an area helps localize the infection 2. DNAse: • Production highly correlated with coagulase 3. Staphylokinase: • Clot dissolution, activates conversion of plasminogen to fibrinolytic plasmin 4. Hyaluronidase: • Hydrolyzes hyaluronic acids that are present in the extracellular matrix of connective tissue 5. Lipase: • Production associated with boils II. Exotoxins 1. Hemolysins-(alpha through delta): Alpha hemolysin or Alpha toxin • Most potent of hemolysins • Dermonecrotic and lethal when injected into rabbits • Causes membrane damage that lyses red blood cells and kills white blood cells by forming pores in eukaryotic membranes ~-hemolysin:
• A sphingomyelinase made by ",,20% of isolates 2. Leukocidin: • Lethal to PMNs, disrupts their membranes through pore formation 3. Epidermolytic toxin or exfoliatin: • Cleaves stratum granulosum layer per splitting desmosomes-no inflammatory response • Scalded skin syndrome in newborns, bullous impetigo in older kids and adults • Toxin is antigenic-circulating antibody confers immunity 4. Toxic shock syndrome toxin (TSST-1): • Symptoms = fever, desquamative skin rash, vomiting, diarrhea, hypotension, multiple system involvement • Induces production of interleukin 1 and tumor necrosis factor by macrophages • Antibody associated with resistance to shock 5. Enterotoxin: • Food poisoning • Types A through E • Heat stable • 1-6 hours post-ingestion; symptoms = increased intestinal peristalis, vomiting, diarrhea, no fever, rapid recovery • Toxin appears to act on neural receptors in UGI tract ~ stimulation of vomiting center • Pyrogenic exotoxins include TSST and the enterotoxins; they are super antigens (like streptococcal pyrogenic exotoxin A or erythrogenic toxin) and cause T-cell proliferation and release of toxic cytokines such as IL-1 and TNF (Continued)
meClical
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Microbiology
Table V-3-2. Major virulence factors of Staphylococcus aureus (cont'd). IV. Resistance to penicillin and other ~-lactam antibiotics 1. (3-Lactamase production • Most often plasmid-encoded (few strains are chromosomal) 2. Intrinsic resistance-(aka methicillin resistance) • Chromosomal • Caused by a mutation in a penicillin-binding protein
Table V-3-3. Identification of Staphylococcus aureus versus Staphylococcus epidermidis. Staphylococcus aureus Coagulase Thermostable nuclease Mannitol fermentation Ribitol teichoic acid Glycerol teichoic acid
Staphylococcus epidermidis
+ + + +
+
STREPTOCOCCUS A. Genus characteristics 1. Streptococci are Gram-positive cocci that form chains.
2. Metabolically, the streptococci are facultative anaerobes because they derive energy from fermentation only (lack cytochromes). a. The principal end product of fermentation is lactic acid, and perhaps because of this they are more acid-tolerant than most bacteria. b. Although streptococci can live in conditions where oxygen is present, they lack catalase. Catalase is a cytochrome-containing enzyme that degrades hydrogen peroxide to oxygen and water. This feature is useful for differentiating the streptococci from other Gram-positive cocci such as the staphylococci. c. Almost all medically important streptococci are auxotrophs, meaning they require
one or more vitamins, amino acids, or nucleic acids for growth and therefore are not free-living in the environment.
B. Classification. The most common classification scheme for the streptococci is based upon their reaction in blood agar. 1. a-Hemolysis-the red blood cells surrounding the colonies are intact, but there is partial breakdown of the heme, resulting in a green (viridans) pigment.
2.
~-Hemolysis-the red blood cells surrounding the colonies are completely lysed. Betahemolytic streptococci are also classified serologically into Lancefield groups (A-O) based on their cell wall carbohydrate. Clinically, the most important group is Group A.
3. y-Hemolysis-no hemolysis or color change of the red blood cells is detected.
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meClical
- -
-------------------------
------------------
Bacteriology: Gram-Positive Cocci
TableV-3-4 Species
Classification
Common Location
Associated Diseases
Streptococcus agalactiae*
Group B
Gastrointestinal tract, vagina, some animal sources, such as raw milk
Early-onset and delayed -onset neonatal disease (meningitis, respiratory distress, sepsis), puerperal sepsis, pharyngitis
Streptococcus bovis
Group D; a-hemolytic or nonhemolytic
Gastrointestinal tract
Endocarditis in patients with gastrointestinal tumors
Streptococcus dysgalactia and Streptococcus equi
Groups C and G; j3-hemolytic
Skin, mucosae
Pharyngitis, erysipelas, impetigo, neonatal meningitis, endocarditis, septic arthritis, wound infections
Streptococcus intermedius group
Groups A, C, F, and G; variable hemolysis
Mouth, gastrointestinal tract, vagina
Sinusitis, endocarditis, brain and liver abscesses
Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguis
Wide variety of Lancefield antigens; viridans streptococci; a-hemolytic
Mouth
Endocarditis
Streptococcus mutans
No Lancefield antigens; viridans streptococci; a-hemolytic
Mouth
Dental caries, endocarditis
Streptococcus pneumoniae*
No Lancefield antigens; a-hemolytic
Upper respiratory tract of 5-70% of general population
Pneumonia, meningitis, otitis media, sinusitis, conjunctivitis
Streptococcus pyogenes*
Group A;
Upper respiratory tract of 5-20% of general population
Pharyngitis, scarlet fever, pyoderma, erysipelas, necrotizing fasciitis, toxic streptococcal syndrome, rheumatic fever, heart disease, glomerulonephritis
Enterococcus faecalis*
Group D; variable hemolysis
Gastrointestinal tract, oral cavity, gallbladder, urethra, vagina
Endocarditis, bacteremia, urinary tract infections, abdominal infections, neonatal sepsis, soft tissue infections
Enterococcus faecium*
Group D; a-hemolytic or nonhemolytic
Gastrointestinal tract
Neonatal meningitis
*Boards' favorites.
~-hemolytic
~-hemolytic
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271
Microbiology
c. (J.-Hemolytic streptococci can be distinguished from each other by their inhibition or growth in the presence of optochin or bile. This group includes Streptococcus pneumoniae and the viridans streptococci. 1. Streptococcus pneumoniae, also known as pneumococci, grows in pairs or short chains. a. It is lysed by optochin or bile and is the most autolytic of bacterial pathogens. At stationary phase, an autolytic enzyme, muramidase, is activated and destroys the bacteria.
In a Nutshell
b. It is a natural transformer since it can take up pieces of DNA from the surroundings and if there is sufficient homology, incorporate it into its genome.
S. pneumoniae • Alpha hemolytic; inhibited by optochin and bile
c. Transmission. Streptococcus pneumoniae is spread person-to-person through aerosol droplets. Twenty to 40% of normals are transiently colonized in their nasopharynx.
• Virulence conferred by polysaccharide capsule
d. Clinical manifestations. It is the most common cause of bacterial pneumonia and also causes otitis media, sinusitis, bronchitis, and bacteremia. It is the most common cause of meningitis in the elderly.
• Clinical correlations: - Pneumonia (#1 cause, especially middle-aged and older adults)
e. Risk factors for infection due to Streptococcus pneumoniae include poverty, a debilitated state of health, the absence of a spleen, and certain diseases such as sickle cell anemia, Hodgkin disease, multiple myeloma, and AIDS.
- Otitis media (#1 cause)
f. The most important virulence factor of Streptococcus pneumoniae is its carbohydrate capsule. Although there are more than 85 capsule types, 23 types cause the most virulence (85%). The capsule is thought to interfere with the opsonizing activity of the alternative complement pathway. Antibody against the capsule is necessary so C3b can attach without being buried.
- Sinusitis - Meningitis (#1 cause in elderly)
g. Prevention. A vaccine of 23 of the polysaccharide antigens exists. It should be given to the elderly, those undergoing splenectomy, and those with a condition predisposing them to Streptococcus pneumoniae disease. A vaccine of 7 of the polysaccharide antigens is administered to infants at 2, 4, 6, and 12-18 months as recommended by the CDC.
- Bacteremia • Certain populations are especially susceptible: elderly, smokers, alcoholics, children, asplenics
h. Treatment. In the past, all Streptococcus pneumoniae organisms were sensitive to penicillin. Penicillin resistance due to transformation with DNA from nonpathogenic streptococci in the oropharynx is becoming a problem. These cases can be treated with vancomycin or erythromycin.
• Vaccine available • Treatment with penicillin but resistance on the rise
2. Viridans streptococci are normal oral flora. a. In contrast to S. pneumoniae, they are not inhibited by optochin or lysed by bile.
Bridge to Cardiovascular and Renal Systems • Rheumatic fever is discussed in the Cardiovascular Pathology chapter of Organ Systems Book 1 (Volume III). • Poststreptococcal glomerulonephritis is discussed in the Renal Pathology chapter of Organs Systems Book 1 (Volume III).
272
mellical
b. They produce dextran, a substance that allows them to adhere to many surfaces. c. They are the major cause of subacute endocarditis (e.g., S. sanguis) in those with
abnormal heart valves. Streptococcus mutans causes dental caries. d. Treatment of choice is penicillin. D.
~-Hemolytic streptococci are further subdivided into groups A through D and F and G based on antibodies to a heat-stable, acid-stable carbohydrate in their cell walls. This antigen is called C carbohydrate or the Lancefield antigen, in honor of Rebecca Lancefield, the scientist who devised this classification system.
1. Group A streptococci (GAS) contains only one species, Streptococcus pyogenes, but it is
the most important streptococcal pathogen. a. It can be distinguished from the other by the antibiotic bacitracin.
~-hemolytic
streptococci because it is inhibited
b. Clinical manifestations of S. pyogenes are characterized as suppurative and nonsuppurative.
- - - - - - - - -
Bacteriology: Gram-Positive Cocci
Table V-3-S. Virulence factors of Streptococcus pyogenes. I. Cell constituents
1. M protein
• Required for virulence-80+ types • Externa-extends from cell envelope as fimbriae; carboxyterminal is attached to PDG and shows little variability; aminoterminal is outside-variable • Prevents phagocytosis by preventing complement opsonization. It has a site that binds factor H, leading to the hydrolysis of C3b to inactive form. • In conjunction with LTA, binds fibrinogen, hiding the C3-binding site • Antibody to a specific M-terminus provides immunity to that M type 2. Lipoteichoic acid or fibronectin-binding molecule • Attached to M protein; binds fibrinogen 3. F protein • Allows binding to fibronectin 4. Hyaluronic acid capsule • Inhibits phagocytosis 5. Cell-bound protease • Cleaves C5a component of complement and inhibits neutrophil chemotaxis II. Extracellular products
1. Hyaluronidase (spreading factor) • Destroys hyaluronic acid-could be important in the spread through tissue during cellulitis 2. Streptolysin 0
• Oxygen labile (inhibited by oxygen)-a hemolysin that is active only in its reduced form • Works by inserting directly into the host cell membrane, forming transmembrane pores • Immunogenic-can use ASO test for evidence of recent strep infection 3. Streptolysin S
• Oxygen stable; made in the presence of serum • Hemolysin causing l3-hemolysis-produced by most group A strains • Non-immunogenic 4. Streptococcal pyrogenic exotoxins (SPE) (erythrogenic toxins) • Rash of scarlet fever • Encoded by bacteriophage • Pyrogenic exotoxins are non specific activators of the immune system (superantigens)
KAPLA~_ I meulca 273
Microbiology
In a Nutshell S. pyogenes (Group A) • Beta hemolytic; sensitive to bacitracin • Many virulence factors, including M protein, capsule, F protein, hyaluronidase, Streptolysins 0 and S, and erythrogenic toxins • Clinical correlations - Pharyngitis (strep throat) - Scarlet fever - Skin: erysipelas, cellulitis, impetigo, necrotizing fasciitis, pyoderma - Secondary diseases: rheumatic fever and acute glomerulonephritis • Treatment with penicillin
(1) Suppurative complications of pharyngitis include otitis media, peritonsillar
cellulitis, peritonsillar and retropharyngeal abscesses, and bacteremic metastatic spread. Other suppurative infections include erysipelas (skin infection), pyoderma (impetigo), scarlet fever, cellulitis, lymphangitis, perianal cellulitis, puerperal sepsis, meningitis, pneumonia, and empyema. (2) Nonsuppurative sequelae occur weeks after initial infection. Inflammation occurs in organs not originally infected. These sequelae include acute glomerulonephritis, in which edema, hypertension, and hematuria occur after pharyngeal or skin infection. Also classified within this group is rheumatic fever, occurring 7-28 days after pharyngitis and results in fever, carditis, and polyarthritis. c. Transmission and epidemiology. Group A strep is an obligate human parasite spread person-to-person by respiratory secretion via droplets, direct contact with the skin, or fomites. Pharyngitis is most common in winter and spring, with the highest incidence among preadolescents. Contaminated milk or eggs have also been the cause of foodborne epidemics of pharyngitis. Impetigo-like skin infections are most prevalent in summer and are often due to the infection of insect bites. Group A strep produces a toxic enzyme called streptolysin 0 (SLO). Patients can be diagnosed in the lab based on the presence of antibodies to SLO. d. Virulence factors. Table V-3-5 summarizes the virulence factors of S. pyogenes. The most important virulence factor to remember is M protein. 2. Group B Streptococcus (Streptococcus agalactiae) is part of normal vaginal and intestinal flora in 25% of a given population. a. It is classified into serotypes based on polysaccharide and surface-protein antigens.
In a Nutshell
b. In contrast to S. pyogenes, S. agalactiae are resistant to bacitracin.
S. agaladiae (Group B)
c. The major virulence factor is an antiphagocytic polysaccharide capsule.
• ~-Hemolytic; resistant to bacitracin
d. Infants are more susceptible to disease from the organism than adults. They aspirate the organism during passage through the birth canal, and if they lack passive resistance from maternal IgG antibody, disease may ensue.
• Antiphagocytic capsule
e. Clinical manifestations include pneumonia, sepsis, and meningitis. Group B streptococci and E. coli are the major causes of these diseases in the neonatal population (under 1 month of age).
• Causes meningitis, sepsis, and pneumonia in neonates (acquired during passage through vaginal canal where organisms are part of normal flora)
f. Risk factors for acquiring pneumonia include: recent colonization of the mother, premature birth, low birth weight, and premature rupture of the membranes. Diabetics are presumably more susceptible to infection because high glucose concentration promotes capsule synthesis.
• *1 cause of neonatal
g. Therapy for S. agalactiae is a penicillinase-resistant synthetic penicillin.
meningitis Enterococci • Variable hemolysis • Normal fecal flora • Cause urinary tract infections in hospitalized patients; rare cause of subacute endocarditis
274
meClical
3. Enterococcus, formerly group D Streptococcus, include the important pathogens Enterococcus faecalis and Enterococcus faecium. a. Both organisms are part of the normal fecal flora. These organisms were classified as group D, ~-hemolytic streptococci; however, hemolysis is not a consistent feature since it is encoded on a plasmid. b. Enterococci can grow in 40% bile and 6.5% sodium chloride, as would be expected of a fecal organism. c. These organisms can cause infection when they spread to the urinary tract. When the intestine is disrupted, they are one of the organisms found in an abscess. Enterococcus species also cause approximately 10% of cases of subacute endocarditis.
- - - - - - - - -- -
--
---
----- - - - - - - - - - -
Baderiology: Gram-Positive Cocci
d. Unlike the streptococci, enterococci exhibit penicillin tolerance since they are inhibited, but not killed, by the antibiotic. Recently, vancomycin-resistant enterococci have appeared. During peptidoglycan synthesis, vancomycin binds to the D-ala-D-ala residues and prevents the construction of the cell wall. Vancomycin-resistant enterococci use D-lactic acid instead of D-ala to form the "peptide" bridges.
Positive Positive Gram-positive cocci
Catalase Test
Staphylococcus species
Coagulase Test
Resistant
Negative
Negative
Staphylococcus aureus
- - - - - - - 1 Novobiocin
Streptococcus species
Resistant
Sheep Blood Agar
a
50%) of non-A-non-B (NANB) hepatitis infections. Screening of blood for HCV has significantly reduced the incidence of transfusion-related hepatitis. HCV develops a chronic carrier in nearly 80% of patients and is associated with cirrhosis and hepatocellular carcinoma. D. Hepatitis D (delta agent) is a defective RNA virus that can replicate only in cells concurrently infected with hepatitis B. This virion requires the presence of hepatitis B enzymes to
replicate. It occurs in Italy and in the Near East. E. Hepatitis E is caused by a single-stranded RNA virus that is similar to Norwalk agent (calicivirus). Clinically, it causes a disease similar to hepatitis A, but it can become fulminant in pregnant women (20% mortality). It is spread via fecal-oral route. It occurs primarily in the Far East.
Table V-11-2. Summary of hepatitis viruses.
Note Not surprisingly, yet another hepatitis virus was recently identified (1996). Hepatitis G (HGV) is an RNA virus associated with both acute and chronic hepatitis. It has a global distribution and is transmissible by transfusion. It appears to be distantly related to hepatitis C.
Virus
HepA
HepB
HepC
HepD
HepE
+RNA Flavivirus Parenteral
-RNA Delta agent Parenteral
+RNA Calicivirus Fecal-oral
Rare
DNA Hepadnavirus Parenteral Perinatal Sexual Rare
Rare
Frequent
Never No
Often Yes
Very often Yes
Very often
In pregnant women Never No
Genome
+RNA Picornavirus Transmission Fecal-oral Fulminant course Chronicity Oncogenic
Note Creutzfeldt-Jakob disease is considered a "prion" disease. Another spongiform encephalopathy, kuru, was associated with cannibalism and is now extinct.
340
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PRIONS Prions do not have genomes or virion structures. They are unique infectious protein particles with a mode of replication that is not entirely known. The major human disease caused by a prion is Creutzfeldt-Jakob disease (C]D; a spongiform encephalopathy [mad cow diseaseD. Outbreaks of CJD were reported with the use of contaminated growth hormone preparations. This disease is progressive and always fatal. It is preceded by dementia and motor/movement disorders. Confirmation of diagnosis can be made at autopsy only.
Virology
Table V-11-3. Recommended childhood and adolescent immunization schedules-United States, 2007. DEPARTMENT OF HEALTH AND HUMAN SERVICES· CENTERS FOR DISEASE CONTROL AND PREVENTION
Recommended Immunization Schedule for Persons Aged 0-6 YearS-UNITED STATES • 2001 Age...
VaccineT Hepatitis B'
Birth Hep8
Rota
Rotavirus' Diphtheria, Tetanus, Pertussis'
DTaP jm ...................... !
Range of recommended ages
DTaP
•
....................... !.......... m.................... m..............
Haemophilus influenzae type b'
Hib
Pneumococcal'
PCV
Inactivated Poliovirus
IPV
Hib
Catch-up immunization
Influenza'
Certain high-risk groups
Measles, Mumps, Rubella' Varicella' Hepatitis A' Meningococcal'·
Recommended Immunization Schedule for Persons Aged 7-18 YearS-UNITED STATES • 2001 Age"
Vaccine,..
Tetanus, Diphtheria, Pertussis1 Human Papillomavirus Meningococcal
2
7-10
years
11-12 YEARS
13-14
years
15
years
16-18
years
see footnote
1 see footnote
2
3
Pneumococcal' Influenza 5 Hepatitis AS Hepatitis 81
Range of recommended ages
-
Catch-up immunization
Certain high-risk groups
Inactivated Poliovirus8 Measles, Mumps, Rubella 9 Varicella 10 This schedule indicates the recommended ages for routine administration of currently licensed childhood vaccines, as of December 1, 2006, for children aged 7-18 years, Additional information is available at http://www.cdc.gov/nip/recs/child-schedule.htm. Any dose not administered at the recommended age should be administered at any subsequent visit, when indicated and feasible. Additional vaccines may be licensed and recommended during the year. Licensed combination vaccines may be used whenever any components of the combination are indicated and other components
of the vaccine are not contraindicated and if approved by the Food and Drug Administration for that dose of the series, Providers should consult the respective Advisory Committee on Immunization Practices statement for detailed recommendations. Clinically significant adverse events that follow immunization should be reported to the Vaccine Adverse Event Reporting System (VAERS), Guidance about how to obtain and complete a VAEAS form is available at http://www.vaers.hhs.govorbytelephone, 800-822-7967.
mettica.
341
Mycology
Fungi are eukaryotic organisms that can be classified as either yeast or molds based on their morphology and their mode of reproduction. The simplest way to review medically important fungi is to divide them into groups based on their clinical presentation: cutaneous mycoses, subcutaneous mycoses, systemic mycoses, and opportunistic mycoses.
INTRODUCTION A. Morphology. Fungi are eukaryotes possessing a cell wall (composed of glucose and mannose polymers called chitin) and a cell membrane (containing ergosterol). Capsules are found only with Cryptococcus neoformans. The fungi are divided into yeasts or molds based on shape and mode of reproduction. 1. Yeasts have round or oval morphology and reproduce by budding. 2. Molds have tubular structures called hyphae. Molds grow by branching and longitudinal extension to form mycelial structures. Hyphae can be either septate (divided into nucleated cells) or nonseptate (coenocytic). 3. Dimorphic fungi grow in the host as a yeast-like form, but grow as molds at room temperature in vitro. B. Reproduction can be sexual and/or asexual. Asexual spores form through mitosis. This
form of reproduction is referred to as an "imperfect" state. Most pathogenic fungi are found only in the imperfect state. C. Immunity. The T-cell response is protective in fungal diseases; antibodies are not, although
they may have some role in serodiagnosis.
DERMATOPHYTOSIS (CUTANEOUS MYCOSES) Dermatophytosis is a mycotic infection of any keratinous structure of the skin and its appendages by Trichophyton (T.), Microsporum (M.), or Epidermophyton (E.). Candida can cause cutaneous infections but will be discussed later under Opportunistic Mycoses. A. Stratum corneum infections are characterized by location. Ringworm or tinea is another name for these cutaneous mycoses. The clinical syndrome includes scaling associated with pruritus. 1. Tinea corporis-body 2. Tinea cruris-groin (jock itch) 3. Tinea pedis-feet (athlete's foot)
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343
Microbiology
4. Tinea manuum-hands 5. Tinea capitis-scalp B. Infections of the hair include tinea capitis (scalp) and tinea barbae (beard). These infections are commonly due to organisms from the genera Trichophyton or Microsporum. Ectothrix is when fungi are present outside of the hair shaft. Endothrix is the term to describe fungi within the hair shaft.
Clinical Correlate
C. Infections of the nail are primarily due to tinea unguium. Trichophyton rubrum and T. mentagrophytes are the two most common etiologic agents.
The major dermatophyte that spreads from person to person is M. audouinii. M. audouinii and M. canis are the two species that fluoresce in ultraviolet light.
D. Diagnosis is made by microscopic examination of skin scrapings using KOH, by fungal cultures, or by UV (Woods) lamp examination (to detect fluorescent tinea capitis caused by M. canis and M. audouinii). E. Treatment consists of topical imidazoles such as miconazole or dotrimazole (only for stratum corneum infections) or oral griseofulvin. Tolnaftate may also be used for skin infections.
SUBCUTANEOUS MYCOSES Subcutaneous mycoses typically result from implantation of the organism by means of some sort of trauma.
Note If a question mentions roses, there is a very good chance the answer is Sporothrix
schenckii.
A. Sporotrichosis is caused by the dimorphic saprophytic fungus, Sporothrix schenckii. Sporotrichosis has worldwide distribution with all ages and both sexes affected. S. schenckii is isolated from soil, living plants, or plant debris. It is classically associated with rose thorns and is often called "rose gardener'S disease:' 1. Clinical disease is generally limited to the skin and regional lymphatics (lymphocuta-
neous sporotrichosis). S. schenckii enters through skin breaks in the fingers or hands, causing a chancre, papule, or subcutaneous nodule with erythema and fluctuance. Nodular, ulcerating lesions appear along lymphatic channels, but the lymph nodes are not commonly infected. Culture of drainage or aspirated material for the presence of S. schenckii is usually diagnostic. 2. Treatment consists of potassium iodides for subcutaneous disease and amphotericin B for relapsing cutaneous disease as well as pulmonary and disseminated forms. B. Mycetoma (Madura foot, maduromycosis) is a local chronic progressive destruction of skin, subcutaneous tissue, fascia, muscle, and bone. 1. Transmission is by soil contamination of a wound. Lesions usually occur on the feet or
hands. 2. Infection results in a suppurative granuloma with multiple sinus tracts, and mycotic grains (granules) are extruded. 3. Mycetoma is caused by infection with several fungi (eumycotic mycetoma) or by higher bacteria (actinomycotic mycetoma). 4. Diagnosis is made by obtaining granules and staining with KOH for fungi and with Gram stain and modified acid fast for the presence of Nocardia organisms.
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SYSTEMIC MYCOSES
In a Nutshell
Systemic mycoses are also called deep mycoses. They can invade organs and are potentially lifethreatening.
Dimorphic Fungi
A. Histoplasmosis (Darling disease) is caused by Histoplasma capsulatum, which is found in
bird and bat droppings. Endemic areas in the central U.S. include the Ohio, Mississippi, and Missouri River valleys. 1. The diphasic pathogen H. capsulatum exists in soil in the mycelial phase and converts to
• Histoplasma • Blastomycoses • Coccidioides • Sporothrix
the yeast phase at 37.0°C, a. The mycelial form has septate branching hyphae-bearing spores at the lateral or terminal positions. b. Microconidia spores are the infectious particles. c. Macroconidia (8-14 mm) develop finger-like appendages over their surfaces (tuberculate macro conidia spores), which is a morphology used to identify this species. 2. Yeast forms are found within macrophages. Methenamine silver stains the cell membrane of this organism. 3. Transmission is mediated by airborne inhalation of microconidia spores that get deposited in alveoli and spread through lymphatics to the regional lymph nodes. Hematogenous spread may lead to metastatic foci of infection in the liver, spleen, etc. Human-to-human spread is infrequent. Within 7-21 days of primary exposure, cellmediated immunity develops with resulting granulomas at infected sites. 4. Clinical manifestations include acute and chronic pulmonary infections that very rarely progress to a disseminated histoplasmosis. (Ninety-five percent of infections are asymptomatic.) a. Acute pulmonary histoplasmosis arises 5-21 days after exposure and is expressed as headache and fever in most or all cases. (1) Chills, cough, and chest pain occur in two thirds of cases, with weakness, weight
loss, myalgia, and fatigue as less common symptoms. (2) Diagnosis is based on clinical suspicion and confirmed by culture and examination of sputum. Complement fixation tests are diagnostic. (3) No treatment is usually required since it is a benign and self-limited disease. b. Chronic pulmonary histoplasmosis occurs in patients with chronic obstructive lung disease. It begins as a benign segmental interstitial pneumonitis with 20% of cases progressing to a chronic cavitary disease. Cavitary disease is treated with a full course of amphotericin B. In some cases, surgical resection is necessary. c. Disseminated progressive histoplasmosis is uncommon, occurring in persons with deficient cell-mediated immunity, chronic underlying debilitating disease, or in patients undergoing corticosteroid or immunosuppressive therapy. (1) Clinical manifestations. A variety of clinical manifestations may arise, including systemic symptoms (fever, chills, anorexia, malaise, weight loss), hepatosplenomegaly (abnormal liver function tests), interstitial pneumonitis, and adrenal or renal involvement. (2) Diagnosis is confirmed by complement fixation and by cultures from blood, bone marrow, CSF, and urine. Tissue may be acquired for histopathology and culture of organisms. (3) Treatment consists of amphotericin B.
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B. Coccidioidomycosis (San Joaquin fever; Valley fever) is due to infection with another dimorphic fungus, Coccidioides immitis. The organism is indigenous to deserts of the Southwestern U.S. and northern Mexico. C. immitis is inhaled as airborne arthroconidia (associated with fresh diggings, dust storms, etc.). Wind causes the spores to become airborne (infections increase during the dry dusty season). 1. C. immitis exists in soil in a mycelial phase. As it matures, alternate cells along a hypha become barrel-shaped (termed arthroconidia). These forms become airborne and are inhaled.
2. In the host, the spore swells up to 50 Il in diameter, becomes spherical (termed spherule), and reproduces to form internal spherical spores called endospores. Each endospore may form a new spherule or, if returned to soil, will revert to the mycelial form. 3. Preparations to identify organisms from tissues include KOH preparations, lactophenol cotton blue stain, and Gomori methenamine-silver stain. 4. Clinical manifestations arise as acute and/or potentially chronic and disseminated infection. a. Acute pneumonitis is subclinical in 60% of cases and is detected only by skin testing. 40% develop an influenza-like syndrome 7-28 days following exposure. (1) These symptoms include fever, malaise, dry cough, eosinophilia, toxic erythema (a fine, generalized erythematous macular rash), and erythema nodosum (particularly in females). (2) Diagnosis is confirmed by sputum culture. Skin tests are not helpful; however, serology for the presence of specific antibodies may be helpful. (3) Treatment is typically not required in adults since the pneumonitis will resolve spontaneously. Amphotericin B is given to infants, debilitated patients, or those at risk for dissemination; miconazole is given as an alternative. b. Disseminated coccidioidomycosis occurs in less than 1% of infected patients. ( 1) Patients at risk include leukopenic or immunosuppressed individuals, those with Hodgkin or AIDS, and certain racial/ethnic groups (African Americans, Filipinos). (2) Disseminated coccidioidomycosis may spread to any organ, causing a toxic state and high fever. (3) Treatment consists of amphotericin B. Fluconazole may also be effective. C. Blastomycosis is caused by Blastomyces dermatitidis, a dimorphic fungus growing as a mycelial form at room temperature and as a yeast form at 37.0°C.1t is endemic in states east of the Mississippi River. Conidia spores are thought to be infectious for humans, converting to the yeast form after inhalation into the lungs. Infections are limited to North America. Infection occurs in normal hosts, usually in occupations associated with soil contact. 1. Acute blastomycosis (pneumonitis) can be asymptomatic or a severe, often fatal, illness. The clinical presentation is marked by an influenza-like syndrome with fever, chills, productive cough, and pleuritic chest pain.
a. Diagnosis is made by sputum smear and cultures that are usually positive. Chest x-ray shows multiple parenchymal nodules or localized consolidation. b. Therapy may not be required since acute blastomycosis is usually a benign, self-limited disease.
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2. Chronic blastomycosis has a variable course of progressive illness. Lungs and skin are most commonly involved. Treatment consists of amphotericin B or ketoconazole combined with surgical excision or drainage of the local lesion. D. Paracoccidioidomycosis is sometimes called South American blastomycosis because It IS
endemic in Latin America (especially Brazil). In infected tissues, the yeast cells have multiple buds sprouting from a single parent cell, giving it a characteristic "pilot's wheel" appearance. Typically, the clinical manifestations are primarily pulmonary; often times it is asymptomatic.
OPPORTUNISTIC MYCOSES The opportunistic fungi are those that cause disease in immunocompromised patients (AIDS, chemotherapy, transplants). Unlike the systemic mycoses, these fungi are sometimes normal flora and are not limited to certain geographic regions.
In a Nutshell Fungal Disease Geography • Ohio, Mississippi, Missouri River valleys
~
Histoplasmosis
• Southwestern ~ Coccidioidomycosis deserts, California • States east of ~ Blastomycosis Mississippi River • Latin America
~
Paracoccidioidomycosis
A. Candida. Candidal infections may be cutaneous or systemic.
1. C. albicans is the major pathogenic species. 2. Candida are normal inhabitants of mucocutaneous body surfaces, soil, hospital environments, and some foods. C. albicans colonizes normal skin and most diseases are endogenous in origin. Occasional person-to-person spread occurs (e.g., newborn thrush). 3. Invasive disease results from host alterations, leading to a change in the commensal status of the organism. Factors important in the invasiveness of Candida include predisposing illnesses (such as diabetes mellitus), damaged mucosal surfaces (such as those caused by indwelling catheters), depression of the immune status (as in immunocompromised individuals), and the use of steroids or antibiotics. 4. Clinical manifestations depend on the site of infection. a. Oropharyngeal infection (includes thrush) is observed as discrete or confluent white patches on the tongue and buccal mucosa. Microscopic examination of scraping reveals pseudohyphae. Therapy consists of nystatin suspension, oral ketoconazole, or mycelex troches. b. Vaginal infection is frequently seen in diabetes mellitus, antibiotic therapy, and in pregnancy. It is associated with thick yellow-white discharge and intense pruritus. Nystatin suppositories are beneficial. Imidazole drugs (topical or oral) are also therapeutic. c. Gastrointestinal colonization causes disease in malnourished or immunocompromised persons, or in persons undergoing prolonged intra-abdominal surgical procedures. Clinical presentation may include diffuse ulcerative and erosive esophagitis, gastritis, or multiple superficial ulcerations of the small and large intestine. Stool and throat cultures may not be diagnostic because of frequent colonization by Candida. Therapy consists of nystatin, ketoconazole, and low-dose amphotericin B. d. Invasive, disseminated infection occurs, particularly in patients with leukemia, lymphoma, and AIDS. The gastrointestinal tract is probably the most common portal of entry. Symptoms include fever, shock, hypotension, and prostration. Renal infection occurs usually from hematogenous spread and may result in renal failure. Endocarditis occurs in drug abusers. e. Chronic mucocutaneous candidiasis represents extensive cutaneous disease that is refractory to treatment and can be disfiguring. 5. Diagnosis is made by demonstration of fungal pseudohyphae in the tissue or by culture and biochemical identification (e.g., urease positive).
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6. Treatment. Systemic treatment includes ketoconazole or fluconazole; amphotericin B is used less frequently. Flucytosine is also valuable in systemic disease. Cutaneous involvement usually responds to topical miconazole. B. Cryptococcosis is due to infection with Cryptococcus neoformans. C. neoformans is an encapsulated yeast that reproduces by budding. C. neoformans is identifiable on Wright stain or India ink (especially helpful due to presence of capsule). C. neoformans is found worldwide in avian feces (particularly pigeon droppings), in soil, fruits, milk, and wood products. Immunosuppression due to malignancy or AIDS predisposes to this disease. 1. Pulmonary disease is common since this is the primary portal of entry. Disease is usually
transient and not severe if the patient is otherwise healthy. 2. Disseminated disease arises most often in immunocompromised individuals.
Note (ryptococcal meningitis has been a Boards' favorite. Think cryptococcus if: • Immunocompromised patient • Meningeal signs • Encapsulated organisms on India ink stain of (SF
a. Central nervous system involvement includes meningitis or lesions that occupy the cerebral white and gray matter space. Diagnosis is made by India ink stain. CSF cryptococcal antigen is found in the CSF and is also diagnostic. b. Treatment consists of amphotericin B alone or in combination with 5-fluorocytosine. During treatment, CSF must be monitored at weekly intervals by culture, India ink, cell count, glucose, and protein. CSF must be monitored every 3-4 weeks for antigen titer. C. Aspergillosis is a disease arising from several species of ubiquitous molds. A. fumigatus is the most common species. Organisms are normal inhabitants of the soil, and spores are readily disseminated in the air.
1. Allergic bronchopulmonary aspergillosis is marked by a hypersensitivity reaction to the fungal antigens. Inhalation of conidia or mycelial fragments may elicit an IgE-mediated hypersensitivity reaction causing bronchospasm. a. Clinical manifestations include episodic wheezing, fixed or transient pulmonary infiltrates, fever, and peripheral eosinophilia. Elevated IgE is usually seen. Positive· skin test to Aspergillus antigenic extract and positive sputum cultures are found in most infectious states. b. Treatment. No therapy is required if the disease is mild. Corticosteroids, however, are helpful in reducing symptoms. 2. Aspergillomas (fungus balls) are the result of colonization of pulmonary cavities (usually secondary to tuberculosis or sarcoidosis). Patients may be asymptomatic, but hemoptysis occurs in the majority of cases. Diagnosis is confirmed by chest x-ray, sputum culture, and/or positive serum precipitin. Surgery is indicated for massive hemoptysis. 3. Invasive aspergillosis usually occurs as an opportunistic infection in immunocompromised patients, with iatrogenic neutropenia. a. Pulmonary involvement is present in 90-95% of cases. Invasive aspergillosis presents as an unremitting fever and pulmonary infiltrate despite therapy with broad-spectrum antibiotics. Necrotizing bronchopneumonia is common. b. Extrapulmonary dissemination to the esophagus, brain, or GI tract (with GI bleeding) occurs in about 25% of cases. c. Diagnosis must demonstrate tissue invasion. Blood cultures are typically negative, whereas sputum culture is positive in only 10% of cases. Serologic diagnosis by serum precipitins (CIE), ELISA, or passive hemagglutination assays may by confirmatory if seroconversion occurs. Chest x-ray and lung biopsy may be necessary to make the definitive diagnosis. Septate branching hyphae will be seen in biopsy specimens.
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d. Treatment. Surgical removal of the aspergilloma may be necessary. Amphotericin B is the therapeutic standard, although itraconazole may be a valuable alternative. D. Zygomycosis (mucormycosis) is most often caused by organisms from the genera Rhizopus and Mucor. These molds are ubiquitous on decaying vegetable matter in soil. They have nonseptate hyphae. 1. Individuals predisposed to invasive zygomycosis disease include those with diabetic
ketoacidosis, leukemia, and lymphoma, antibiotic and steroid use, and infants and children with malnutrition. 2. Clinical manifestations a. Rhinocerebral disease is the most common presentation. It typically occurs in diabetics with ketoacidosis and is an infection of the nasal mucosa, palate, sinuses, and/or orbit, whereby progressive neurologic deficits ensue as the organism invades to the base of the brain. b. Pulmonary disease is usually the consequence of inhalation of spores in a patient with leukemia or lymphoma. 3. Treatment involves control of underlying disease such as diabetes, surgical debridement, and amphotericin B.
In a Nutshell Fungi Forms In Vivo - Coccidioides
---7
spherules
-Histoplasma
---7
intracellular yeast
-Blastomyces buds
---7
broad-based
-Cryptococcus -Candida
---7
large capsule
---7
pseudo hyphae
-Aspergillus
---7
branching septate hyphae
-Mucor! Rhizopus
---7
nonseptate hyphae
E. Pneumocystis jiroveci (formerly carinii) was originally thought to be a protozoa because of its morphologic stages and sensitivity to antiprotozoal drugs. However, rRNA homologies suggest that it is a fungus. 1. Clinical manifestations. In individuals with normal cell-mediated immunity, infection is
asymptomatic. Defects in cell-mediated immunity, such as AIDS, cause trophozoites to invade the alveoli and cause interstitial pneumonia. 2. Diagnosis is made by the demonstration of organisms in endobronchial biopsy or bronchial lavage with methenamine-silver or immunofluorescent stain. 3. Treatment for pneumonia consists of trimethoprim-sulfamethoxazole or pentamidine. Steroids are indicated for severe pneumonitis. Prophylactic and suppressive therapy consists of trimethoprimsulfamethoxazole, with dapsone or pentamidine as a second choice.
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Protozoa
Protozoa are unicellular eukaryotic organisms. They have a true nucleus, mUltiple chromosomes, and organelles. All protozoa have endoplasmic reticulum and Iysosomes, whereas some have mitochondria and Golgi apparatus. They usually reproduce asexually in the human host.
INTRODUCTION Protozoa are classified into four groups.
Note Table V-13-l. Protozoa. Classification
Common Name
Examples
Sarcodina
Amoeba
Acanthamoeba, Entamoeba, Naegleria
Sporozoa
Sporozoans
Babesia, Cryptosporidium, Isospora, Plasmodium, Pneumocystis, Toxoplasma
Mastigophora
Flagellates
Dientamoeba, Giardia, Leishmania, Trichomonas, Trypanosoma
Ciliata
Ciliates
Balantidium
Although it behaves like a protozoa, Pneumocystis is now classified as a fungus. It was discussed in Mycology, the previous chapter.
Note
INTESTINAL AND MUCOCUTANEOUS PROTOZOA Intestinal and mucocutaneous protozoa include Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis, Isospora belli, and Cryptosporidium parvum. Of these organisms, Giardia lamblia and Entamoeba histolytica have the simplest life cycles. The environmental form is called the cyst. Transmission of these organisms is fecal-oral, and results from the ingestion of contaminated water or food. Once in the intestine, the organisms excyst and the intestine is colonized by the motile, feeding trophozoite. This form is responsible for the pathology associated with disease. Trichomonas vaginalis is a sexually transmitted flagellated organism that undergoes binary fission. It has no cyst stage. Isospora belli and Cryptosporidium parvum both have complex life cycles that include sexual and asexual stages. A. Giardia lamblia is the infectious agent of giardiasis, acquired by the ingestion of fecalcontaminated water or food. 1. Clinical manifestations. Disease is caused by trophozoite attachment to the wall of the
small intestine.
Most lumen dwelling parasites infect the gastrointestinal tract and cause diarrhea. Blood and tissue protozoa cause a variety of symptoms and may be fatal.
Note You should suspect Giardia in campers or hikers who present with diarrhea, bloating, flatulence, etc. KAPLA~.
meulCa
I
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a. Manifestation of G. lamblia colonization ranges from asymptomatic infection to inflammation with diarrhea, cramps, bloating, flatulence, malaise, weight loss, and occasional steatorrhea from impaired fat absorption.
Note
b. Disease usually resolves within 3-4 weeks, although a minority of patients may have a subclinical course and remain chronic carriers.
Giardia trophozoites
c. Infection is usually more severe in children and in immunosuppressed adults.
have a characteristic "face-like" appearance.
2. Diagnosis is confirmed by the presence of cysts in formed stools of the patient. Trophozoites appear in diarrheal stools. Trophozoites from jejunal biopsy or duodenal secretions are usually superior for diagnosis. 3. Treatment consists of metronidazole or furazolidone.
B. Entamoeba histolytica is the etiologic agent for amebiasis. E. histolytica is spread by the fecal-oral route and is more common in areas with poor sanitation.
Clinical Correlate The liver abscess is characterized by hepatomegaly, right upper quadrant pain, fever, and weight loss.
1. Clinical manifestations. The organisms infect the colon of humans, invading and lysing intestinal epithelium with subsequent ulceration. a. Clinical disease may be mild with diarrhea, abdominal cramps, nausea, vomiting, and flatulence. b. More severe (invasive) disease results in dysentery, severe abdominal pain, dehydration, and bloody stools. c. Severe infection can also result in perforation of the bowel wall, resultant peritonitis, and liver abscess formation.
2. Diagnosis is made by the presence of E. histolytica cysts with four or less nuclei in stool specimens; E. coli has up to eight nuclei. Colonoscopy reveals punctate hemorrhagic areas on colonic mucosa rich with E. histolytica. Serologic methods are useful in invasive intestinal disease and in extraintestinal disease. 3. Treatment. Symptomatic amebiasis is treated with metronidazole (Flagyl) followed by iodoguinol. C. Trichomonas vaginalis causes vaginitis in women. Infection in men is frequently asymp-
tomatic but may lead to prostatitis or urethritis. 1. Clinical manifestations in women are dependent upon the physiologic status of vaginal
pH, flora, and the intensity of the infection. Symptoms include dyspareunia, dysuria, pruritis, and a copious discharge that is yellow and frothy. Symptoms worsen when vaginal pH is more alkaline. 2. Diagnosis is made by the identification of motile organisms from wet preparation of genital secretions. 3. Treatment consists of metronidazole for both the patient and her partner. D. Isospora belli has a complex life cycle. Oocysts are ingested from human fecally contaminated water or food. In the duodenum and small intestine, sporozoites are released that invade epithelial cells and multiply asexually to produce merozoites. Merozoites can reinfect the small intestine or undergo sexual differentiation into macrogametocytes and microgametocytes. Fertilization leads to the formation of oocysts, which are expelled in feces. 1. Clinical manifestations. Isospora may also be isolated in the stools of asymptomatic persons. Clinical disease is usually self-limiting after 1-2 weeks in an immunocompetent
host. Symptoms include abdominal pain, low-grade fever, diarrhea, and malabsorption. Increasing incidence of symptomatic infection with Isospora belli is seen in AIDS patients.
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2. Diagnosis is made by the identification of oocysts shed in stool. 3. Treatment consists of folate antagonists-trimethoprim-sulfamethoxazole or pyrimethamine and folinic acid. Immunocompromised patients require maintenance therapy. E. Cryptosporidium parvum is acquired by ingestion of oocysts. The oocysts release sporozoites that invade epithelial cells and multiply asexually to produce merozoites. Merozoites may reinfect cells in the jejunum and ileum and a sexual process results in the formation of oocysts, which are found in the stool. 1. Clinical manifestations. Cryptosporidium is increasingly recognized as a cause of traveler's and seasonal diarrhea. Disease presents with watery diarrhea, anorexia, and weight loss that clears in 10 days to 2 weeks. However, in immunodeficient patients, especially those with AIDS, it can be a life-threatening chronic disease. 2. Diagnosis is made by finding the oocyst in stools using acid fast stain, or through antigen detection.
Note On the exam, you are most likely to see Isospora and Cryptosporidium as causes of diarrhea in AIDS patients.
3. Treatment. Currently, no effective treatment exists for Cryptosporidium.
BLOOD AND TISSUE PROTOZOA Blood and tissue protozoa include Plasmodium species, Babesia species, Leishmania species, Trypanosoma species, and Toxoplasma gondii. A. Plasmodium species are intracellular parasites with a complex life cycle. The sexual phase occurs in the Anopheles mosquito and is transmitted to humans by the bite of the female; the asexual phase occurs in humans. Malaria affects 200 to 400 million people per year and kills approximately 1% of them. It is mainly a tropical disease. However, in the United States, it is the most common imported acute febrile illness. 1. Malaria in humans is caused by one of four species of Plasmodium.
a. Plasmodium falciparum causes malignant tertian malaria. Plasmodium falciparum causes the most deaths from malaria. It can infect all ages of red blood cells, leading to a higher level of parasitemia. Also, infected erythrocytes adhere to vascular endothelium, clogging the capillaries. This leads to thrombosis and tissue ischemia. (Fever spikes are irregular.) b. Plasmodium vivax causes benign tertian malaria. It is found widely distributed in the tropics and some temperate areas. There are 48-hour fever spikes. (1) The form acquired from the mosquito, the sporozoite, can remain dormant in the liver for extended periods. (2) Treatment requires both a blood schizonticide and a tissue schizonticide to eradicate the exoerythrocytic stage. (3) The form released from liver cells, the merozoite, uses the Duffy blood group antigen to enter reticulocytes.
Note P. vivax infects only young RBCs (reticulocytes), whereas
P. malariae infects only old erythrocytes. P. falciparum species are less discriminating and infect RBCs of all ages.
c. Plasmodium ovale causes benign tertian malaria, and is found only in West Africa. It can infect populations that are Duffy negative because its merozoites use a different receptor. Like Plasmodium vivax, it may remain dormant in the liver as well. This necessitates treatment with both tissue and blood schizonticides. There are 48-hour fever spikes.
d. Plasmodium malariae has rare focal distribution in the tropics. It causes quartan malaria, and without treatment can persist for decades, although the primary attack
iiiectical
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Microbiology
In a Nutshell
resolves in 3 weeks to six months. It invades old erythrocytes only. There are 72-hour fever spikes.
Plasmodium life Cycle Infected mosquito injects sporozoites
~
Sporozoites taken up by hepatocytes
+
Merozoites form and infect RBCs
/\
Form gametocytes
Schizonts develop into merozoites
Taken up by mosquito where fertilized to form zygote
1
a. An infected mosquito bites a human, taking a blood meal and injecting sporozoites from its salivary gland into the blood of the host. b. Sporozoites travel through the blood to the liver and multiply asexually in hepatic cells to form merozoites (exoerythrocytic stage). Upon the rupture of infected liver cells, organisms are released into the bloodstream to infect erythrocytes. ( 1) P. ovale and P. vivax may persist in the liver. (2) They may remain quiescent in the liver for up to 5 years, converting to merozoites, and can then invade RBCs and cause an acute malaria attack.
Form trophozoites that become schizonts
Merozoites are released and infect other RBCs
2. The life cycle involves a sexual stage (sporogony) in the mosquito and an asexual stage (schizogony) in humans.
1
+
Form haploid sporozoites that can migrate to salivary glands and are injected during blood meal
(3) The incubation period is about two erythrocytic cycles and is dependent upon the malaria type. c. Merozoites enter RBCs and develop into ring trophozoites. The ring trophozoites further develop into amoeboid trophozoites that become schizonts. d. Schizonts contain merozoite daughter cells. Release of the merozoites leads to periodic paroxysms of disease due to the resultant parasitemia. e. Some merozoites develop into macrogametocytes (females) and microgametocytes (males). The mosquito ingests gametocytes with a blood meal from the infected host. f. Gametocytes enter the mosquito gut, where the male initiates exflagellation followed by fertilization. The zygote invades the gut wall of the mosquito to form an oocyst within 24 hours following ingestion. Sporozoites are formed and then released into the stomach. Sporozoites then migrate to salivary glands and are injected into the human during a blood meal. 3. Clinical manifestations. All species can cause anemia, dehydration, electrolyte abnormalities, splenomegaly, and acute splenic rupture. a. Periodic fever and chills arise and last up to one hour followed by diaphoresis. Nausea, anorexia, vomiting, and malaise are clinical complications that follow. b. P. falciparum causes the greatest morbidity and mortality due to the greater degree of parasitemia and adherence to vascular endothelium. Complications can include the following: (1) Blackwater fever characterized by intravascular hemolysis, icterus, hemoglobinuria, and acute renal failure.
Note
(2) Cerebral malaria characterized by headache, confusion, rapid coma, and convulsions. Mortality from cerebral malaria is between 20 and 30%.
The gametocytes of P. falciparum have a
characteristic "banana-shaped" appearance; other species have spherical gametocytes.
4. Diagnosis is made by blood smears stained with Giemsa. Early treatment is critical and depends on the species. 5. Treatment depends on the stage of the illness and the infecting organism. a. Chloroquine is the treatment of choice unless the infecting organism is chloroquineresistant P. falciparum. b. Alternate drugs for chloroquine-resistant P. falciparum include quinine, quinidine, mefloquine, pyrimethamine, and artemisinin. Tetracycline is usually added to the above drugs.
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c. Primaquine is a tissue schizonticide required to eliminate tissue merozoites in Plasmodium vivax and Plasmodium ovale infection. Ingestion by individuals with a glucose-6-phosphate dehydrogenase (G6PD) deficiency leads to hemolysis. 6. Risk factors. Genetic factors influence host susceptibility to malarial infection. a. In West Africa, heterozygotes for the sickle cell trait have a selective advantage because the growth of the trophozoite is inhibited by low oxygen tension. b. Individuals lacking the Duffy blood group antigen are not susceptible to Plasmodium vivax infection. c. Glucose-6-phosphate dehydrogenase (G6PD) deficiency increases resistance to Plasmodium falciparum infection. B.
Babesia microti is an erythrocytic protozoan that is responsible for the human disease babesiosis.
Note If you see "Nantucket," think babesiosis.
1. Transmission and epidemiology. Babesia microti is transmitted via Ixodes dammini, the
same tick vector as in Lyme disease, so coinfections are likely. Babesia primarily infects animals; humans are incidental hosts. The species has a rodent reservoir. Babesiosis is common in the Northeastern United States, especially on islands off the northeast coast (e.g., Nantucket). Babesiosis causes severe disease or death if the patient is asplenic. 2. Clinical manifestations. B. microti elicits symptoms 1 to 3 weeks after the tick bite. Typical symptoms include malaise, fatigue, fever, generalized myalgia, nausea, sweats, arthralgia, and possibly depression. Symptoms may wax and wane for several weeks and may be mistaken for malaria. Travel history may be important diagnostically. 3. Diagnosis is made by an appropriate clinical history and by analysis of blood smears for the presence of Babesia within erythrocytes. The life cycle occurs exclusively in red blood cells, and there are no schizonts. Immunoblot of anti-Babesia serum antibodies is also diagnostic. 4. Treatment. B. microti infection is usually self-limited. Therefore, therapy is not indicated except for symptom abatement (e.g., aspirin given for fever reduction). When infection is severe or the patient is known to be asplenic, the drugs of choice are clindamycin and quinine.
In a Nutshell Review of the Tick-Borne Diseases • Babesiosis (Babesia) • Lyme disease (Borrelia) • Endemic relapsing fever (Borrelia) • Rocky Mountain spotted fever (Rickettsia) • Ehrlichosis (Ehrlichia) • Tularemia (Froncisella tularensis)
C. Leishmania species cause zoonotic infections that are transmitted by the bite of the phle-
botomine sandfly. Leishmaniasis occurs primarily in India, Africa (Old World), and Central and South America (New World). The parasite invades the host's reticuloendothelial cells and resides in the phagolysosomes. 1. Clinical disease may be expressed as cutaneous, mucocutaneous, or visceral forms.
a. Cutaneous disease is usually self-limited. Infectious species for cutaneous disease include L. mexicana, L. tropica, L. major, and L. braziliensis. b. Mucocutaneous disease (espundia) is a chronic syndrome that attacks the nasal cartilage. Mucocutaneous disease is difficult to treat and is caused by L. braziliensis. This infection may lead to death from asphyxiation or superinfection. c. Visceral disease (kala azar) principally affects the liver, spleen, lymph nodes, bone marrow, and entire reticuloendothelial system. L. donovani is the etiologic agent. 2. Diagnosis is made by clinical findings, finding parasites in slit-skin smears taken from nonulcerated parts of a cutaneous lesion, or in the case of visceral leishmaniasis, by isolation of L. donovani from biopsy samples from spleen, bone marrow, liver, or lymph nodes.
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3. Treatment of cutaneous leishmaniasis depends upon the location and extent of the lesion. Pentavalent antimonials can be used to treat large, disfiguring lesions. Visceral and mucocutaneous leishmaniasis are treated with the pentavalent antimonials sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime), amphotericin B, or pentamidine. D. Trypanosoma is the genus of protozoan responsible for African and American trypanosomiasis. 1. African trypanosomiasis, African sleeping sickness, is caused by Trypanosoma brucei gambiense in Western and Central Africa and by Trypanosoma brucei rhodiense in Eastern Africa. Both species are transmitted by the bite of an infected tsetse fly, and lead to CNS involvement and death if not treated. a. Clinical manifestions (1) Disease is first expressed as a chancre appearing at the site of inoculation. (2) Parasitemia arises 2-3 weeks later, invading the lymphoid-macrophage system and leading to fever, rash, headache, lymphadenopathy, and mental status changes. (3) Once the organism spreads to the CNS, the disease progresses with anorexia, lassitude, fatigue, wasting, and eventually stupor, coma, and death. b. Diagnosis. African sleeping sickness is diagnosed by a combination of history and clinical findings. Giemsa-stained smears of peripheral blood can detect parasites, especially in the hemolymphatic stage. The CSF should be examined for abnormalities involving cell count and protein concentration. Sediment from the centrifuged CSF can yield organisms in individuals with CNS involvement. c. Treatment. Infection is difficult to cure. Suramin or pentamidine are the drugs of choice for treatment of patients with hemolymphatic disease, but with normal CSF. The drug of choice for disease with CNS involvement is melarsoprol. 2. Chagas disease, or American trypanosomiasis, is caused Trypansoma cruzi. It is a zoonotic infection transmitted by the reduviid bug or "kissing bug." The organism enters through the mucous membranes or breaks in the skin.
Clinical Correlate Worldwide, Chagas disease is one of the most common causes of heart disease.
a. Clinical disease is caused by the invasion of the lymphoid-macrophage system, endocrine glands, myocardium, and neural tissue. b. Diagnosis of Chagas disease is made by peripheral blood smear demonstrating the presence of trypomastigotes. Xenodiagnosis is sometimes used, where laboratory triatomine bugs are first allowed to feed on the patient. The stools of the bugs are then examined for the presence of parasites. c. Treatment of acute disease consists of nifurtimox, a derivative of nitrofuran. No effective therapy exists for chronic disease.
E. Toxoplasma gondii, the etiologic agent of toxoplasmosis, occurs worldwide. 1. There are three forms of the organism: the trophozoite, which is the invasive form; the tissue cyst, which contains intracystic organisms; and the oocyst in which the sporozoites are formed.
2. Transmission to humans usually occurs via secondary hosts. a. The organism undergoes the sexual cycle in the intestine of cats, the definitive host, to form oocysts that are passed in the feces. b. Ingested oocysts invade the intestine of intermediate hosts and disseminate hematogenously to form pseudocysts in tissue.
356
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c. Cysts can occur in all tissues, including muscle, brain, and eye.
d. Tissue cysts can then be ingested by humans in raw or undercooked meat from intermediate hosts.
Note
e. Alternatively, infection can be acquired from ingestion of the oocysts in cat feces.
If you see an HIVjAIDS patient with "ring enhancing lesions" on MRI, think toxoplasmosis. (fhese same lesions in a 72-year-old smoker, think metastatic lung cancer.)
3. Clinical manifestations. Intact cell-mediated immunity usually restricts infection to the asymptomatic pseudocyst stage in adults. a. Primary infection may be associated with a mild mononucleosis-like illness. b. In immunodeficient patients (AIDS, treated cancer patients), disease usually arises as an acute infection originating from a chronic, quiescent infection that is activated due to altered immunity. CNS disease may be expressed as a meningoencephalitis or as a mass lesion with seizures. c. Congenital infection may result if the mother acquires primary infection during early pregnancy. Chorioretinitis, diffuse intracranial calcifications, hydrocephaly, anemia, and seizures are associated with congenital disease. 4. Diagnosis is confirmed by the observation of trophozoites in tissue by Giemsa stain. Serology of blood and CSF is also diagnostic. S. Treatment consists of pyrimethamine and sulfadiazine (if the disease is severe) or spiramycin or clindamycin.
Note Toxoplasma is the 'T' in the ToRCHeS acronym for congenital infections: • Toxoplasmosis • Rubella • CMV • HSV, HIV • Syphilis
KAPLAlf I medlea 357
Helminths
Helminths, or worms, are eukaryotic, multicellular organisms. Most do not multiply in their host. This characteristic affects the disease state in the host. If a host has a few worms, there is little disease. In the small proportion that have a high worm burden, there is severe disease. Parasitic helminths can be divided into two phyla: Platyhelminthes, the flatworms, and Nemathelminthes, the roundworms.
PLATYHELMINTHES Flatworms are dorsoventrally flattened, and have no body cavity. Plathyhelminthes share several features. All are hermaphrodites except for schistosomes. They have complex life cycles with development in different hosts. Finally, severe disease occurs only in a small percent of infected patients with large worm burdens or forms of the parasite in ectopic sites. Plathyhelminthes can be subdivided into cestodes (tapeworms) and trematodes (flukes). A. Cestodes (tapeworms) are long worms composed of strobila (ribbons of composed segments). The scolex (head) with sucking disks allows the worm to burrow into the intestinal wall of the definitive host. Posterior to the scolex are multiple segments called proglottids, each with both male and female reproductive organs. Each proglottid will develop into a sack of eggs that eventually breaks off from the main worm and opens, releasing eggs that will be passed in the feces of the definitive host. Humans may serve as either a definitive or intermediate host. A definitive host harbors the adult form of the worm. An intermediate host does not support the adult form. When an intermediate host is infected fecal-orally with cestode eggs, the larvae that hatch will migrate to various tissues and encyst. This can result in severe disease.
Note In the U.s., worm diseases are rare. On the USMLE, worm questions are equally rare. So, don't spend too much time on this chapter! Enterobius vermiculoris is the most common helminth infection in the United States.
1. Taenia saginata (beef tapeworm) and Taenia solium (pork tapeworm) are transmitted
to humans by the ingestion of larvae in poorly cooked beef and pork, respectively. Adults attach and live in the small intestine. Infection is usually asymptomatic or produces mild symptoms. a. Clinical manifestations. Severe infection is marked by abdominal pain, nausea, diarrhea, and weight loss. Coiled organisms may obstruct the appendix, pancreatic duct, or biliary duct. b. Diagnosis is made by the identification of eggs or proglottids in the stool. c. Treatment consists of niclosamide or praziquantel.
2. Cysticercosis is due to infection with the eggs of Taenia solium, most likely acquired from other humans. a. Clinical manifestations. When humans act as an intermediate host for Taenia soiium, severe disease often results.
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(1) Larvae penetrate the intestine to the circulation and form cysts anywhere in the body, especially the brain.
(2) The encysted form, called the cysticercus, is viable for up to 5 years. When alive, cysticerci cause virtually no inflammatory response, but may compress adjacent structures, causing symptoms especially in the eye or the base of the brain. (3) When they degenerate, a granulomatous reaction develops, and there is calcification of the organism that can be seen on x-ray. (4) Symptoms depend on the number and location of the cysts, and may not appear until years after infection. CNS symptoms include vomiting, headache, visual changes, seizures, and psychiatric disorders. b. Diagnosis of cysticercosis is through biopsy of subcutaneous nodules, brain biopsy, serology, and characteristic CT scan. c. Treatment. Symptomatic CNS disease is treated with praziquantel and steroids. 3. Diphyllobothrium latum (fish tapeworm) is transmitted by ingestion of the larvae in freshwater fish. D. latum is found primarily in the Great Lakes area and Scandinavia.
Note
a. Clinical manifestations. It is usually asymptomatic, but can cause diarrhea and vitamin BI2 deficiency with megaloblastic anemia.
Niclosamide has traditionally been the drug of choice in treating cestodes. However, praziquantel is being used with increasing frequency.
b. Diagnosis is made by the presence of eggs or proglottids in the feces. c. Treatment consists of nic10samide or praziquantel. 4. Echinococcus granulosus infection results when humans inadvertently ingest the eggs of the canine tapeworm.
a. Clinical manifestations occur when the ingested eggs hatch and the larvae migrate to multiple tissues, forming cysts. Cysts are usually asymptomatic but can enlarge and cause mechanical problems. Sixty percent of cysts occur in the liver. b. Diagnosis is made from characteristic x-ray or serology. c. Treatment consists of surgical removal. Care must be taken not to burst the cyst because anaphylaxis can result.
B. Trematodes (flukes) are flatworms that are not segmented. They have a primitive digestive tract and often have suckers for attachment to the host. Flukes are divided into two categories: tissue flukes and blood flukes. 1. Tissue flukes. Tissue flukes are leaf-shaped and have at least two intermediate hosts. The
first host of a tissue fluke is a snail. Infection is acquired by ingestion of immature forms. a. Clonorchis sinensis, the liver fluke, is endemic in Asia. Following ingestion of infected fish, the organism excysts in the duodenum and journeys to the bile ducts. (1) Clinical manifestations. Acute infection produces epigastric pain. Chronic
infection predisposes the host to cholangitis and cholangiocarcinoma. (2) Diagnosis is made by finding the characteristic eggs in the stool. (3) Treatment consists of praziquantel. b. Paragonimus westermani, the lung fluke, is transmitted by the ingestion of cysts in crabs or crayfish. The lung fluke is primarily found in Asia, South Central America, Africa, and India. When ingested, the larvae excyst in the small intestine and pass through the diaphragm into the lung.
360
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(1) Clinical manifestations. During migration, cough and abdominal pain may be experienced. Chronic infection is characterized by lung abscesses with hemoptysis. Migration to the brain may induce vomiting and seizures. (2) Diagnosis is made by finding eggs in the sputum or in feces. (3) Treatment consists of praziquantel. 2. Blood flukes. Blood flukes are composed of Schistosoma species. Infection is caused by the penetration of intact skin by a larval form. a. Schistosoma species. Over 200 million people are infected worldwide; 5 to 10% of these are symptomatic. Three species of schistosomes infect humans: Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium. All schistosomes require a developmental cycle in a snail. Humans become infected when they come in contact with contaminated water. (1) Transmission occurs when a form called the cercariae penetrates the skin. The organism migrates through lymphatic or venous channels until adult worms lodge in the portal (Schistosoma mansoni and Schistosoma japonicum) or pelvic veins (S. haematobium). Eggs secrete enzymes to work their way out of blood vessels and into the lumen of the bladder or colon. (2) Clinical manifestations. Acute schistosomiasis due to S. mansoni causes fever, anorexia, weight loss, abdominal pain, and lymphadenopathy. Chronic disease due to S. mansoni is characterized by portal hypertension, splenomegaly, and variceal bleeding. After a heavy infection with S. haematobium, the host develops hematuria. S. haematobium infection has been linked to bladder cancer. (3) Diagnosis is made by finding eggs in the urine (S. haematobium) or stool (other species). (4) Treatment consists of praziquantel.
In a Nutshell Platyhelminthes (Ratworms)
I Cestodes (Tapeworms) Segmented
~
'\ Trematodes (Flukes) Nonsegmented
~
• Taenia saginata Tissue flukes • Clonorchis • Taenia so!ium sinensis • Diphyllobothrium • Paragonimus tatum westermani • Echinococcus Blood flukes • Schistosoma species
C. Nematodes are roundworms that are not segmented and have an outer proteinaceous cuti-
cle. Parasitic nematodes may be grouped into the intestinal nematodes and filarial worms. Diagnosis of nematode infection is usually made by visualizing the characteristic eggs in the stool. Treatment is usually mebendazole. 1. Intestinal nematodes a. Ascaris lumbricoides, the cause of ascariasis, infects up to 1 billion people worldwide. Infection is spread by ingestion of infective eggs from contaminated soil or food. Larvae are released in the intestine and penetrate intestinal blood vessels to the lung, where they move through alveolar capillaries and are coughed up and swallowed. They mature in the small intestine, where the adult can live for up to 2 years. Eggs are passed in the feces and need 10 to 15 days in moist soil or water to develop into the infective form. Eggs are very resistant to the environment and can survive up to 7 years. (1) Clinical manifestations. Eighty-five percent of disease is asymptomatic. (a) During the migration of larvae through the lungs, Ascaris can cause pneumonia with eosinophilia. (b) Intestinal manifestations range from vague abdominal pain to obstruction. (2) Diagnosis is made by finding eggs in the stool. (3) Treatment consists of pyrantel pamoate or mebendazole.
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Microbiology
b. Trichuris trichiura (whipworm). Humans are the principal host, and infection is spread by the ingestion of embryonated ova. Larvae hatch in the small bowel, penetrate the villi, then move into the lumen of the cecum. (1) Clinical manifestations. In very heavy infection, usually in children, the mucosa becomes edematous and friable. Diarrhea and even rectal prolapse may develop. (2) Diagnosis is made by finding the characteristic eggs in the stooL (3) Treatment consists of mebendazole. c. Ancylostoma duodenale, the Old World hookworm, and Necator american us, the New World hookworm, infect almost one quarter of the world's population. Eggs from an infected individual hatch in soil or water and become free-living larvae. Within 1 week, they molt and become infective larvae that can survive for up to one year in soil. Infective larvae penetrate the skin and travel to the lungs via the blood. Once in the lungs they are coughed up and swallowed and establish residence in the small intestine. There, they can survive for 2 to 10 years.
(1) Clinical manifestations. Adult worms receive 25 to 30% of their nutrition from blood. This can lead to anemia, depending on the infecting species and the number of organisms. Pruritis can occur at the site of penetration. During migration through the lungs, hookworms can cause pneumonia with eosinophilia. (2) Diagnosis is made by finding the characteristic eggs in the stooL (3) Treatment consists of mebendazole. d. Enterobius vermicularis, the pinworm, causes infections that are equally prevalent in developed and developing countries. Humans are the only known host. Transmission is through ingestion of an infective egg that hatches and develops into an adult. Gravid females migrate at night to the perianal or perineal regions and deposit eggs. (1) Clinical manifestations. In sensitized individuals, the outer layer of the egg can cause severe perianal and perineal pruritis. (2) Diagnosis is made by finding the characteristic eggs on cellophane tape ("Scotch tape" technique). (3) Treatment consists of a single dose ofmebendazole. e. Strongyloides stercoralis, the threadworm, has a more complex life cycle than the other nematodes. Eggs hatch before leaving the intestine, so larvae are passed in feces. In soil, they develop into the infective stage. Infection occurs when larvae penetrate the skin and migrate to the intestine via the lungs. Unlike all other nematodes, autoinfection is possible. The change to the infective form can occur in the intestine or on perianal skin; reinfection ensues. (1) Clinical manifestations. During acute infection, when the larvae enter through the skin, pruritis to urticaria may occur. When larvae are migrating through the lungs, a mild cough may be present. Upon reaching the intestine, there may be intermittent watery diarrhea and abdominal pain. Disseminated infection, which can be fatal, occurs in the immunosuppressed and in those on steroids or chemotherapy. (2) Diagnosis is made by finding the larvae in stool or through duodenal aspiration. (3) Treatment consists of thiabendazole.
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f. Trichinella spiralis, the agent of trichinosis, is acquired from the ingestion of living organisms in inadequately cooked pork or wild carnivore meat, such as certain species of bears. (1) Clinical manifestations. Disease is initially expressed as abdominal pain and diarrhea. A second phase occurs when larvae invade muscle, resulting in fever, periorbital edema, eosinophilia, myalgia, and conjunctival petechial hemorrhages. (2) Diagnosis is confirmed by the presence of cysts in biopsy tissue. (3) Treatment consists of mebendazole. Corticosteroids can be added in patients with severe symptoms. 2. Tissue nematodes a. Wuchereria bancrofti is the main etiologic agent of filariasis, also known as elephantiasis. Filariasis is transmitted by mosquito bite. The larvae are deposited on the skin and enter the bite wound and then the lymphatics, where they mature into adults. Filariasis is found primarily in Africa, Southeast Asia, South America, and the South Pacific. (1) Clinical manifestations are marked by inflammatory responses accompanying the death of the adult worms and the molting of younger worms. Lymphatic obstruction occurs from granuloma formation and fibrosis, leading to marked lymphedema, especially of the lower extremities and scrotum. Cellulitis, dermatitis, and lymphangitis also arise and are associated with fever.
In a Nutshell Nematodes (Roundworms)
/~ Intestinal
Tissue
•Ascaris • Trichuris •Ancylostoma • Necator • Enterobius • Strongyloides • Trichinella
• Wuchereria • Onchocerca • Toxocara
In a Nutshell Worm Clues
(2) Diagnosis is confirmed by finding microfilariae in the blood or lymphatics.
• Brain cysts, seizures
---7
Taenia solium
(3) Treatment consists of diethylcarbamazine for micro fIlariae and adult worms. Corticosteroids may be necessary during marked inflammatory responses.
• Liver cysts
---7
• Megaloblastic anemia/B 12 deficiency
---7
Echinococcus Diphyllobothrium latum
• Biliary trad disease, cholangiocarcinoma
---7
Clonorchis sinensis
• Hemoptysis
---7
• Portal hypertension, esophageal vances
---7
Paragonimus westermani Schistosoma mansoni
• Hematuria, bladder cancer
---7
• Redal prolapse
---7
b. Onchocerca volvulus causes onchocerciasis or "river blindness:' It is transmitted by the bite of the blackfiy, which resides alongside flowing streams or rivers in Africa, and Central and South America. Onchocerciasis is second only to trachoma as the main cause of infectious blindness. (1) Clinical manifestations are expressed as subcutaneous nodules, a papular ery-
thematous pruritic rash, and blindness. (2) Diagnosis is made by the observation of microfIlaria in a skin biopsy and anterior chamber of the eye. (3) Therapy consists of ivermectin and surgical excision of nodules. c. Toxocara canis causes toxocariasis (visceral larva migrans). It is found in dogs, particularly puppies, and is transmitted by the ingestion of eggs in dirt soiled with feces. (1) Clinical manifestations are characterized by visceral involvement of liver, lung, brain and eyes, hemorrhage, necrosis, and eosinophilic granulomas. (2) Diagnosis is usually based on clinical evidence unless a biopsy of viscera (e.g., liver) reveals larvae. (3) Treatment consists of corticosteroids and antihelminthic agents such as diethylcarbamazine and albendazole.
• Microcytic anemia (blood loss)
Schistosoma haematobium
Trichuris trichiura ---7 Ancylostoma and Necator
• Perianal pruritis (Scotch tape)
---7
Enterobius vermicularis
• Autoinfedion
---7
• Elephantiasis, lymphedema
---7
• Blindness
---7
Strongyloides stercoralis Wuchereria bancrofti Onchocerca volvulus
KAPLA~. I meulCa
363
Antimicrobial Agents
This comprehensive chapter reviews the drugs used to treat the pathogenic microorganisms described in the previous chapters. The outline is divided into antibacterials (antibiotics), antimycobacterials, antifungals, antihelminthics, and antiviral agents. For each drug, the mechanism of action, indications for use, and side effects and toxicity are reviewed.
ANTIBACTERIAL AGENTS Antibiotics are chemicals that may be produced entirely by microorganisms or that may be modified (semisynthetic) to broaden the spectrum of activity, increase the chemical stability, or improve the pharmacokinetic properties. Some antibiotics inhibit bacterial growth (bacteriostatic); others kill organisms (bactericidal); and some possess both properties in a dose-dependent manner. Antibiotics are usually classified according to bacterial specificity or mechanism of action. A. Overview of classes of antibiotics 1. Cell wall synthesis inhibitors. Bactericidal agents that interfere with the synthesis of
bacterial cell walls make microorganisms vulnerable to changes in the osmolarity of the environment. The cell wall contains complex, cross-linked peptidoglycans, which conveys rigidity. The cross-linking occurs across peptide chains as a result of transpeptidation and is catalyzed by enzymes inhibited by antibiotic. a. AlII3-lactam antibiotics include
~-lactam
rings:
In a Nutshell Cell Wall Synthesis Inhibitors
• Penicillins • Cephalosporins
13-ladams
• Carbapenems
(1) Penicillins andcephalosporins. These drugs act as analogs ofD-alanyl-D-alanine.
They prevent the final step in cell wall synthesis by inhibiting the transpeptidase enzyme responsible for cross-linking and peptidoglycan synthesis. The penicillins and cephalosporins differ primarily in that the penicillins are derivatives of 6-aminopenicillanic acid, whereas the cephalosporins are characterized by substituent groups added to 7-aminocephalosporanic acid. Differences in susceptibility of Gram-positive and Gram-negative organisms depend on structural differences in cell walls, the presence of different binding proteins, the nature of peptidoglycans, and the activity of autolytic enzymes.
• Monobadams • Vancomycin • Bacitracin • Cycloserine
(2) Carbapenems (3) Monobactams b. The drugs listed below inhibit cell wall synthesis at early stages within cell cytoplasm; drugs must penetrate the cell membrane to be effective. (1) Vancomycin blocks the growing end of peptidoglycan. (2) Bacitracin blocks dephosphorylation of lipid carrier.
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365
Microbiology
(3) Cycloserine prevents D-alanine additions to form pentapeptides. 2. Protein synthesis inhibitors. These drugs affect the function of bacterial ribosomes, thereby inhibiting protein synthesis. a. The protein synthesis inhibitors that interact with the 30S ribosomal subunit include the following: (1) Aminoglycosides are bactericidal agents that block initiation of protein synthesis, causing accumulation of protein synthetic initiation complexes. Because tetracyclines do not inhibit bacterial cell wall synthesis, they are effective against cell wall-deficient organisms and bacterial variants that may develop during treatment with cell wall-inhibiting antibiotics. (2) Tetracyclines act as bacteriostatic agents that inhibit binding of aminoacyltransfer RNA (tRNA) to mRNA-ribosome complex.
In a Nutshell Protein Synthesis Inhibitors • Aminoglycosides • Tetracyclines • Spectinomycin • Erythromycin • Lincomycin • Clindamycin • Chloramphenicol
(3) Spectinomycin blocks initiation. b. The protein synthesis inhibitors that interact with the 50S ribosomal subunit are: (1) Erythromycin is a macrolide antibiotic that contains a large lactone ring to which sugars are attached. Erythromycin inhibits bacterial protein synthesis by blocking release of the uncharged tRNA from the 50S ribosomal subunit. It is prescribed for patients allergic to penicillins. (2) Clindamycin is a lincosamide that has an unknown mechanism thought to be similar to that of erythromycin (inhibits 50S subunit of ribosome). Whereas clindamycin's Gram-positive antibacterial spectrum is similar to that of erythromycin, it has broader coverage, including anaerobes. (3) Chloramphenicol is a bacteriostatic agent that acts by binding the 50S subunit of the bacterial ribosome to inhibit peptidyltransferase action. This binding is readily reversible and is inhibited by the macrolide and lincosamide antibiotics. 3. Antimetabolites. Folate antagonists are bacteriostatic agents that interfere with bacterial synthesis or reduction of folate. a. Sulfonamides arrest cell growth by inhibiting the bacterial synthesis of folic acid. Sulfonamides are structural analogs of the folic acid precursor, para-aminobenzoic acid (PABA). They competitively inhibit dihydropterate synthetase, the enzyme that directs the incorporation of PABA and a pteridine moiety into dihydropteroic acid. Organisms that do not synthesize folic acid because they obtain it from other sources (e.g., humans) are unaffected. b. Trimethoprim is a structural analog of the pteridine portion of dihydrofolate reductase and acts as a competitive inhibitor of this enzyme, which converts dihydrofolate to tetrahydrofolate (active form offolic acid). Tetrahydrofolate is required as a methyl donor in the synthesis of adenine, guanine, and thymine.
366
meCtical
Antimicrobial Agents
Penicillins
Amidase H
\
I
H
H
i
1/ S ,......- CH 3
R-N-C-C
C ..... I ,CH 3 C-N-C......- H
I B I A
t
O.?'
...
COOH
Lactamase Substituted 6-aminopenicillanic acid Cephalosporins
o II
H
H
H
I
1/S,
-C-N-989
R1
t
A
......-H
Y'H
C-N C O.?' '9~
. . . CH 2-R2
COOH Substituted 7-aminocephalosporanic acid Monobactams
o
H
H
H
II
I
.
1
R-C-N-C-C- CH3
IB I
C-N O.?'
'
R:-C-Cr ~ N~NH2 I O-C(CH3h
S03H
I
COOH Substituted 3-amino-4-methylmonobactamic acid (aztreonam)
HO I
H
H
,
1
Carbapenems
HC-C-C~ /" I B I ~ S-R H3C
.?'C-N
o
NH
II
R:-CH 2-CH 2-NH-CH
COOH Substituted 3-hydroxyethylcarbapenemic acid (imipenem) Clavulanic acid
Figure V-1S-1. Structure of four [3-lactam antibiotics and clavulanic acid. The ring in each structure is the [3-lactam ring. (Reprinted with permission from Katzung BG (ed): Basic and Clinical Pharmacology 6th ed. East Norwalk, Connecticut, Appleton and Lange, 1995, p 681.)
meCtical
367
Microbiology
4. Cell membrane inhibitors act directly on the cell membrane to affect permeability and lead to leakage of intracellular compounds. a. Cell membrane inhibitors that disrupt cell membranes of Gram-negative bacteria (bactericidal) include: (1) Polymyxin, which binds to phospholipids and alters cell permeability
(2) Colistin b. Cell membrane inhibitors that interact with membrane sterols in fungal cells include: (1) Amphotericin B, which binds to ergosterol-altering cell membranes. (2) Nystatin (same mechanism as amphotericin) (3) Fluconazole, clotrimazole, and ketoconazole inhibit ergosterol synthesis. 5. Nucleic acid synthesis inhibitors a. Fluoroquinolones (e.g., ciprofloxacin) and nalidixic acid inhibit DNA gyrases (topoisomerases) necessary for supercoiling of DNA. b. Rifampin binds to and inhibits DNA-dependent RNA polymerase present in bacteria.
(4)
(1 )
Cell-wall synthesis Cycloserine Vancomycin teichoplanin Bacitracin Penicillins Cephalosporins Monobactams
(4)
"'" ,_.----:= (5)
-'C3(Jl'-{3Ur---\3U~
Erythromycin (macrolides) Chloramphenicol Clindamycin
Protein synthesis (30S inhibitors)
PABA
Tetracycline Spectinomycin Streptomycin Gentamicin tobramycin (aminoglycoside) Amikacin
(2)
Figure V-15-2. Antimicrobial sites of bactericidal action on microorganisms. The five mechanisms are: (1) inhibit cell wall synthesis; (2) damage cell membrane; (3) modify nucleic acid/DNA synthesis; (4) modify protein synthesis; and (5) modify metabolism within the cytoplasm. (THFA = tetrahydrofolic acid; DHFA = dihydrofolic acid; and PABA = para-aminobenzoic acid.) (Modified with permission from BrodyTM, Larner J, Minneman KP, Neu He: Human Pharmacology: Molecular to Clinical, 2nd ed. St. Louis, Missouri, Mosby-YearBook, 1994, p 618.)
368
meClical
Antimicrobial Agents
B. Efficacy 1. Successful antimicrobial therapy depends on achieving inhibitory or bactericidal activity
at the site of infection without significant toxicity to the host. 2. In most infections, the normal local and systemic host defense mechanisms playa crucial role in the final elimination of the pathogen. 3. The degree of in vitro susceptibility of bacterial strains to particular antibacterial agents is estimated by determining the minimum inhibitory concentration (MIC), the lowest concentration of antibiotic that prevents growth, and the minimal bactericidal concentration (MBC) , the lowest concentration of antibiotic that kills all the organisms in an in vitro MIC assay. C. Resistance. Microorganisms are capable of acquiring resistance to antimicrobial agents by
genetic changes that are passed on from generation to generation. 1. Spontaneous chromosomal mutations produce a genetically altered bacterial population that is resistant to the drug action, survives, and gives rise to a new drug-resistant population. 2. Drug resistance is usually acquired, not by chromosomal change, but by R-factors from other bacteria in the form of extrachromosomal DNA pieces that contain resistancemechanism information. R-factors are plasmids that carry genes for resistance to one or more antibiotics. Plasmid transfer accounts for over 90% of antibiotic resistance.
Note The Kirby-Bauer disk diffusion method is commonly used to determine antibiotic sensitivity. The organism is exposed to disks saturated with different antibiotics. After growth, the degree of growth inhibition around each disk is measured and the susceptibility/resistance to the antibiotic is determined.
3. Changes in drug permeability. Tetracyclines are able to accumulate in susceptible bacteria; in resistant bacteria, resistance occurs by increasing the energy-dependent efflux of tetracyclines. 4. Drug deactivation. The principal mechanism of resistance to the penicillins and cephalosporins is by B-Iactamase action. Aminoglycosides and chloramphenicol are inactivated by acetylation or other enzymatic modification. 5. Decreased drug conversion to active compound. The antifungal drug, flucytosine, must be converted in vivo to fluorouracil, which is further metabolized to the active form of the drug. Fungi become resistant to flucytosine by losing enzyme activity along the activation pathway. 6. Altered metabolic pathway may occur in bacteria resistant to sulfonamides and in fungi resistant to flucytosine. Some sulfonamide-resistant bacteria can use preformed folic acid (e.g., from mammalian cells). 7. Altered amount of drug receptor. Some organisms become resistant to penicillins and cephalosporins by synthesizing altered penicillin-binding proteins and to fluoroquinolones by altered DNA gyrase activity. 8. Decreased receptor affinity for drug. Resistance to erythromycin may be associated with alteration of a specific protein on the 50S subunit of the bacterial ribosome necessary for drug binding. 9. Evaluations of the effectiveness of antimicrobial agents must use the results of clinical trials as well as the in vitro activity of the drug against potential pathogens. Combinations of antibiotics are often used to broaden coverage with mixed or unknown types of infection, to prevent or delay the emergence of bacterial resistance to the drugs, and to achieve therapeutic synergy.
KAPLA~.
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I 369
Microbiology
BETA-LACTAMS AND OTHER CELL WALL SYNTHESIS INHIBITORS A. Penicillins (Table V-IS-I) are ~-lactam antibiotics similar to the cephalosporins. The development of large-scale production has led to structural modifications of the original penicillin G, with formation of derivatives with greater effectiveness against a variety of infections. 1. Penicillin G (benzyl penicillin)
a. Pharmacologic properties ( I) The cyclic amide structure, called a ~-lactam, binds to a transpeptidase and prevents peptidoglycan cross-linkage essential for completion of cell wall synthesis. Enzymatic or acid hydrolysis of the ~-lactam results in loss of antibiotic activity. (2) Penicillins also increase bacterial cell wall breakdown by activating bacterial autolysins. (3) They possess bactericidal action that affects only growing cells during cell wall synthesis. (4) Gastric acid hydrolyzes penicillin G and only 30% of the active drug is absorbed.
Table V-IS-I. Penicillins. Drug Acid labile Benzyl penicillin (penicillin G)
Route
Bacterial Specificity
Given orally; poor absorption when given intramuscularly and intravenously
Used against most Gram-positive cocci and some Gram-negative; effective against Neisseria; hydrolyzed by acid and ~-lactamase
Phenoxymethyl penicillin (penicillin V) Acid stable Ampicillin
Penicillinaseresistant penicillins Methicillin Oxacillin, cloxacillin, and dicloxacillin Nafcillin Broad-spectrum activity Carbenicillin Ticarcillin Piperacillin
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Given orally only
Similar lability but less effective than penicillin G; preferred for upper respiratory tract infections
Given orally and parenterally
Used against all staphylococci, many E. coli, and H. injluenzae strains (others now resistant); lactamase-vulnerable but stable in combination with clavulanic acid (Augmentin); similar to ampicillin but higher blood levels and fewer gastrointestinal problems
Poor orally; must be given parenterally Fair oral absorption; given intramuscularly and intravenously Poor orally; given parenterally
Spectrum is similar to penicillin G but less potent. Used against ~-lactamase-containing staphylococcal infections Most active against resistant S. aureus
Poor orally; given intramuscularly Lactamase sensitive; active against and intravenously Pseudomonads, Proteus Given intravenously Given intravenously
Available with clavulanic acid; more potent against Gram-negative organisms
Antimicrobial Agents
(5) Crystalline penicillin G given intramuscularly results in therapeutic peak plasma levels that last for 2-3 hours. It may be given intravenously when large doses are required. (6) Repository forms are used to prolong antibiotic levels by delaying the rate of systemic absorption from parenteral sites. Procaine salt of penicillin G is absorbed more slowly after intramuscular administration, attaining therapeutic peak plasma levels lasting 24 hours. Benzathine penicillin G maintains low drug levels for up to 4 weeks after a single intramuscular injection; it is used for prophylaxis of group A streptoccocal infection and for treatment of syphilis. (7) Penicillin is 55% bound to serum proteins and is widely distributed throughout the body. Significant cerebrospinal fluid penetration is seen only with meningeal inflammation. (8) Seventy to eighty percent excreted in urine (i.e., 10% by glomerular filtration, 90% by renal tubular secretion); renal tubular secretion can be blocked by concurrent administration of the weak organic acid, probenecid. Probenecid depresses the tubular secretion of penicillin, allowing it to have a longer half-life. b. Indications for use (1) Gram-positive cocci, including Streptococcus pneumoniae and Streptococcus pyo-
genes (2) Gram-negative cocci, including Neisseria meningitidis (3) Gram-positive bacilli, including Bacillus anthracis, Clostridium perfringens, Listeria monocytogenes, and Corynebacterium diphtheriae
(4) Treponema pallidum, the causative agent of syphilis c. Side effects and toxicity of penicillin are generally few, but in some patients, allergic
reactions may occur. (1) Hypersensitivity reactions occur in up to 10% of patients. Symptoms include
rashes, urticaria, fever, serum sickness, Stevens-Johnson syndrome, and anaphylaxis due to antibody formation to degradation products. Cross-sensitivity with cephalosporins also exists. (2) Diarrhea (3) Jarisch-Herxheimer reaction (flu-like symptoms, including fever, chills, and myalgia) can occur in secondary syphilis within the first few hours following penicillin G therapy. d. Resistance mechanisms (1) Bacteria containing penicillinase enzymes (~-lactamases) in their periplasmic space can inactivate penicillin G by opening the ~-lactam ring. It is a common
resistance mechanism among staphylococci and Gram-negative bacteria. Methicillin-resistant organisms (e.g., many Staphylococcus epidermidis organisms) are resistant even to penicillins not sensitive to penicillinase; these organisms are usually treated with vancomycin. (2) Penicillin penetrates the bacterial cell envelope and attaches to a number of penicillin-binding proteins (PBPs) on the bacterial cytoplasmic membrane. Bacterial resistance may also result from altered affinity and number PBPs.
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2. Penicillin V a. Pharmacologic properties. It is effective against oral bacteria; most used penicillin in dentistry. It is available only in oral form. It is more resistant to gastric acid destruction and has greater gastrointestinal absorption than penicillin G. b. Indications for use (1) Penicillin V is less active than penicillin G, especially against Gram-negative bacteria (e.g., Neisseria, meningococcal meningitis). (2) It is used only when an oral form is desired for susceptible organisms. 3. Broad-spectrum penicillins a. Ampicillin (1) Pharmacologic properties are similar to penicillin G (both are destroyed by ~ lactamase), but ampicillin is acid stable and has increased activity against Gramnegative organisms (e.g., Haemophilus injluenzae, Escherichia coli, Proteus, Salmonella.) Ampicillin is less active than penicillin G is against Gram-positive cocci. It is available for oral and parenteral administration. Resistance mechanisms include inactivation by penicillinase or altered properties of PBPs. (2) Indications for use include some gonococcal infections, upper respiratory infections (e.g., H. injluenzae, S. pneumoniae, S. pyogenes), urinary tract infections (e.g., E. coli, Enterococcus, Proteus mirabilis), meningitis (e.g., H. injluenzae, S. pneumoniae, N. meningitidis), and Salmonella and Shigella infections. It is also preferred in Listeria infections. (3) Side effects and toxicity are similar to penicillin G, but rashes are more common and an idiosyncratic reaction occurs in patients with mononucleosis that causes a maculopapular rash. It can also cause pseudomembranous colitis. b. Amoxicillin is a parahydroxyl derivative of ampicillin. (1) Pharmacologic properties are similar to ampicillin, but there is better intestinal absorption and less gastrointestinal disturbance. Amoxicillin is hydrolyzed by ~ lactamases but is stable in combination with ~-lactamase inhibitor, davulanic acid (Augmentin). Amoxicillin is resistant to gastric acid destruction and can be taken orally. It attains higher peak serum levels than ampicillin after similar oral dosage; this is the major difference between the two agents. The in vitro antibacterial spectrum of amoxicillin is identical to ampicillin except it is less effective against Shigella. (2) Indications for use include infections of the skin, soft tissue, and lower urinary and respiratory tracts, caused by nonpenicillinase-producing strains of staphylococci, streptococci, H. injluenzae, E. coli, and P. mirabilis; uncomplicated anogenital and urethral gonococcal infections; and otitis media. (3) Side effects and toxicity include lower incidence of nausea, vomiting, and diarrhea than with ampicillin, and hypersensitivity reactions. c. Amoxicillin plus davulanic acid (Augmentin) (1) Pharmacologic properties are similar to amoxicillin, but the combination with
clavulanic acid broadens coverage to include ~-lactamase-producing organisms, including H. injluenzae. Clavulanic acid covalently reacts with ~-lactamase and prevents the enzyme from hydrolyzing the ~-lactam ring. Augmentin is administered orally only.
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(2) Indications for use include severe otitis media, sinusitis, pneumonia (e.g., H. inJluenzae, Moraxella catarrhalis), and animal bites (Pasteurella multocida). (3) Side effects and toxicity are similar to amoxicillin given alone except that there is a greater incidence of nausea and vomiting. d. Ampicillin with sulbactam (a penicillinase inhibitor) (1) Pharmacologic properties are similar to ampicillin. Sulbactam broadens coverage to include ~-lactamase-positive organisms and some anaerobes. There is parenteral administration only.
Note Clavulanic acid and sulbactam are inhibitors of [3-lactamase that can greatly broaden the spectrum of penicillins.
(2) Indications for use include intra-abdominal infections where anaerobic coverage is desired and severe urinary tract infections (UTIs), including those caused by enterococci. (3) Side effects and toxicity are similar to ampicillin. 4. Antipseudomonal penicillins a. Carbenicillin ( 1) Pharmacologic properties. Carbenicillin is administered parenterally or orally. It is excreted unchanged by the kidney. There is increased antibacterial activity against Pseudomonas species and other Gram-negative organisms such as Enterobacter and Proteus. (2) Indications for use. The main indication is treatment for systemic infection with Pseudomonas aeruginosa. Resistance develops rapidly; thus, it should almost always be used with an aminoglycoside. (3) Side effects and toxicity. Carbenicillin is generally well-tolerated, but occasional hypersensitivity reactions occur, including hypokalemia, sodium overload, and platelet dysfunction. b. Ticarcillin (1) Pharmacologic properties are similar to carbenicillin. It can be given in lower doses by parenteral route only. It is excreted by the kidney. Resistance involves inactivation by penicillinases.
(2) Indications for use. It is more active than carbenicillin against E. coli, Enterobacter, and Proteus. (3) Side effects and toxicity are similar to carbenicillin. c. Ticarcillin with c1avulanic acid (Timentin) ( 1) Pharmacologic properties are similar to ticarcillin; clavulanic acid broadens coverage to include ~-lactamase-producing organisms. The drug is used only parenterally. (2) Indications for use include hospital-acquired infections (nosocomial infections) requiring Pseudomonas coverage and broad Gram-positive protection. (3) Side effects and toxicity are the same as with ticarcillin. d. Piperacillin (1) Pharmacologic properties. Piperacillin is more active than ticarcillin or carbenicillin against Pseudomonas. It is also active against Proteus, Enterobacter, and
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In a Nutshell Carbenicillin, ticarcillin, and piperacillin are penicillins with broad-spectrum coverage used primarily in the treatment of pseudomonal infections (along with an aminoglycoside).
Klebsiella. Administration is by the parenteral route. Toxicity and resistance mechanism are similar to ticarcillin and carbenicillin, but these drugs are more resistant to penicillinase. (2) Indications for use. These agents should be used with an aminoglycoside against serious Pseudomonas infections. 5. Penicillinase-resistant penicillins (antistaphylococcal penicillins) a. Methicillin (1) Pharmacologic properties. Methicillin is penicillinase-resistant; it provides
increased resistance to hydrolysis of the ~-lactam ring by staphylococcal penicillinase and other ~-lactamases. It is used only parenterally because of poor oral absorption and sensitivity to gastric acid. Fifty percent of the drug is excreted unaltered in urine. It is not effective against Gram-negative organisms. (2) Indications for use include penicillin-resistant S. au reus infections. Methicillinresistant Staphylococcus infections should be treated with vancomycin. (3) Side effects and toxicity include allergic reactions, reversible bone-marrow depression, phlebitis, and nephrotoxicity. Because the incidence of interstitial nephritis is greater than with other penicillins, methicillin is not the agent of choice in most cases. b. Oxacillin, dicloxacillin, and cloxacillin are used mainly against infections with lactamase-producing staphylococci.
~
(1) Pharmacologic properties. Spectrum is similar to penicillin G but less potent. Because these drugs are resistant to gastric acid destruction, they may be administered orally or parenterally. High concentrations of these drugs are found in bile and in pleural and amniotic fluids. There is rapid renal excretion. These drugs are less effective than ampicillin against Gram-negatives. Dicloxacillin is the most active of the oxacillins against S. aureus, but it is less active than nafcillin. (2) Indications for use involve penicillin G-resistant staphylococci. (3) Side effects and toxicity include rash, urticaria, pruritus, hypersensitivity reactions, and hepatitis. c. Nafcillin (1) Pharmacologic properties. Nafcillin is administered parenterally (variable oral
absorption). There is variable inactivation by gastric acid and biliary excretion. (2) Indications for use. Nafcillin is useful in treating staphylococcal infections and mixed infections with penicillin-resistant staphylococci and streptococci. (3) Side effects and toxicity. Nafcillin is generally well-tolerated, but allergic reactions, gastrointestinal discomfort, and mild phlebitis may occur. B. Cephalosporins originated from a Cephalosporium fungus and have been found to inhibit
Staphylococcus aureus. As with the penicillins, semisynthetic cephalosporins now exist for clinical use and are classified as first-, second-, third-, or fourth-generation drugs. 1. Mechanism of action is similar to that of the penicillins; cephalosporins block terminal cross-linking of the bacterial cell wall peptidoglycan and activate cell wall autolytic enzymes.
2. The nucleus of the cephalosporins consist of a ~-lactam ring fused to a six-membered ring similar in structure to the nucleus of the penicillins.
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Antimicrobial Agents
3. Resistance to the cephalosporins is by ~-lactamases and by lack of antibiotic penetration in Gram-negative organisms. 4. The first-generation cephalosporins are broad-spectrum agents with activity against Gram-positive organisms (except Enterococcus and methicillin-resistant Staphylococcus) and Gram-negative bacteria (including E. coli, Klebsiella, Proteus). 5. Anaerobic bacteria, including Bacteroides fragilis, are sensitive to the second-generation agents (e.g., cefoxitin), which are less potent than first-generation cephalosporins but have broader Gram-negative coverage. Haemophilus, Neisseria, E. coli, Klebsiella, and Proteus are sensitive to the second-generation drugs. Gram-positive infections are generally treated with this drug class. 6. The third-generation drugs can penetrate the central nervous system (CNS) and, with the exception of cefoperazone, are active against bacterial meningitis. These third-generation agents are resistant to j3-lactamases. Gram-negative organisms are susceptible, whereas Gram-positives are poorly treated by third-generation agents. 7. Fourth-generation cephalosporins, much like third-generation drugs, have good activity against Gram-negative bacteria. However, this generation has strong activity against Gram-positives, similar to first-generation drugs. 8. Pharmacologic properties are similar to the penicillins but somewhat less potent, requiring higher dosages. a. Most cephalosporins are excreted by the kidney (glomerular fIltration and tubular secretion). b. Those with acetylated residues (cephalothin, cephapirin) are metabolized in the liver, and their metabolites are renally excreted. c. Some are highly protein bound (e.g., cephalothin, cefamandole, cefoxitin, and
cephapirin). d. Variable susceptibilities to
~-lactamases
9. Indications for use include upper respiratory infections, acute otitis media, urinary tract infections, skin and soft tissue infections with S. aureus, prophylaxis before surgery, systemic infections with bacteria sensitive to these agents, and life-threatening infections before specific organisms are identified (due to broadness of antimicrobial spectrum). 10. Side effects and toxicity
a. Allergic reactions may occur, including urticaria, rash, fever, and eosinophilia. There is a 5-10% cross-reactivity between cephalosporins and penicillins in those hypersensitive to penicillins. b. There may also be nausea, vomiting, diarrhea, and elevated liver function test values (pseudomembranous colitis). c. Hypoprothrombinemia (with cefamandole, cefoperazone, moxalactam)
d. Disulfiram-like reaction with alcohol moxalactam)
(with cefamandole, cefoperazone,
e. Nephrotoxicity f. Positive direct Coombs test 11. Specific agents (Table V-15-2) KAPLA~.
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In a Nutshell • First generation
Table V-15-2. Cephalosporins.* Similar to penicillin and ampicillin
• Second generation Extended Gram-negative coverage, including anaerobes • Third generation
Excellent Gram-negative coverage; cross blood-brain barrier
Drug First generation Cephalothin Cefazolin Cephalexin Cephradine Cefaclor Second generation Cefamandole Cefuroxime
Cefoxitin
Third generation Cefotaxime Ceftizoxime Ceftriaxone Cefixime (oral agent)
Cefoperazone Fourth generation Cepirome Cefepine
Route and Bacterial Specificity These drugs inhibit most Gram-positive organisms except enterococci. They are used to treat respiratory infections in children.
This drug is more active than first-generation drugs against Haemophilus species, some E. coli, Klebsiella, and other Enterobacteriaceae. This drug inhibits Gram-positive organisms, is excellent against Haemophilus and Neisseria species, and has greater resistance to ~-lactamase than does cefamandole. This drug is less active against Gram-positive organisms, but its high ~-lactamase stability and inhibitability to Enterobacteriaceae and 85% of anaerobic bacteria make it useful for aspiration pneumonitis and intra-abdominal and intrapelvic infections. These drugs are excellent against Gram-positive streptococci, including S. pneumoniae and Haemophilus and Neisseria species. This drug is similar in action to cefotaxime and ceftizoxime. It is used to treat nosocomial infections, Lyme disease, and gonorrhea. It precipitates in the bladder and may cause diarrhea. CefIxime inhibits streptococci, Haemophilus species, Neisseria, Moraxella, and many Enterobacteriaceae. It is used to treat respiratory infections. Cefoperazone has an Antabuse-like action and it changes prothrombin activity. It is less ~-lactamase stable but is active against Pseudomonas. These drugs have increased activity against Grampositive bacteria, inhibit Pseudomonas species, and are not labile to some ~-lactamases.
*Note: Cephalosporins are similar to the penicillins in structure; they also have similar activities, which have increased with each "generational" modification in structure.
C. Monobactams: aztreonam. As the name implies, monobactams have a single ~-lactam ring. L Pharmacologic properties. Aztreonam interferes with cell wall synthesis. Excretion is
mostly urinary. Tissue levels are excellent. 2. Indications for use. Aztreonam is used to treat Gram-negative infections; it provides no Gram-positive or anaerobic coverage (narrow spectrum). 3. Side effects and toxicity include swelling and local irritation, mild LFT abnormalities, and anaphylactic reactions (but little cross-reactivity with other ~-lactams).
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Antimicrobial Agents
D. Carbapenems: imipenem
1. Pharmacologic properties a. Imipenem is a synthetic ~-lactam antibiotic. b. It inhibits cell wall synthesis. c. Imipenem is marketed in combination with cilastatin, a renal dipeptidase inhibitor, which prevents renal tubular metabolism and accumulation of nephrotoxic metabolites. d. Imipenem has broad-spectrum coverage, including penicillinase-producing Grampositives, Gram-negatives, P. aeruginosa, and anaerobes. It is the most potent and broadest-spectrum ~-lactam currently on the market. 2. Indications for use. Use is essentially limited to nosocomial infections. 3. Side effects and toxicity include nausea, vomiting, and diarrhea; allergic reactions; and seizures. E. Other cell wall inhibitors 1. Vancomycin a. Pharmacologic properties (1) Vancomycin is a bactericidal agent that inhibits cell wall synthesis by binding D-
Ala-D-Ala, thus preventing its incorporation into peptidoglycan. (2) It is poorly absorbed after oral administration. (3) It is active against Gram-positive bacteria but not against Gram-negative organisms. b. Indications for use (1) Vancomycin is given orally to treat pseudomembranous colitis caused by the
anaerobe Clostridium difficile. (2) It is given intravenously for treatment of methicillin-resistant staphylococci (MRSA) and penicillin-resistant pneumococci. (3) It is useful in patients allergic to penicillins and cephalosporins in the treatment of Gram-positive infections. (4) Finally, vancomycin is used with gentamicin for Streptococcus faecalis or Streptococcus viridans endocarditis or for serious infections in patients with penicillin allergy. c. Side effects and toxicity. Rapid infusion can cause facial and neck erythema ("red man" syndrome). There may also be ototoxicity (rare), phlebitis, and nephrotoxicity. 2. Bacitracin is a cyclic polypeptide that inhibits cell wall synthesis by blocking the dephosphorylation of the phospholipid that transports peptidoglycan subunits across the cytoplasmic membrane. Because of nephrotoxicity, use is limited to topical application.
In a Nutshell Uses of Vancomycin
• Methicillin-resistant staphylococci • Enterococcal infections in the penicillin-allergic patient • Pseudomembranous colitis (C difficile) • Penicillin-resistant S. pneumoniae
3. Cycloserine is a broad-spectrum antibiotic that inhibits D-alanine incorporation into peptidoglycan. Its use is limited to treatment of tuberculosis (TB).
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PROTEIN SYNTHESIS INHIBITORS A. Aminoglycosides 1. Overview. Binds the 30S ribosomal subunit and causes change in the codon:anticodon
recognition sites. Bactericidal action results from the irreversible inhibition of the initiation of protein synthesis. a. Pharmacologic properties. All aminoglycosides are administered parenterally because of poor intestinal absorption and are used against aerobic Gram-negative bacteria. b. Side effects and toxicity (1) Ototoxicity and renal impairment are side effects of all aminoglycosides and are usually dose dependent, whereas hypersensitivity reactions are idiosyncratic. (2) Increased nephrotoxicity is seen when arninoglycosides are given concurrently with amphotericin, furosemide, and cephalosporins. In addition, elderly patients or patients with shock, dehydration, or pre-existing renal disease are more susceptible to the nephrotoxicity. (3) Aminoglycoside doses must be reduced in renal insufficiency. (4) Resistance develops rapidly to all aminoglycosides mainly by plasmid-encoded enzyme inactivation via acetylation, phosphorylation, or adenylation. 2. Gentamicin a. Pharmacologic properties (1) Gentamicin is active against Enterobacter, E. coli, Klebsiella, Proteus,
Pseudomonas, Neisseria, Serratia, and Shigella. (2) It is active against Streptococcus viridans and Streptococcus faecalis when combined with penicillin or ampicillin. b. Indications for use ( 1) Gentamicin is used for serious infections with susceptible Gram-negative bacteria. (2) It is also used topically in burns infected with Pseudomonas and for ocular infections. 3. Tobramycin
Note Tobramycin is the most active aminogJycoside against Pseudomonas.
a. Pharmacologic properties. Tobramycin demonstrates an antibacterial spectrum including aerobic Gram-negative bacilli such as Pseudomonas, Klebsiella, Enterobacter, Proteus, Citrobacter, and Providencia. b. Indications for use. Tobramycin is used with penicillins, cephalosporins, and alone to treat infections in all sites except the CSF. 4. Streptomycin a. Indications for use (1) Although primarily reserved for treatment of TB (Mycobacterium tuberculosis), it is active against many other microbes and is often used in combination with a [3lactam antibiotic in life-threatening diseases such as K. pneumoniae pneumonia. (2) It may be used with penicillin to treat streptococcal endocarditis. (3) Streptomycin is ineffective against anaerobic infections because there is an oxygen-dependent transport step required for its penetration into the cell.
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Antimicrobial Agents
b. Side effects and toxicity. Ototoxicity and nephrotoxicity are more common than with most other aminoglycosides. 5. Neomycin a. Pharmacologic properties. Neomycin is active against E. coli, Enterobacter, Klebsiella, Proteus, and some S. aureus species. b. Indications for use (1) Neomycin is used as a topical application for superficial skin infections. (2) It is also given orally to patients with hepatic coma because it reduces the number of ammonia-producing bacteria in the gastrointestinal tract. (3) Finally, it is given as an oral prophylaxis to prepare the bowel prior to intestinal surgery. c.
Side effects and toxicity. Neomycin is the most toxic aminoglycoside with doserelated ototoxicity and nephrotoxicity; it is not for parenteral administration. There may be diarrhea and malabsorption following oral administration.
6. Amikacin has the widest antibacterial spectrum of the aminoglycosides. a. Pharmacologic properties include resistance to most bacterial enzymes that inactivate other aminoglycosides. It is susceptible to inactivation by acetylation. Amikacin is active against E. coli, P. aeruginosa, Proteus, Klebsiella, Enterobacter, Serratia, Acinetobacter, Citrobacter, Providencia, and M. tuberculosis. b. Indications for use. Amikacin is used mainly for treatment of organisms resistant to other aminoglycosides. It is the drug of choice for burns infected with resistant Pseudomonas. B. Tetracyclines. Tetracyclines (four rings) are broad-spectrum bacteriostatic antibiotics. They bind at the 30S ribosomal subunit and block protein synthesis by interfering with the interaction of aminoacyl-tRNA and the mRNA-ribosome complex.
1. Tetracycline a. Pharmacologic properties (1) Variable gastrointestinal absorption after oral administration occurs by forming insoluble complexes in the gut with calcium or other ions. The presence of food, milk, metallic salts, or antacids results in poor absorption. (2) Intravenous administration is used for serious infections, malabsorption syndromes, and critically ill patients. (3) Tetracycline diffuses readily into most tissues and fluids. 4) The drug's broad antibacterial spectrum includes a wide variety of Gram-positive and Gram-negative bacteria. (5) Resistance to tetracycline develops in direct proportion to usage. Resistance mechanisms include tetracycline-resistant ribosomes, bacterial production of enzymes that degrade the antibiotic, and decreased permeability of the bacterial cell surface to the drug (plasmid mediated). b. Indications for use (1) Tetracycline is useful for the following infections: Rickettsia (Rocky Mountain spotted fever, Q fever), Chlamydia (lymphogranuloma venereum; psittacosis, Chlamydophila pneumoniae), Francisella tularensis, Vibrio cholerae (cholera), Borrelia (Lyme disease), Ureaplasma, and Mycoplasma pneumoniae.
In a Nutshell Tetracyclines can be used to treat the following organisms:
• Rickettsia • Chlamydia • Francisella tularensis • Vibrio cholerae • Mycoplasma pneumoniae (second choice after erythromycin)
• Ureaplasma • Borrelia • Severe acne
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(2) Tetracycline is an alternative treatment for infections with L. monocytogenes and N. gonorrhoeae.
(3) It is also used as a treatment for chronic severe acne (topical or oral administration). c. Side effects and toxicity (1) Intravenous administration can produce thrombophlebitis and hepatotoxicity. (2) Fetal and neonatal tooth discoloration make it contraindicated during pregnancy, nursing, and in children under 8. (3) Gastrointestinal disturbances, including esophageal ulceration, occur in 10% of patients. (4) Dry mouth, hoarseness, stomatitis, glossitis, pharyngitis, enterocolitis, and proctitis can occur. (5) There can be hepatotoxicity with prolonged administration of high doses; pregnant women are more susceptible. (6) Pseudotumor cerebri, elevation of blood urea nitrogen (BUN), and photosensitivity also occur. 2. Demedocycline demonstrates variable gastrointestinal absorption affected by food and milk ingestion. This antibiotic is similar to tetracycline, but photosensitivity limits its use. It has been used to treat inappropriate secretion of antidiuretic hormone (SIADH) because it renders kidney tubules insensitive to ADH. 3. Doxycycline and minocycline a. Pharmacologic properties are like those of the naturally occurring tetracyclines. Resistance mechanisms are similar to other tetracyclines. (1) Doxycycline and minocycline are more fat soluble and penetrate bacteria better
than other tetracyclines when changes in drug penetration and resistance develop. (2) They are completely absorbed from the gastrointestinal tract; their absorption is not as affected by food or milk as with tetracycline. (3) Some species of B. fragilis are more susceptible to doxycycline and minocycline than to tetracycline. (4) Unlike other tetracyclines, doxycycline is primarily excreted in feces and can be used in patients with renal insufficiency. b. Indications for use (1) Doxycycline can be administered to patients with renal insufficiency without
exacerbating azotemia. (2) Similar to tetracycline, minocycline is more active and more toxic than doxycycline. (3) These drugs are commonly used to treat sexually transmitted diseases; they are very effective against both Chlamydia infections and gonorrhea. They are also used in the treatment of Lyme disease. c. Side effects and toxicity involve vestibular disturbances, including dizziness and nausea. C. Spectinomycin. Spectinomycin inhibits protein synthesis by interacting with the 30S ribo-
somal subunit. Clinical use is limited to the treatment of N. gonorrhoeae in patients allergic to penicillin.
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D. Macrolides and lincosamides. The antibacterial action of these agents is through binding to the 50S subunit of the bacterial ribosome and interfering with protein synthesis. 1. Erythromycin a. Pharmacologic properties (1) Because most of the drug is inactivated by acid, it is administered orally with an enteric coating that dissolves in the duodenum. Most of drug is concentrated in the liver and excreted in bile. (2) Erythromycin diffuses readily into all body fluids except the brain or CSF. (3) Erythromycin has an antimicrobial spectrum similar to penicillin G and is active against Gram-positive bacteria including Listeria, Staphylococcus aureus, S. pneumoniae, S. viridans, S. faecalis, Clostridium, Corynebacterium, and Actinomyces. (4) Erythromycin is active against Mycoplasma pneumoniae, Treponema, Chlamydia, Rickettsia, and Legionella pneumoniae. (5) Resistance is now becoming more of a problem as most nosocomial staphylococcal infections are now resistant. Mechanisms include failure of the organism to take up antibiotic and plasmid-encoded decreased binding to the 50S ribosomal subunit. b. Indications for use (1) Erythromycin is used in patients with penicillin allergy; it is an alternative to penicillin for susceptible pathogens. (2) It is the drug of choice for the treatment of Legionnaires disease and M. pneumoniae. (3) It is a prophylaxis for endocarditis and recurrent rheumatic fever. (4) It is an alternative to penicillin in treating syphilis. (5) Long-acting macrolides such as azithramycin and clarithramycin are replacing erythromycin as preferred therapeutics. (6) It is an alternative to tetracycline in treating chlamydial infections. (7) A topical preparation of erythromycin is used in the treatment of acne.
In a Nutshell Erythromycin is the first choice for treatment of Legione//a and M. pneumoniae. It is a backup choice in the treatment of syphilis (penicillin is first choice) and Chlamydia (tetracycline is first choice). It is also used widely as an alternative to penicillin in penicillin-allergic patients.
c. Side effects and toxicity
(1) Gastrointestinal disturbances are common. (2) There may be cholestatic jaundice. (3) Like the aminoglycosides, sensorineural hearing loss may occur with large doses. (4) Drug interactions interfere with the drug-metabolizing cytochrome P-450 enzyme system, producing increased digoxin effect (decreased metabolism, increased absorption) and increased theophylline effect (decreased metabolism). Drug interactions also increase the effects of carbamazepine, warfarin, and cyclosporine. 2. Clindamycin a. Pharmacologic properties (1) It is widely distributed to bones, fluids, and tissues but shows poor CNS pene-
tration. The spectrum of activity is similar to that of erythromycin.
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(2) It is the drug of choice for serious infections caused by the anaerobic organisms, B. fragilis, Fusobacterium, and Peptococcus. (3) It is active against common Gram-positive pathogens, including staphylococci and streptococci, but it is not active against most Gram-negative organisms. (4) Clindamycin is excreted mainly through the liver, bile, and urine. (5) Clindamycin reacts with the 50S ribosomal subunit and interferes with amino acid transfer to the growing peptide chain (mechanism unknown). b. Indications for use. Clindamycin's most important use is in the treatment of severe anaerobic infections caused by Bacteroides and other anaerobes. (1) Primary lung abscesses with susceptible pathogens and aspiration pneumonia (2) Intra-abdominal sepsis and intrapelvic infections (3) Orthopedic infections with susceptible pathogens (4) Acne (topically)
In a Nutshell Clindamycin • Used to treat anaerobic infections (e.g., Bacteroides) • Causes pseudomembranous colitis
c. Side effects and toxicity (1) Clindamycin can produce pseudomembranous enterocolitis, now described as antibiotic-associated colitis (AAC), resulting from suppression of intestinal organisms and proliferation of the anaerobe, Clostridium difficile. C. difficile is treatable with vancomycin.
(2) Like the aminoglycosides, it increases neuromuscular blockade in the presence of neuromuscular blocking agents . E. Chloramphenicol was isolated from cultures of Streptomyces and is the first completely synthetic antibiotic.
1. Pharmacologic properties a. A potent inhibitor of microbial protein synthesis, chloramphenicol binds to the 50S subunit of bacterial ribosome to block the action of peptidyltransferase. b. Chloramphenicol is bacteriostatic for many bacteria and rickettsiae. c. It can be administered parenterally or orally with good CNS penetration. d. It is inactivated by conjugation to glucuronide in the liver (90%), and its metabolites are rapidly excreted in the urine. e. It is clinically active against many strains of Gram-positive and Gram-negative bacteria, Rickettsia, anaerobes, and Mycoplasma. f. Gram-negative bacteria develop resistance via a factor acquired by conjugation, which induces acetyltransferase enzyme to inactivate chloramphenicol by acetylation. Resistant staphylococci contain an inducible form of chloramphenicol acetyltransferase for this purpose. 2. Indications for use a. Chloramphenicol is used as a treatment for acute typhoid fever and other serious Salmonella infections, particularly in developing nations; infections with ampicillinresistant H. influenzae, especially meningitis (some third-generation cephalosporins are also effective); and meningitis caused by N. meningitidis and Streptococcus pneumoniae in patients hypersensitive to penicillin. b. The drug is used as an alternative treatment of rickettsial infections when sulfonamides and tetracyclines cannot be used.
382
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c. In general, use is limited by toxicity; it is used only for serious infections when other agents are not viable alternatives. 3. Side effects and toxicity a. Pancytopenia, which is dose-related and often reversible, may occur.
In a Nutshell Protein Synthesis Inhibitors Bind to 30S ribosomal subunit: • Aminoglycosides
b. Aplastic anemia may occur in 1/30,000 patients. It is not dose-related and may occur months after drug use.
• Spectinomycin
e. Hemolytic anemia may occur in patients with glucose-6-phosphate (GGPD) deficiency.
• Tetracyclines
d. Nausea, vomiting, glossitis, stomatitis, diarrhea, and enterocolitis may occur.
Bind to 50S ribosomal subunit:
e. "Gray baby syndrome" occurs, especially in premature infants of mothers on chloramphenicol due to an infant's immature liver function (lacking glucuronide synthetase); it is potentially fatal. f. Chloramphenicol inhibits cytochrome P-450 and prolongs drug action when normally metabolized by this system.
• Erythromycin • C1indamycin • Chloramphenicol
FOLATE ANTAGONISTS A. Sulfonamides are rarely used alone because a large number of other drugs covering the same bacterial spectrum are available. However, the presence of certain opportunistic infections with AIDS has renewed interest in the sulfonamides for use against organisms that must synthesize folic acid. 1. Pharmacologic properties a. All are structurally similar to PABA and compete with PABA for enzyme sites on dihydropteroate synthetase to inhibit folic acid synthesis. b. Their action is bacteriostatic. e. A large fraction is bound to plasma proteins and distributed throughout body water. It penetrates CSF and crosses the placenta.
d. Sulfonamides are acetylated in liver and eliminated by glomerular filtration. e. Resistance mechanisms include: (1) Increased bacterial synthesis of PABA to overcome competitive inhibition or
through structural changes in the enzyme. (2) Inactivation of the drug by acetylation (3) Acquired ability to use preformed folate 2. Indications for use a. Usage has diminished due to resistance and the availability of penicillins. However, in combination with the antimalarial drug, trimethoprim, sulfonamide inhibits Pneumocystis jiroveci and Isospora belli, opportunistic infections of AIDS patients. b. Sulfonamides are used to treat acute urinary tract infections caused by susceptible strains of E. coli, Klebsiella, Enterobacter, Staphylococcus aureus, Proteus, and Staphylococcus saprophyticus. e. They are the drugs of choice for treatment of Nocardia.
d. Sulfadiazine is used with pyrimethamine for the treatment of toxoplasmosis and chloroquine-resistant falciparum malaria. KAPLA~.
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e. They are alternative drugs for the treatment of chancroid, trachoma, and inclusion conjunctivitis (tetracycline or erythromycin is first choice). f. They are used as an alternative treatment for lymphogranuloma venereum (tetracycline is first choice). 3. Side effects and toxicity a. Hypersensitivity rashes, eosinophilia, and angioedema; kernicterus (displaces bilirubin from albumin) and increased serum bilirubin levels in neonates; Stevens-Johnson syndrome (rare); and hematologic disorders such as agranulocytosis, aplastic anemia, thrombocytopenia, and hemolytic anemia in G6PD deficiency. b. Drug interactions can occur, resulting in potentiation of oral hypoglycemics, warfarin, and phenytoin by displacing them from albumin. 4. Individual agents (Table V-lS-3) Table V-lS-3. Sulfonamides. Agent
Important Features
Clinical Usage
Sulfisoxazole
Short-acting agent; rapidly absorbed and eliminated; low incidence of crystalluria
Sulfadiazine
Short-acting agent; high incidence of crystalluria; must hydrate patient
Sulfamethoxazole
Intermediate-acting agents; similar to sulfisoxazole but slower absorption and excretion; high risk of crystalluria
Sulfasalazine
Very poor gastrointestinal absorption
Main usage for urinary tract infections; also active against lymphogranuloma venereum and chancroid; can be used to treat Nocardia Best sulfonamide for treatment of meningitis; used for treatment of nocardiosis; used in combination with pyrimethamine for treatment of toxoplasmosis Usage similar to sulfisoxazole; marketed in combination with trimethoprim (Bactrim, Septra) for treatment of urinary tract infections, shigellosis, prostatitis; combination also used for treatment of P. jiroveci Used for treatment of ulcerative colitis and regional enteritis Used in ophthalmic infections Used to prevent colonization and infection of wounds and burns Same as silver sulfadiazine
Sulfacetamide Most often used as a topical solution Silver sulfadiazine Topical solution Mafenide Sulfadoxine
Topical solution; inhibits carbonic anhydrase; may cause metabolic acidosis Very long-acting; risk of severe reactions, such as Stevens-Johnson syndrome, limits use
Used in combination with pyrimethamine for treatment and prophylaxis of chloroquineresistant Plasmodium falciparum malaria; has also been used in prophylaxis of AIDS-related pneumonia caused by P. jiroveci
B. Trimethoprim-sulfamethoxazole 1. Pharmacologic properties. As indicated, these drugs are a combination of a sulfa drug
with an antimalarial drug, also known as co-trimoxazole, consisting of five parts sulfamethizole to one part trimethoprim. a. The synergistic combination acts as an inhibitor of two sequential steps in the synthesis of folic acid. b. Sulfamethoxazole inhibits PABA incorporation into folic acid, and trimethoprim inhibits the reduction of dihydrofolic acid to tetrahydrofolic acid.
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2. Indications for use. These drugs are used in the treatment of acute and chronic urinary tract infections, including prostatitis, shigellosis, salmonellosis carriers, respiratory infections caused by H. infiuenzae and Streptococcus pneumoniae, Pneumocystis jiroveci (treatment and prophylaxis) in immunosuppressed hosts, and as alternatives to chloramphenicol and ampicillin in treating typhoid and paratyphoid fevers. It is also used for the treatment of Isospora belli. 3. Side effects and toxicity a. These include hypersensitivity reactions, rarely, Stevens-Johnson syndrome; gastrointestinal disturbances with glossitis and stomatitis; and nephrotoxicity. b. Blood dyscrasias include megaloblastic anemia, leukopenia, and thrombocytopenia. e. Patients with AIDS being treated for P. jiroveci often have reactions that include fever, rash, and, sometimes, pancytopenia.
NUCLEIC ACID INHIBITORS A. Fluoroquinolones: ciprofloxacin and norfloxacin are newer antibiotics with a unique action on enzymes that catalyze the direction and extent of DNA supercoiling. 1. Pharmacologic properties
In a Nutshell Uses of Sulfonamides • Urinary tract infections (+/trimethoprim depending on severity) • Pneumocystis jiroved pneumonia (with trimethoprim) • Isospora belli (with trimethoprim) • Nocardia infections • Toxoplasmosis (with pyrimethamine) Chloroquine-resistant falciparum malaria (with pyrimethamine)
a. Fluoroquinolones are structurally related to nalidixic acid, an older quinolone. b. They possess rapid oral absorption and are mostly excreted in urine. e. Mechanism involves inhibition of bacterial DNA gyrases.
d. They are the only oral agents effective against Pseudomonas. 2. Indications for use. Ciprofloxacin and norfloxacin are used in Gram-negative infections. a. Norfloxacin is effective in UTls, including resistant and recurrent organisms, and in prostatitis resulting from E. coli infections. b. Ciprofloxacin is effective for treating UTls, gonorrhea, diarrheal diseases, and soft tissue infections. It has been shown to be effective against respiratory infections (resulting from Haemophilus or Streptococcus pneumoniae), in bronchitis, and in Pseudomonas infections in cystic fibrosis patients. 3. Side effects and toxicity. Adverse reactions are mostly gastrointestinal with rash and crystalluria (rare). B. Nitrofurantoin 1. Pharmacologic properties a. Nitrofurantoin is effective against many Gram-positive and Gram-negative bacteria, but not against Pseudomonas. b. Its mechanism of action is through the damage of bacterial DNA. e. It is rapidly absorbed and undergoes urinary excretion. It shows no systemic antibacterial activity.
2. Indications for use. Nitrofantoin is limited solely as a urinary antiseptic for acute or chronic UTIs. 3. Side effects and toxicity a. Gastrointestinal upset KAPLAlf I me dlea 185
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Clinical Correlate
b. Pulmonary inftltrates and fibrosis (may be fatal)
Antimicrobials to Avoid
c. Peripheral neuropathy (especially in patients with renal impairment)
in Patients with G6PD Deficiency
d. Hemolytic anemia (should not be used in patients with G6PD deficiency)
• Nitrofurantoin • Primaquine • Sulfa Drugs • Chloramphenicol • Dapsone
Note Mycobacteria were discussed in detail in the Mycobacteria and Actinomycetes chapter of this section.
ANTIMVCOBACTERIAL AGENTS M.tuberculosis invades many organs and requires prolonged therapy. Because single-drug therapy for a long period (weeks) allows the growth of resistant mutants, TB must be treated simultaneously with two or more drugs. Standard therapy for pulmonary and extrapulmonary TB may be either isoniazid and rifampin for 9 months or isoniazid and ethambutol for 8 months. In cases of infection with drug-resistant TB, overwhelming disseminated TB (miliary), or TB meningitis, three-drug regimens are often used. Atypical Mycobacterium infections (e.g., Mycobacterium avium-intracellulare) are usually treated with multidrug regimens. A summary of these agents is provided in Table V-lS-4. A. Treatment of tuberculosis. The number of cases in the U.S. has increased dramatically in part due to immigration, AIDS, and the number of homeless individuals.
Table V-lS-4. Drugs used to treat tuberculosis and leprosy. Drug
Adverse Effects
Isoniazid
Elevation of hepatic enzymes, peripheral neuropathy, hepatitis, CNS effects, and increased phenytoin concentration Orange-red discoloration of secretions, urine, tears, and contact lenses; hepatitis, drug fever, flu-like symptoms, and thrombocytopenia; interferes with methadone, warfarin, medroxyprogesterone, theophylline, dapsone, and ketoconazole Gastrointestinal upset, elevation of liver enzymes, rash, arthralgia, and hyperuricemia Optic neuritis (everything appears green), decreased visual acuity, and skin rash Ototoxicity, nephrotoxicity, hypokalemia, and hypomagnesemia Abdominal cramps, gastrointestinal upset, insomnia, headache, photosensitivity, and hypersensitivity reactions; drug interactions with warfarin and theophylline Auditory and renal toxicity, vestibular toxicity (rare), hypokalemia, and hypomagnesemia Gastrointestinal upset, bloating, liver enzyme elevation, metallic taste, and hypothyroidism (especially if on para-aminosalicylic acid [PAS]) Psychosis, depression, seizures, rash, headache, and increased phenytoin concentrations Gastrointestinal upset, elevated liver enzymes, sodium loading, decreased digoxin, and increased phenytoin levels Orange-brown skin discoloration, gastrointestinal complaints, and rare visual disturbances Anemia, rash, and methemoglobinemia
Rifampin
Pyrazinamide Ethambutol Streptomycin Ciprofloxacin
Amikacin Ethionamide Cycloserine Para-aminosalicylic acid Clofazimine Dapsone
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iileilical
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1. Isoniazid (INH) is a primary agent in all treatment measures. a. Pharmacologic properties (1) Inhibits biosynthesis of mycolic acids (mycobacterial cell wall components).
(2) INH is absorbed well after oral administration and demonstrates 20% CSF penetration. (3) The drug is metabolized in the liver by acetylation, and the speed of acetylation-and consequently INH's half-life-is genetically determined (fast versus slowacetylators). (4) There is no activity against atypical mycobacteria other than M. kansasii. b. Side effects and toxicity (1) Peripheral neuropathy is a most common side effect, but it can be prevented by the administration of pyridoxine (vitamin B6 ). (2) Hepatotoxicity usually occurs in a mild transient form during the initial 1-2
months of therapy. Risk increases with age and pre-existing liver disease. (3) Rash, fever, and eosinophilia may occur. (4) Induction of antinuclear antibody (ANA) and lupus-like syndrome may occur. (5) Bone-marrow depression and arthritis are rare effects. 2. Rifampin
Clinical Correlate Drug-induced, lupus-like syndrome with a positive ANA is seen with INH, hydralazine (antihypertensive), and procainamide (antiarrhythmic). Symptoms abate with drug withdrawal. This is a USMLE favorite side effect.
a. Pharmacologic properties (l) Rifampin is a bactericidal agent with activity against intra- and extracellular TB organisms. It is also active against certain Gram-positive bacteria (e.g., S. au reus) and certain Gram-negative bacteria (e.g., N. meningitidis). It is used prophylactically for people exposed to meningitis caused by H. inJluenzae or meningococci. It eliminates nasal carriage of methicillin-resistant S. aureus and N. meningitidis.
(2) The mechanism of action involves the inhibition of DNA-dependent RNA polymerase, thereby blocking RNA synthesis. (3) There is oral absorption with metabolism in the liver and then enterohepatic circulation. (4) Resistance emerges if rifampin is used alone against TB. (5) Rifampin is also active against atypical mycobacteria and M. leprae. b. Side effects and toxicity rarely occur. (l) Rifampin may color urine, saliva, sputum, and tears orange-red, but this discoloration is harmless.
(2) Rash, fever, nausea, vomiting, jaundice, and hepatotoxicity may occur. (3) Blood dyscrasias include leukopenia, thrombocytopenia, and anemia. (4) There is stimulatory interaction with the cytochrome P-450 system. This decreases the oral anticoagulant effect, may render oral contraceptives ineffective, decreases the glucocorticoid effect, decreases digitoxin levels, decreases quinidine levels, and decreases oral hypoglycemic and barbiturate effects. This also decreases methadone levels, which may precipitate withdrawal.
In a Nutshell Rifampin is secreted into many body fluids and may discolor them (orange-red). It also stimulates the cytochrome P450 system, resulting in increased elimination of anticoagulants, digitoxin, oral contraceptives, methadone, barbiturates, etc.
meClical
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B. Treatment of leprosy. Mycobacterium leprae is responsible for the development of leprosy. 1. Dapsone is an organic sulfone but is unlike the sulfonamides.
a. Pharmacologic properties (1) Dapsone inhibits folate synthesis with a mechanism like that of the sulfonamides; it is bacteriostatic for M. leprae. (2) Gastrointestinal absorption from the upper gastrointestinal tract is 90% complete. It is widely distributed and active in the skin. It is acetylated in the liver like isoniazid, and it is excreted as the glucuronide and sulfate conjugates. b. Indications for use. Dapsone is the preferred drug in the treatment of leprosy, usually in combination with either rifampin or clofazimine. Because leprosy is a chronic disease, therapy is usually continued for several years. c. Side effects and toxicity (1) Hemolysis occurs and can be severe with G6PD deficiency. (2) Methemoglobinemia can occur. (3) Nausea, vomiting, anorexia, and headache can occur. (4) Dapsone can cause fatal mononucleosis-like syndrome. 2. Rifampin (see above) 3. Clofazimine a. Clofazimine is a phenazine dye that inhibits DNA template function. b. Clofazimine is bactericidal against M. leprae; it is also active against M. avium intra-
cellulare. c. It is absorbed gastrointestinally and accumulates in tissues.
ANTIVIRAL AGENTS Viruses are obligate intracellular parasites that depend on the metabolism of the host cell. Therefore, agents that inhibit or kill viruses are also likely to injure the host cells that harbor them. Developmental approaches in the design of antiviral agents have focused on the selective inhibition of enzyme systems unique to virus-infected cells. However, at this time, very few agents have been found to be both effective and safe. A. Treatment of herpesviruses 1. Acyclovir requires phosphorylation to be effective. It is phosphorylated efficiently by a virus-specific thymidine kinase and becomes trapped within virus-infected cells. Guanine analog (acycloguanosine) is converted to the triphosphate, the active form of the drug, which inhibits virus-specific DNA polymerase.
a. Pharmacologic properties (1) Acyclovir can be used orally or intravenously.
(2) It is only slightly protein bound. It is well distributed, including in the CNS. (3) Acyclovir inhibits multiplication of various herpesviruses, including varicellazoster, herpes simplex types I and II, and Epstein-Barr virus. It is not effective against cytomegalovirus (CMV).
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meClical - - - - - - - - - - -
----
Antimicrobial Agents
b. Indications for use (1) It is used as a topical treatment for herpes simplex infections with little skin penetration. (2) Intravenous treatment is required for mucosal, cutaneous, and disseminated herpes simplex infections and treatment of herpes zoster or chickenpox in immunocompromised hosts. (3) Oral administration can lessen the severity of genital herpes if administered early in the attack, but intravenous therapy is necessary for severe infections. c. Side effects and toxicity
(1) Nephrotoxicity may occur. (2) Encephalopathy, bone marrow depression, and abnormal hepatic function may occur. 2. Vidarabine, also called ara-A, is an adenosine arabanoside that is phosporylated within cells and inhibits viral DNA polymerases. a. Pharmacologic properties (1) Vidarabine is available for intravenous and topical administration.
(2) It inhibits viral DNA polymerase. (3) It should be administered intravenously with a lot of fluid. b. Indications for use (1) It is used in the treatment of ocular herpes simplex keratoconjunctivitis. (2) It is used in the parenteral treatment of herpes simplex encephalitis (if acyclovir resistant). (3) It is used in the treatment of herpes zoster infections in patients with suppressed immunologic response. c. Side effects and toxicity
(1) Ophthalmic preparation is less irritating than idoxuridine, but it may cause lacrimation, burning, conjunctival and corneal edema, and photophobia. (2) Systemic infusion may result in nausea, vomiting, diarrhea, hallucinations, ataxia, psychoses, and tremor. 3. Ganciclovir a. Pharmacologic properties. Ganciclovir is a synthetic analog of guanosine whose mode of action is similar to that of acyclovir; i.e., it inhibits viral DNA polymerase after being converted to the triphosphate form in the infected cell.
In a Nutshell
b. Indications for use (1) Available only in intravenous form, ganiclovir is active against CMV and other herpesviruses.
Ganciclovir ~ CMV
(2) It is primarily used for CMV infections in immunocompromised patients. (3) It is more toxic than acyclovir, so use is restricted to CMV.
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c. Side effects and toxicity
(1) Bone marrow suppression with granulocytopenia occurs in up to 40% of patients and thrombocytopenia occurs in 20%. (2) Hepatotoxicity may occur. 4. Foscarnet a. Pharmacologic properties. Foscarnet inhibits viral DNA polymerase. Unlike acyclovir and ganciclovir, foscarnet does not need to be activated by a viral kinase. b. Indications for use. It is effective in CMV retinitis and herpesvirus infections that are resistant to ganciclovir and acyclovir, respectively. c. Side effects and toxicity are more common with foscarnet than ganciclovir.
(1) Nephrotoxicity may occur. (2) Bone marrow suppression may occur but is less than that seen with ganciclovir. 5. Idoxuridine (IdUR) is used only topically. a. Pharmacologic properties (1) As a thymidine analog, it can be used to treat herpes simplex keratitis and dendritic ulcers. (2) It is phosphorylated in cells; triphosphate derivative is incorporated into both viral and mammalian DNA. (3) It renders DNA more susceptible to breakage with altered viral proteins secondary to faulty transcription. (4) It is restricted to topical application because of liver and bone marrow toxicity.
In a Nutshell Neuraminidase inhibitors (zanamivir, oseltamavir) are now available for the treatment of influenza and are active against influenza A and B.
b. Indications for use. Idoxuridine is used in the treatment of herpes simplex infections of cornea, conjunctiva, and eyelids, including herpes keratitis. c. Side effects and toxicity include conjunctival irritation, photophobia, edema of the eyelids and cornea, and corneal epithelium punctuate defects. B. Treatment of respiratory viral infections 1. Amantadine and rimantadine are used for the treatment and prophylaxis of influenza A. They are ineffective against influenza B. a. Pharmacologic properties (1) Amantadine is completely absorbed from the gastrointestinal tract after oral
administration, and 90% is excreted unmetabolized in urine. Rimatadine is metabolized extensively in the liver and only 25% is excreted unchanged in urine. (2) The drugs inhibit replication of orthomyxoviruses (specifically influenza A) by interfering with viral protein uncoating. They are basic amines that elevate the pH of the cytoplasmic vesicle in which the virus resides and thus inhibit fusion of viral peptomeres with the vesicle membrane, the prelude to uncoating. (3) Rimantadine has a longer plasma half-life than amantadine. b. Indications for use (1) Prophylactic administration of the drug to high-risk patients in the presence of an influenza A virus epidemic
390
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Antimicrobial Agents
(2) Treatment shortens the duration of influenza symptoms in patients with influenza A if administered within the first 12-24 hours. (3) Amantadine is used in treating symptoms of Parkinson disease because it causes the release of dopamine and other catecholamines from neuronal storage sites and delays reuptake of these neurotransmitters into synaptic vesicles. c. Side effects and toxicity (1) Common CNS reactions include irritability, tremor, slurred speech, ataxia,
depression, insomnia, lethargy, and dizziness. (2) Drug interactions between amantadine and anticholinergic drugs such as benztropine mesylate (Cogentin) may cause confusion and hallucinations in patients. (3) Rimantadine has fewer side effects than amantadine.
In a Nutshell Current HIV therapy consists of two nucleoside reversetranscriptase inhibitors (AZT) and one protease inhibitor. This highly active retroviral therapy (HAART) results in a J- in viral RNA, an i in C04+ count, and a J- in opportunistic infections.
In a Nutshell
2. Ribavirin is a synthetic purine nucleoside analog active against respiratory syncytial virus. a. Pharmacologic properties. Ribavirin undergoes phosphorylation, interfering with nucleotide synthesis, viral mRNA production, and viral protein production (mechanism unknown). b. Indications for use. Aerosols are used to treat respiratory syncytial virus in infants and children.
Antivirals • Acyclovir
-7
Inhibits viral DNA polymerase. Used to treat HSV, VZV, and EBV.
• Vidarabine (ara·A)
-7
Inhibits viral DNA polymerase. Used primarily in invasive HSV infections resistant to acyclovir (e.g., encephalitis).
• Ganciclovir
-7
Inhibits viral DNA polymerase. Used to treat (MV infections.
Foscarnet
-7
Inhibits viral DNA polymerase. Used to treat ganciclovirresistant (MV retinitis in imrnunocornprornised patients.
Idoxuridine (ldUR)
-7
Inhibits enzymes involved in DNA synthesis. Used topically for treatment of herpetic keratitis.
Amantadine, Rimantadine
-7
Interfere with influenza A virus penetration and uncoating. Used in treatment and prophylaxis of influenza A.
c. Side effects and toxicity include anemia and conjunctivitis. C. Treatment of human immunodeficiency virus (HIV). Because antiviral drugs inhibit virus
replication after infection has occurred, they are most effective when given at an early stage of infection. Current practice is to use these drugs in asymptomatic or mildly symptomatic persons in whom the CD4+ cell number drops below SOO/Ill or to use them as prophylaxis in individuals accidentally exposed to HIV or born of HIV + mothers. 1. Zidovudine (AZT) a. Pharmacologic properties (1) AZT is a thymidine analog that terminates DNA transcription by viral reverse transcriptase, preventing viral replication. (2) AZT penetrates the CNS and undergoes hepatic and renal excretion. b. Indications for use. AZT is used in the treatment of AIDS and various stages of HIV infection (studies suggest prolonged survival in HIV-positive patients). c. Side effects and toxicity. Side effects are numerous, but toxicity associated with bone marrow suppression is the most serious problem. ( 1) Anemia, which may be severe and require transfusions, can occur. (2) Megaloblastic erythrocyte changes and granulocytopenia may occur within weeks of therapy, but using lower doses of AZT decreases side effects. (3) Liver function abnormalities are exacerbated by acetaminophen, and bone marrow toxicity is exacerbated by ganciclovir. (4) Less severe adverse effects of AZT include headache, insomnia, diarrhea, rashes, and fever.
(Continued)
2. Dideoxycytidine (ddC) and dideoxyinosine (ddI) also inhibit reverse transcriptase. They have activity against AZT-resistant HIV. Both drugs cause peripheral neuropathies.
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3. 3-Thiacytidine (3TC) is an inhibitor of reverse transcriptase. It is most often used in conjunction with AZT. Pancreatitis is the major adverse side effect, particularly in infants. Ribavirin
~
Interferes with nucleic acid synthesis (DNA and RNA). Used to treat infections caused by respiratory syncytial virus. Also effective against Lassa fever.
Zidovudine (All)
~
Inhibits reverse transcriptase. Used in treatment of HIV.
ddC, ddl, and
~
Same mechanism as AIr. Used in AZT-resistant cases.
3TC Protease inhibitors (ritonavir, saquinovir, indinavir)
Interferons (ot, ~, 'I)
~
~
Newer agents used in HIV treatment. Block viral protease required for normal viral protein synthesis. Interfere with viral protein synthesis. Used in treatment of hairy cell leukemia, Kaposi sarcoma, and condylomata acuminatum.
4. Protease inhibitors such as ritonavir, saquinovir, and indinavir are the newest drugs used in the treatment of HIV. They interfere with the viral protease that cleaves the polypeptide chain translated from the polycistronic viral mRNA, thus, the gag-pol polyprotein is not cleaved and functional reverse transcriptase is not produced. These drugs are usually used in conjunction with reverse transcriptase inhibitors. Adverse reactions most commonly seen are diarrhea, abdominal discomfort, and nausea. D. Human interferons are naturally occurring glycoproteins produced in response to viral infection. They inhibit viral replication and promote antiviral responses. Three types of interferon are produced: interferon a. (produced by monocytes), interferon ~ (produced by fibroblasts), and interferon y (produced by T lymphocytes). 1. Pharmacologic properties
a. Human interferons (IFNs) are small glycoproteins produced by nucleated cells of the body in response to viruses (especially double-stranded nucleotides). b. Once released, IFNs inhibit viral multiplication in other cells by inducing cellular enzymes that block protein synthesis and therefore viral protein synthesis. (I) The induction of a protein kinase phosphorylates elongtion factor 2, thereby preventing the initiation of protein synthesis. (2) A kinase induced in the cell synthesizes an adenine trinucleotide that activates an endonuclease that cleaves mRNA. c. The interferons have been produced by recombinant DNA techniques and can be generated in large amounts. d. IFNs are administered intramuscularly, subcutaneously, or intravenously. They are not effective orally. 2. Indications for use. IFNs are used in the treatment of hairy cell leukemia, Kaposi sarcoma in patients with AIDS; condylomata acuminatum (genital warts); and herpes keratoconjuctivitis, in combination with other antiviral agents. 3. Side effects and toxicity a. A flu-like syndrome is experienced during the first few days of treatment, including fever, chills, headache, nausea, and vomiting. b. There may also be bone marrow suppression, neurotoxicity, elevated liver function tests, and nephrotoxicity.
ANTIFUNGAL AGENTS A. Polyene antibiotics. Polyene antibiotics are effective against both filamentous and yeast-like fungi, including Histoplasma, Blastomyces, Coccidioides, Cryptococcus, Candida, Aspergillus, Mucor, and Rhizopus species. The polyenes have no activity against dermatophytes or bacteria. Polyenes interact with ergosterols in the cytoplasmic membrane of fungi, leading to rapid leakage of small molecules and fungal death. 1. Amphotericin B
a. Pharmacologic properties (1) Systemic infections are treated by slow intravenous infusion; amphotericin B
cannot be administered orally.
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meClical
Antimicrobial Agents
(2) The drug binds to serum
~-lipoproteins
and tissue membranes.
(3) There is poor eNS penetration; intrathecal administration may be required for fungal meningitis. (4) The drug re-enters the circulation slowly from tissue depots with a small portion excreted in bile (major route) or urine over weeks. b. Indications for use (1) Amphotericin B is a broad-spectrum antifungal agent used for treatment of sys-
temic fungal infections. It is active against Histoplasma, Cryptococcus, Candida, Blastomyces, and Aspergillus. (2) The drug is also used in the treatment of mucocutaneous leishmaniasis and amebic meningoencephalitis (freshwater amebae). c. Side effects and toxicity (1) Low therapeutic index (small test dose usually given)
(2) Fever, chills, nausea, vomiting, headache, diffuse pain, and hypotension (3) Nephrotoxicity, including decreased glomerular fIltration rate, azotemia, renal tubular acidosis, and hypokalemia (4) Normochromic, normocytic anemia from bone-marrow suppression 2. Nystatin (mycostatin) a. Pharmacologic properties (1) Its structure and mechanism of action are similar to amphotericin B, but it is more toxic than amphotericin B. (2) It is used primarily in topical preparations but can be taken orally for oral and esophageal candidiasis. It is not for parenteral administration. (3) It is not absorbed from skin, mucous membranes, or the gastrointestinal tract. b. Indications for use include candidal infections of skin, mucous membranes, and vagina; and prophylaxis to prevent intestinal fungal overgrowth in patients on chemotherapy. c. Side effects and toxicity include nausea and vomiting (rare). B. Imidazoles. These agents inhibit 14-alpha-demethylase and block the synthesis of fungal
cell membrane ergosterol, leading to increased membrane permeability and loss of essential nutrients. 1. Miconazole and clotrimawle
Note
a. Pharmacologic properties. Miconazole and clotrimazole are topically active antifungals.
Aspergillus is very resistant to
b. Indications for use. The drugs are used in the treatment of ringworm and vulvovaginal candidiasis. Intravenous miconazole is rarely used because of toxicity.
these imidazole antifungals. Aspergillosis is treated with amphotericin B.
c. Side effects and toxicity
(1) Intravenous miconazole most commonly causes nausea, phlebitis, anemia, thrombocytopenia, and pruritus. (2) Hyponatremia occurs in 50% of patients. (3) Arthralgias, anaphylaxis, acute psychosis, and hyperlipidemia may also occur.
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Note
2. Ketoconazole, itraconazole, and fluconazole
Ketoconazole absorption is favored by low pH; absorption is therefore decreased by antacids and H2 blockers. Fluconazole is unaffected by pH.
a. Pharmacologic properties (1) Administered orally
(2) Ketoconazole is metabolized in the liver. b. Indications for use (1) Ketoconazole is used to treat coccidioidomycosis, histoplasmosis, blastomycosis, paracoccidioidomycosis, and mucocutaneous candidiasis. (2) Itraconazole is used in blastomycosis, histoplasmosis, and aspergillosis.
(3) Fluconazole is used in treatment of cryptococcal meningitis in AIDS patients and in severe cases of candidiasis.
c. Side effects and toxicity are rare. C. Miscellaneous antifungal agents
1. Flucytosine a. Pharmacologic properties (1) The mechanism of action involves conversion to 5-fluorouracil in fungal cells, then metabolism to 5-fluorodeoxyuridylic acid, an inhibitor of thymidylate synthetase (pyrimidine antimetabolite). (2) It is well absorbed orally. (3) CSF levels are 80% of serum levels. (4) Eighty percent of the drug is excreted in urine by glomerular filtration.
b. Indications for use (1) Flucytosine is administered with amphotericin B in the treatment of cryptococcal meningitis and systemic candidiasis.
(2) It is rarely used alone because of rapid development of resistance.
c. Side effects and toxicity (1) Hematologic effects include anemia, leukopenia, and thrombocytopenia. (2) Nausea, vomiting, diarrhea, and enterocolitis can occur.
2. Griseofulvin a. Pharmacologic properties (1) Griseofulvin inhibits fungal mitosis by interacting with microtubules to cause mitotic spindle disruption. (2) It must be taken orally to be effective. (3) It binds to keratin and is deposited in skin, hair, and nails (keratin-precursor cells), where it is taken up by the fungus. b. Indications for use. Griseofulvin is used in the treatment of dermatophytic infections with Epidermophyton, Microsporum, and Trichophyton species.
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c.
Side effects and toxicity (1) CNS effects include headache, lethargy, confusion, fatigue, syncope or vertigo
(less often), gastrointestinal disturbances, and hepatotoxicity. (2) Hematologic effects include leukopenia and granulocytopenia with long-term and high-dose therapy. (3) Drug interactions involve reduction in warfarin activity (increased metabolism) and decreased barbiturate absorption from the alimentary tract. 3. Trimethoprim-sulfamethoxazole and pentamidine are used in the treatment of Pneumocystis jiroveci pneumonia. a. Trimethoprim-sulfamethoxazole is a first-line drug used in both prophylaxis and therapy in immunocompromised patients. b. Pentamidine is administered by aerosol for treatment of P. jiroveci pneumonia. It can be used intramuscularly for the treatment of leishmaniasis and trypanosomiasis.
ANTI PROTOZOAL AGENTS Therapy for protozoal infections include chemotherapeutic techniques, control of the insect vector, eradication of the infection reservoir, and improvement of sanitation and living conditions. Antihelminthic agents expel worms from the gastrointestinal tract and eliminate helminths that have migrated into body tissues. A. Antimalarial drugs are usually classified according to the stage of the life cycle that they affect. They include treatment of the acute attack, drugs to effect radical cure, drugs for prophylaxis, and those used to prevent transmission. 1. Chloroquine is very effective against erythrocytic forms but not against the liver stages. a. Pharmacologic properties (1) Chloroquine inhibits the ability of parasites to digest hemoglobin and thus interferes with their viability. (2) Chloroquine also intercalates into the DNA of the parasite and causes cessation of DNA synthesis. b. Indications for use (1) Chloroquine is used in prophylaxis and treatment of susceptible Plasmodium organisms. (2) It is the drug of choice for treatment of malaria, although the increasing incidence of resistant P. falciparum strains limits its use. (3) It is used as a treatment for extraintestinal amebiasis. (4) It is occasionally used in rheumatoid arthritis and SLE because of its marked anti-inflammatory action. c.
Side effects and toxicity (1) Mild pruritus, nausea, headache, and vomiting may occur. (2) Hematologic abnormalities may occur. (3) There may be quinidine-like effects and fatal arrhythmias with high doses. (4) There may be retinopathy with long-term use. KAPLA~. I meulca 395
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2. Primaquine a. Pharmacologic properties (1) Primaquine is an 8-aminoquinoline compound that causes hemolysis in patients
with "primaquine sensitivity:' a genetic deficiency of erythrocyte G6PD. It may cause methemoglobinemia with cyanosis. (2) It binds to DNA and interferes with cell division. (3) Primaquine is a liver schizonticide and is also effective against the gametocytic form. b. Indications for use. Primaquine is used for prophylaxis. It is also the only drug known to provide a radical cure of malaria caused by P. vivax or P. ovale; it kills the exoerythrocytic form of the parasite. 3. Quinine a. Pharmacologic properties (1) Quinine is an alkaloid with local anesthetic and irritant actions. Its mechanism of action is unknown, but it binds to malarial pigment, hemozoin, and may intercalate in the DNA. (2) It is effective against the erythrocytic stage (blood schizonticide) of all species of Plasmodium. (3) It undergoes hepatic metabolism with renal excretion of metabolites. (4) A weak analgesic, antipyretic agent, it has a depressant action on the heart and an oxytoxic effect on the gravid uterus. (5) It has a curare-like effect on skeletal muscle. b. Indications for use (1) It is an alternative to chloroquine when chloroquine resistance occurs. (2) It is used alone or with tetracycline, pyrimethamine, and a sulfonamide to treat chloroquine-resistant strains of P. falciparum. (3) It is used for the treatment of nocturnal leg cramps and myotonia congenita. (4) It is used for the treatment of babesiosis. c. Side effects and toxicity (1) Cinchonism includes tinnitus, headache, blurred vision, nausea, and diarrhea.
Urticaria and pruritus often occur with use. (2) "Blackwater fever" results from acute hemolysis, hypoprothrombinemia, thrombocytopenic purpura, and agranulocytosis with kidney failure. (3) Because of a curare-like effect, it may worsen symptoms of myasthenia gravis. (4) Excessive levels of quinine may cause hypotension, cardiac arrhythmias, deliriurn, and coma. 4. Mefloquine a. Pharmacologic properties (1) A chloroquine derivative, it is a blood schizonticide active against P. falciparum
and P. vivax. (2) Well absorbed orally, its long half-life is probably due to enterohepatic recycling.
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(3) It is thought to interfere with transport of nutrients from the host. (4) It is excreted through feces. b. Indications for use (1) It is the only antimalarial agent that is effective alone against multiresistant strains. (2) It is used prophylactically for travelers to areas where chloroquine-resistant strains are endemic (e.g., Southeast Asia, South America, and sub-Saharan Africa). c. Side effects and toxicity
(1) Gastrointestinal side effects are most common. (2) Use is contraindicated in pregnancy and for patients on channel blockers, and other antiarrhythmics.
~-blockers,
calcium-
5. Pyrimethamine a. Pharmacologic properties (1) Pyrimethamine is similar in structure to trimethoprim. Both are potent dihydrofolate reductase inhibitors of folic acid synthesis. (2) When used with a sulfonamide, it inhibits two consecutive steps in the formation of folic acid from PABA in parasites. b. Indications for use. Pyrimethamine is given in combination with quinine in the treatment of acute malarial attacks with P. falciparum and in the treatment of toxoplasmosis (in combination with a sulfonamide). c. Side effects and toxicity. There may be significant hematologic abnormalities at increased dosages. 6. Tetracycline and doxycycline may be used in the prophylaxis and treatment of malaria. B. Agents used in the treatment of amebiasis. Amebiasis refers to an infection caused by
Entamoeba histolytica. Dysentery with diarrhea and abdominal pain are common features, although infected individuals may remain asymptomatic while continuing to spread the disease. The cecum and colon are sites of primary involvement, but liver abscesses may develop. 1. Metronidazole
a. Pharmacologic properties (1) A reduced nitro group acts as an electron acceptor to deprive cells of required
reducing equivalents; it may also cause DNA strand breakage with subsequent functional impairment. (2) There is good absorption after oral administration; it is given intravenously or orally. (3) It has both gastrointestinal luminal and systemic activity; it is used for both intestinal amebiasis and amebic liver abscesses (with chloroquinine). (4) It is usually given with a drug acting luminally, such as diloxanide furoate. (5) It undergoes hepatic metabolism. (6) There is good CSF penetration. b. Indications for use (1) It is used in the treatment of all amebic infections except asymptomatic intestinal amebiasis.
Note Metronidazole, in addition to its antiparasitic activity, is also useful for treating anaerobic bacterial infections.
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(2) It is the agent of choice in the treatment of trichomoniasis and giardiasis. (3) There is general anaerobic coverage, such as in postsurgical abdominal and pelvic infections with Bacteroides fragilis and in flare-ups of intestinal diverticulitis. (4) It is a good agent for treating most brain abscesses. c. Side effects and toxicity ( 1) Gastrointestinal effects include nausea, vomiting, anorexia, and epigastric distress. (2) CNS effects are rare but include seizures, neuropathy, and ataxia. (3) A disulfiram-like reaction may occur; thus patients should not consume alcohol. (4) Because of metabolic interactions, metronidazole effects are diminished when given with phenobarbital, but it can decrease warfarin metabolism. (5) It should be avoided in pregnant women, especially during the first trimester. 2. Diloxanide furoate a. Pharmacologic properties (1) Diloxanide is an insoluble ester whose mechanism of action is unknown. (2) It exerts a luminal amebicidal action directly in the bowel lumen, but it has no systemic effects. (3) It is absorbed after oral administration. b. Indications for use (1) Diloxanide is the drug of choice for the treatment of asymptomatic cyst carriers. (2) It is not effective against hepatic abscesses. c. Side effects and toxicity. Diloxanide is generally well-tolerated; flatulence is the most common side effect. 3. Chloroquine a. Pharmacologic properties (1) Liver concentration is 100 times that of plasma concentration.
(2) It achieves only low concentration in the intestinal wall, so it is not useful in treating intestinal amebiasis. b. Indications for use. Chloroquine is used in the treatment of extraintestinal amebiasis, especially hepatic amebiasis, and malaria. C. Agents used in the treatment of giardiasis. Giardiasis is a gastrointestinal tract infection caused by the flagellate Giardia lamblia. Most people with giardiasis are asymptomatic and excrete cyst forms in stool; those with acute disease usually pass trophozoites with frank diarrhea.
1. Metronidazole a. Pharmacologic properties. Metronidazole is favored in the treatment of urogenital trichomoniasis and intestinal and extraintestinal amebiasis infections. (1) It is readily absorbed and penetrates all tissues. (2) The mechanism of action involves entry into cells of the organism, where it is chemically reduced, and these products cause death by interacting with DNA and interfering with cell division.
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Antimicrobial Agents
b. Side effects and toxicity include nausea, headache, dry mouth, and a disulfiram-like effect if alcohol is consumed. 2. Quinacrine a. Pharmacologic properties (1) Quinacrine is an acridine derivative.
(2) It may accumulate in tissues with chronic administration. (3) Its mechanism of action is unknown.
b. Indication for use. It is used in the treatment of giardiasis. c. Side effects and toxicity include nausea and vomiting; headache and dizziness; rash,
urticaria, or yellow discoloration of the skin; optic nerve damage. It is contraindicated in pregnancy. D. Agents used in the treatment of leishmaniasis. Leishmaniasis results from invasion of the reticuloendothelial system by Leishmania transmitted to humans by sandtlies. Few drugs are uniformly effective in leishmaniasis. Drugs employed include sodium stibogluconate and pentamidine. E. Agents used in the treatment of trypanosomiasis. The two types of trypanosomiasis are African sleeping sickness, caused by the bite of a tsetse fly infected with T. gambiense; and
South American Chagas disease, caused by the bite of reduviid bugs infected by Trypanosoma cruzi. 1. Melarsoprol
a. Pharmacologic properties (1) Melarsoprol is a trivalent arsenic compound that reacts with sulfhydryl groups to
inactivate enzymes. (2) It is administered by slow intravenous infusion. (3) It enters the CNS.
b. Indications for use include African trypanosomiasis meningoencephalitis. c. Side effects and toxicity include: gastrointestinal and abdominal pain and vomiting,
encephalopathy (may be fatal), hypotension, albuminuria, rash and angioedema, arthralgia, peripheral neuropathy, and myocardial damage. 2. Nifurtimox
a. Pharmacologic properties (1) It is a nitrofuran derivative.
(2) It is orally administered. (3) It is rapidly metabolized and excreted by the kidney. b. Indications for use. It reduces parasitemia in the acute stage of Chagas disease (T. cruzi infections). c. Side effects and toxicity include generally mild anorexia, nausea, vomiting, abdominal
pain, myalgia, and skin rashes. 3. Pentamidine is an ethionate and is effective against tissue forms of Leishmania.
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4. Suramin a. Indications for use (1) It is used in prophylaxis against African trypanosomiasis. (2) It is used in the treatment of early African trypanosomiasis (without eNS involvement). b. Side effects and toxicity include nausea, vomiting, and colic; photophobia and paresthesias; rash and urticaria; albuminuria and hematuria. 5. Other drugs used in treatment include melarsoprol and nifurtimox. F. Agents used in the treatment of trichomoniasis. Trichomoniasis most commonly refers to vaginal infections caused by Trichomonas vaginalis. Trichomonads may persist in extravaginal foci, particularly the urethra, and diagnosis is easily obtained by microscopic examination of a hanging drop preparation containing fresh exudate from the vagina, semen, or prostatic fluid. Metronidazole is the drug of choice in trichomoniasis.
G. Agents used in the treatment of toxoplasmosis. Toxoplasmosis is a common infectious disease caused by the protozoan Toxoplasma gondii. This organism is usually transmitted to humans by ingestion of raw or inadequately cooked meat. Treatment of toxoplasmosis includes pyrimethamine and a sulfonamide.
ANTIHELMINTHIC AGENTS A summary of agents is presented in Table V-IS-S. A. Nematodes (roundworms) 1. Mebendazole
a. Pharmacologic properties (1) A broad-spectrum antihelminthic agent, it is effective only against intestinal
nematodes. (2) It interferes with the microtubule synthesis of the parasite. (3) It has poor gastrointestinal absorption. b. Indications for use. It is used in treatment of Trichuris trichiura and Enterobius vermicularis. It is also effective against Ascaris lumbricoides, Ancylostoma duodenale, and
Necator american us. c. Side effects and toxicity (1) Transient abdominal pain and diarrhea occur. (2) Because it is poorly absorbed, there is no systemic toxicity. (3) Teratogenic effects are possible. 2. Thiabendazole a. Pharmacologic properties (1) Thiabendazole is readily absorbed after oral administration.
(2) It has broad-spectrum activity against nematodes. (3) It inhibits the helminth-specific enzyme, fumarate reductase, although this may not be the primary mode of action.
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(4) It may interfere with microtubule synthesis.
Antimicrobial Agents
b. Indications for use (1) Thiabendazole is the drug of choice in treating Strongyloides stercoralis and
cutaneous larva migrans. (2) It is the primary therapy for trichinosis. e. Side effects and toxicity. The most common side effects are dizziness, anorexia, nau-
sea, and vomiting; occasionally, fever, erythema multiforme, and the Stevens-Johnson syndrome are seen. 3. Ivermectin a. Pharmacologic properties (1) Causes parasitic immobilization by increasing GABA-mediated transmission (2) Does not cross the blood-brain barrier b. Indications for use. It is the drug of choice for treatment of onchocerciasis (Onchocerca volvulus) and is being used in several nematode infections. c. Side effects and toxicity include fever, rashes, headache, dizziness, and pruritis. B. Trematodes (flukes)
1. Praziquantel a. Pharmacologic properties (1) It is rapidly absorbed with oral administration; there is extensive first-pass
metabolism. (2) It penetrates the CSF. (3) It increases the membrane permeability of susceptible worms to calcium, resulting in paralysis of the parasite. b. Indications for use (1) Praziquantel is the agent of choice for all trematode infections, including all forms of schistosomiasis. (2) It is also active in cestode infections, such as Taenia saginata, Taenia solium, Diphyllobothrium latum, and Hymenolepis nana. e. Side effects and toxicity include abdominal pain, nausea, headache, dizziness, occa-
sional fever, rashes, and eosinophilia.
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Table V-I5-5. Antihelminthic drugs. Helminth
Preferred Drug
Roundworms (nematodes)
Mebendazole Thiabendazole Mebendazole Thiabendazole Mebendazole Mebendazole Praziquantel
Enterobius vermicularis Strongyloides stercoralis Ascaris lumbricoides Trichinella spiralis Tapeworm (cestodes) (Taenia saginata, Taenia solium) Hookworm (Necator americanus) Whipworm (Trichuris trichiura) Flukes (trematodes) Schistosoma haematobium Schistosoma mansoni Schistosoma japonicum
Pyrantel Mebendazole
Praziquantel Praziquantel Praziquantel
2. Oxamniquine
a. Pharmacologic properties. Oxaminiquine has schistosomicidal activity against both mature and immature worms. b. Indications for use. It is used primarily for Schistosoma mansoni infections. c. Side effects and toxicity include dizziness and somnolence. C. Cestodes (tapeworms)
1. Nidosamide a. Pharmacologic properties (1) There is very little gastrointestinal absorption following oral administration.
(2) It has prominent activity against most cestodes, by inhibiting anaerobic metabolism, parasitic oxygen, and glucose uptake. b. Indications for use (1) Niclosamide is used in treating tapeworm infections from T. saginata, D. latum,
andH. nana. (2) It has some effectiveness in treating T. solium infections; however, there is some risk of cysticercosis, as the drug does not kill ova. Praziquantel is the drug of choice for cysticercosis. c. Side effects and toxicity are mild malaise, abdominal pain, and nausea.
2. Praziquantel is used in the treatment of cysticercosis.
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SECTION VI
Immunology
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Basic Immunology
The immune system is an intricate collection of organs, tissues, cells, and soluble factors that allow individuals to defend against harmful agents such as viruses, bacteria, and tumor cells. The immune system includes the primary or central lymphoid organs in which the leukocytes develop, the secondary or peripheral lymphoid organs and tissues in which immune responses occur, and the leukocytes circulating in the blood. This chapter reviews the cells and organs that compose the immune system and the basic characteristics of immune responses, including antigens, antibodies and T-cell receptors, and mechanisms of regulation. Further, T-cell and B-cell activation, the cellular interactions necessary for cell-mediated (e.g., T-cell) and humoral (e.g., antibodies) responses will be examined, as well as the cytokines that influence these processes. Finally, complement and inflammation will be reviewed, along with important immunologic laboratory methods.
GENERAL PROPERTIES OF IMMUNE RESPONSES The immune system is composed of innate and adaptive immune responses. The innate system is thought of as a nonspecific protection against infection and includes barriers such as the skin and epithelial lining of the gut, as well as cell surface receptors for bacterial proteins termed "toll-like" receptors. Adaptive immunity consists of all responses that lead to the development of specific antibodies and antigen-specific T lymphocytes.
In a Nutshell Primary lymphoid Organs
LYMPHORETICULAR SYSTEM
• Bone marrow
The lymphoreticular system (Figure VI -1-1) is composed of the primary lymphoid organs, in which hematopoiesis and lymphopoiesis occur, and secondary lymphoid organs, in which immune responses occur. Primary lymphoid organs in children and adults include the bone marrow and thymus; in the fetus, the spleen and liver are also primary organs. Hematopoiesis occurs in the bone marrow, producing mature erythrocytes, platelets, monocytes, granulocytes, and B cells, as well as precursors for T cells, NK cells, dendritic cells, and mast cells. The T cells finish maturation in the thymus, and all other cells finish maturation in the periphery. The major secondary or "peripheral" lymphoid organs and tissues include the lymph nodes, spleen, and the mucosa-associated lymphoid tissue system (MALTS). MALTS includes the gut-associated lymphoid tissue (GALT), bronchus-associated lymphoid tissue (BALT), and submucosal lymphoid tissues of the genitourinary tract. The purpose of secondary lymphoid organs is to trap and present antigen to circulating lymphocytes and then stimulate adaptive immune responses. These secondary tissues or organs protect all surfaces and fluids of the body. Extracellular fluid, or lymph, is filtered through lymph nodes and MALT, and the primary filter for the blood is the spleen and to some extent the liver.
• Thymus
Secondary lymphoid Organs • Lymph nodes • Spleen • Tonsils • Mucosa-associated lymphoid tissue (MALT) - Gut-associated lymphoid tissue (GALT) - Bronchus-associated lymphoid tissue (BALT)
iiie&ical
405
Immunology
Waldeyer ring lymph nodes, } - tonsils, and adenoids (BALT) Thymus ---F-----'-?.,....,.,r.-_+--- Lung X~...-\--- Lymph nodes
Bone marrow
>-"~~a-+--+-- Spleen :~I*'\--+--t--
Lamina propria
Mesenteric -+-I-+~~..6'r--T---T-t- Peyer patch (GALT) lymph nodes Lymph --"o,;1~t--:-.... nodes
b--'--+---"6~
Urogenital
Figure VI-1-1. The Iymphoreticular system.
Note Bone marrow is the source of pluripotent stem cells (precursors of all white blood cell types as well as RBCs) and the site of B-cell maturation.
Note Early contact with antigen in primary lymphoid organs tends to produce tolerance. Later contact in peripheral lymphoid organs tends to produce activation of both T and B cells.
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A. Bone marrow structure and function. Bone marrow is a primary lymphoid organ because it is the site of hematopoiesis and B-cell maturation, as well as the site of origin of the stem cells involved in T-cell production. 1. Bone marrow structure. The bone marrow is a very large tissue comprising 3-5% of body mass in humans. It is found in the axial skeleton, cranium, ribs, and long bones.
There are two functional components of approximately equal size: the vascular and adipose portion, and the hematopoietic portion. The latter is involved in the formation of blood cells, which are all derived from a single hematopoietic stem cell. 2. Hematopoietic cell differentiation (Figure VI-1-2). All blood cells are derived from pluripotent hematopoietic stem cells that differentiate into myeloid and lymphoid progenitor cells. a. Progenitor cells differentiate after the by appropriate stimuli in certain anatomic sites, such as primary lymphoid organs (thymus and bone marrow) and secondary lymphoid organs (lymph nodes). Stimuli include colony-stimulating factors, erythropoietin, thymosin, and antigen (both self and foreign). Polycolony-stimulating factors include stem cell factor, IL-3, GMCSF, and IL-6. b. After maturation in the thymus or bone marrow, lymphocytes leave these sites and migrate to the spleen, lymph nodes, and MALT (secondary lymphoid organs), where further development occurs under the influence of antigens and cytokines.
Basic Immunology
PRE-B-CELL
PRE-B-CELL
INITIATES H CHAIN GENE REARRANGEMENT
EXPRESSES CYTOPLASMIC IMMUNOGLOBULIN HEAVY CHAINS OF M CLASS; LACKS MEMBRANE Ig
IMMATURE B LYMPHOCYTE
EXPRESSES MEMBRANE IgM AND IgD, HIGH DENSITY OF RECEPTORS FOR COMPLEMENT COMPONENTS TOLERIZABLE
MATURE B LYMPHOCYTE
EXPRESSES MEMBRANE IgM AND IgD, HIGH DENSITY OF RECEPTORS FOR COMPLEMENT COMPONENTS INDUCEABLE
PLASMA CELL
ANTIBODYSECRETING
HELPER T CELL (CD4)
CYTOTOXIC T CELL (CDS) PRE-T-CELL ENTERS THYMUS CORTEX-MEDULLA NK CELL PLURIPOTENTIAL STEM CELL
NEUTROPHIL
MACROPHAGE MONOCYTE EOSINOPHIL MYELOID STEM CELL
BASOPHIL
PLATELET MEGAKARYOCYTE ERYTHROCYTE COLONY-FORMING PRECURSORS
ERYTHROBLAST
Figure VI-1-2. Hematopoietic cell differentiation.
B. Thymus structure and function. Precursors of thymic (T) lymphocytes travel from the bone marrow to the thymus, where early differentiation and maturation take place, including T-cell receptor expression and clonal deletion of autoreactive T cells. Mature T lymphocytes leave the thymus to seed the secondary lymphoid organs in thymic-dependent regions, where they can be activated and undergo their final maturation into effector T cells that function in immune responses. 1. Development of the thymus. The thymus is derived from the third and fourth pharyn-
geal pouches and is found in the mediastinum. a. The development of the thymus begins with epithelial (endodermal) outgrowths of the third and fourth pharyngeal pouches. b. Subsequently, this epithelial reticulum is infiltrated by pre-T cells and other mesodermal elements. c. The thymus reaches its maximum weight at puberty and then slowly involutes.
d. There is a significant amount of programmed cell death (apoptosis) that occurs in the thymus. This is a reflection of the elimination of autoreactive lymphocytes during differentiation in this organ (tolerance by clonal abortion). 2. Organization of the thymus. The stroma of the thymus consists of a prominent connective tissue capsule, which invaginates into the parenchyma as septa and divides the thymus into lobules. The parenchyma is organized into a cortex and medulla. a. Cortex is composed of tightly packed differentiating thymocytes surrounded by a meshwork of epithelial cells and macrophages. KAPLA!!._
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Immunology
In a Nutshell Thymus Cortex • Darker peripheral zone • Extensive population of immature T cells, epithelial cells, and macrophages Thymus Medulla • Lighter central zone • Larger number of epithelial cells and mature large- and medium-sized T cells • Hassall corpuscles
Clinical Correlate DiGeorge syndrome is the absence of the thymus, resulting in a severe deficit of T cells. Afflicted individuals have no T-cell-mediated immunity. DiGeorge syndrome is often accompanied by hypoplasia of the parathyroid glands with resultant tetany. Early death from severe infection often occurs. Babies with DiGeorge syndrome have low-set ears and develop tetany shortly after birth due to a lack of parathormone. The immune deficiency becomes apparent a few months later when maternallgG disappears from the circulation.
Clinical Correlate Each node receives lymph from a defined and limited region of the body. Neoplasms can metastasize via these nodes. For example, a common metastatic site for breast cancer is the axillary nodes.
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b. Medulla consists of epithelial cells and mature T cells. The medulla exhibits a paler staining than the cortex as a result of the large reticular cells and the presence of larger lymphocytes that are not as densely packed. The medulla also contains the characteristic Hassall corpuscles, which are composed of concentrically arranged dead and dying reticular cells, macrophages, neutrophils, and nuclear material whose origin is unknown. 3. Blood supply. Arteries from the connective tissue capsule and septa enter the thymus at the level of the corticomedullary junction. These arteries branch into capillaries, which loop up to the periphery and turn back to the medulla to form venules, which leave the septa. a. Thymic capillaries have a nonporous endothelium with a thick basement membrane. b. Epithelial cells surround thymic vessels and constitute an incomplete barrier, which separates the blood from the thymocytes. The thymic reticulum is composed of branching epithelial cells that are joined one to another by desmosomes. 4. Thymus is large at birth in relation to other organs; it increases in size until puberty, when involution begins. Thymectomy in young animals results in poor development of the other lymphoid tissues and the absence of cell-mediated immunity. This can be reversed by a thymic graft. Congenital absence of the thymus (i.e., DiGeorge syndrome) results in poor development of peripheral lymphoid tissues and the absence of cell-mediated immunity. 5. Thymosin, a family of lymphokines that stimulate thymus-dependent zones in the peripheral lymphoid tissues, is produced by thymic epithelium. C. Lymphatics. More plasma is flitered from capillary beds into the tissues of the body than is
reabsorbed back into the venous end of those capillary beds. This excess fluid moves through the tissues or organs as interstitial fluid and is collected in small lymphatic vessels throughout the tissues as lymph. These small lymphatics fuse into larger afferent lymphatics that enter lymph nodes. The efferent lymphatics leaving one node may become the afferent lymphatics entering another node in a cluster. Eventually, most efferent lymphatics fuse into the large thoracic duct that extends the length of the thorax and empties into the left subclavian vein, returning the lymph fluid to the blood. D. Lymph nodes are highly organized secondary organs that are the most common site for an adaptive immune response because they fliter the lymph that washes through the body tissues. During an infection, lymph nodes may increase two to five times in size. They are encapsulated, kidney-shaped structures that have a concave side with a hilum. The blood vessels and nerves enter and exit at the hilum, and the efferent lymphatic exits from this region. Beneath the stromal capsule, the parenchyma is organized into a cortex, paracortex, and medulla. 1. The stroma consists of a dense connective tissue capsule that surrounds each lymph node
and sends collagenous trabeculae into the node to divide its parenchyma into incomplete compartments (Figure VI-1-3). a. Reticular cells produce reticular fibers that anastomose with the trabeculae and form an extensive network. b. The delicate reticulum filters the lymph and suspends the lymphocytes and macrophages. c. This stromal organization and function facilitates cell-to-cell and antigen-receptor interactions.
Basic Immunology
2. Cortex. Beneath the capsule (except at the hilum) lies a cortex, which is composed of B cells in a diffuse lymphatic tissue network that intermingles with the subcapsular and peritrabecular sinuses (macrophages). T cells are found predominantly in the paracortex. a. Lymphatic sinuses are the lymphatic passageways within lymph nodes. They are lined with flat endothelial cells in the area of the capsule and are partially lined by reticular fibers and macrophages elsewhere. The sinuses receive lymph brought by afferent vessels and transport it toward the medulla. b. Germinal centers may be present in the nodules of the cortex (see Figure VI-I-3). They are composed mostly of B cells, some T cells, and macrophages and are transient structures that are only present during an immune response. Antigen stimulation increases the number and development of germinal centers. In germinal centers, B cells develop into plasma cells in response to specific antigens. 3. The medulla of the lymph node occupies the center of the organ. It contains medullary cords composed of lymphoid tissue that extend from the cortex. Medullary sinuses, like those in the cortex, transmit the lymph toward the hilum, where it exits through the efferent lymphatic. 4. Function. Lymph nodes serve as filters trapping foreign particles from the lymph before circulating to other areas of the body.
In A Nutshell Germinal center of follicle (Clones dividing)
Cortex Paracortex (T-cell rich) Medulla
Primary follicle (8-cell rich)
• Lymph nodes filter tissue fluids. • Spleen filters blood-borne antigens.
Efferent lymphatic (Memory cells exit)
Afferent lymphatic (Ag enters)
Clinical Correlate Figure VI-1-3. Lymph node.
a. Lymph enters the node via afferent lymphatic vessels, which are located on the convex side of the organ (see Figure VI -1-3). b. From the afferent lymphatics, lymph passes through the subcapsular sinus to the peri trabecular sinuses. It then enters the medullary sinuses and exits the lymph node via the efferent lymphatic vessels at the hilum. E. The spleen is a peripheral lymphoid organ in the upper left quadrant of the abdominal cavity, which acts as a filter for blood, clears old and defective erythrocytes (RBCs), and provides protection from blood-borne pathogens.
Patients with sickle cell disease undergo gradual splenic infarction. This predisposes them to septicemia, particularly caused by Streptococcus pneumoniae. Other opportunists include Salmonella, meningococci, and H influenzae.
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1. The stroma of the spleen consists of the following:
a. A dense connective tissue capsule that contains smooth muscle cells b. Trabeculae that branch off of the capsule and partially partition the parenchyma of splenic pulp c. A delicate meshwork of reticular connective tissue that filters the blood 2. Splenic parenchyma consists of white and red pulp.
Note In the splenic white pulp, Band T lymphocytes are segregated. The central portion of PALS are rich in T cells, whereas the marginal zone are populated by B cells.
a. White pulp consists of lymphatic tissue arranged in sheaths around arterioles (periarteriolar lymphoid sheath) and in nodules (marginal zones). Antigenic stimulation increases the amount of white pulp. (1) The periarteriolar lymphocyte sheaths (PALS) are accumulations of diffuse
lymphatic tissues that are rich in T cells. The marginal zone is occupied mostly by B cells. (2) B lymphocytes cluster peripherally in the PALS to form primary follicles. After antigenic stimulation, these follicles develop into secondary follicles with germinal centers containing rapidly dividing B cells. (3) At the marginal zone, dendritic cells trap and process antigen and migrate to PALS to present it to antigen-specific cells of the immune system. b. Red pulp consists primarily of erythrocyte-filled sinusoids and macrophages in a reticular fiber network; most filtration occurs here. (1) The sinuses vary in size and are separated by pulp (i.e., Billroth) cords. (2) RBCs and platelets are exposed to macrophages in the pulp cords; macrophages phagocytize worn-out or damaged cells. (3) Splenic sinusoids are lined by loosely arranged endothelial cells, which have numerous fenestrations. These sinusoids are surrounded by a poorly developed basal lamina. (4) Phagocytosis is carried out primarily by macrophages located outside the sinusoids. The macrophages extend finger-like projections into the sinusoids, which push through the discontinuous basement membrane between the endothelial cells. 3. Blood supply. Arteries from the hilum of the spleen pass along trabeculae to enter the periarteriolar sheath. a. These vessels branch like brush bristles to form penicillar arteries, which continue in capillaries and open blindly in the red pulp, connecting directly to venous sinusoids. b. The venous sinusoids are specialized, large-caliber spaces that are bordered by elongated endothelial cells and held in place by circularly arranged reticular fibers. c. Venous sinusoids drain into veins that eventually exit at the hilum.
F. Gut-associated lymphoid tissue (GALT) is not encapsulated and is present in the submucosa and lamina propria in the gastrointestinal tract. It is the site of immune responses to injested microbes and some food antigens. 1. Structure. Lymphoid tissue in GALT includes the large follicular aggregates in the small
intestine called Peyer patches, the lamina propria beneath the mucosal epithelia in the villi, and the intraepitheliallymphocytes (IELs) found between mucosal epithelial cells along the surface of the villi.
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2. Function a. The lymphoid tissues lining the intestinal tract are well exemplified by the Peyer patches. Structurally unique antigen-presenting cells, called M cells, are located in mucous membranes. They endocytose microbes and antigens and present specific epitopes to T lymphocytes located between follicles and in the lamina propria. With Tcell help, the B cells become activated and form germinal centers, where they differentiate into 19A-secreting plasma cells. b. The 19A dimers react with a polyimmunoglobulin receptor on intestinal epithelial cells. The dimer (held together by the J chain) is internalized and crosses the epithelial cell cytosol. It is then proteolytically cleaved from the poly-Ig receptor and excreted into the lumen of the intestine. A small portion of the Ig receptor remains attached to the dimer; this is called the secretory component. The function of this small peptide is to protect the antibody molecule from enzymatic hydrolysis in intestinal fluid.
Clinical Correlate Peyer patches and other lymphoid tissues will be hypoplastic in individuals with Bruton hypogammaglobulinemia. Lymphoid tissue is almost totally absent in patients with severe combined immunodeficiency.
G. Bronchus-associated lymphoid tissue (BALT) includes the lymphoid tissue beneath the respiratory mucosa and the aggregates of nodular lymphatic tissue called tonsils. These are structurally similar to lymph nodes and contain deep crypts that allow antigen to be trapped, degraded, and processed by antigen-presenting cells, such as macrophages and dendritic cells. 1. Organization of the tonsils. Tonsils are peripheral lymphoid organs composed of aggre-
gates of nodular and diffuse lymphatic tissues that protect epithelial surfaces. The lymphoid tissues are located beneath the epithelium in the underlying connective tissues. a. Lymphatic nodules contain aggregates of B lymphocytes, which differentiate into plasma cells to produce antibodies for the humoral immune response. b. T cells are found primarily in diffuse lymphatic tissues. 2. Types of tonsils. Three important examples of tonsillar tissue are the palatine tonsils, the lingual tonsils, and the pharyngeal tonsil. a. Palatine tonsils are located bilaterally in the oropharynx. (1) They are composed of dense lymphoid tissue, which forms a band oflymphatic nodules with germinal centers. These are intermingled with diffuse lymphatic tissue beneath the stratified squamous epithelium.
Clinical Correlate Tonsillitis involves the formation of abscesses in the crypts.
(2) A dense connective tissue capsule often separates the tonsil from subjacent tissues. (3) Each tonsil has numerous epithelial invaginations, or crypts, that contain desquamated epithelial cells, lymphocytes, and bacteria in their lumina. b. Lingual tonsils are smaller and more numerous aggregates of lymphoid tissues located at the base of the tongue. (1) They are covered by the stratified squamous epithelium on the dorsum of the tongue. (2) Each aggregate possesses a single crypt. c. Pharyngeal tonsil is an unpaired accumulation of lymphoid tissue located on the
posterior wall of the nasopharynx. (1) It is usually covered by a pseudostratified columnar epithelium with cilia and goblet cells; however, the epithelium can be obscured by an infiltration of lymphocytes or by metaplasia (seen in smokers). The pharyngeal tonsils, or hypertrophic pharyngeal tonsils, are often referred to as adenoids. (2) Instead of forming crypts, the overlying epithelium occurs in a series of folds.
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H. Lymphocyte recirculation 1. Lymphocytes are capable of high levels of recirculation, continuously moving from the blood out into the tissues and then returning via the lymphatics. This movement is facilitated by cell adhesion molecules (CAM).
2. The lymphocytes bear in their membranes specific CAMs, called selectins, which interact specifically with addressins (another type of CAM) that are found in the lymphatic vasculature, particularly in the postcapillary venules. It is here that specialized cells with a plump cuboidal shape are found; these are called high endothelial venules due to the height in the region caused by the cuboidal cells. 3. As T cells home to lymph nodes, they make contact with antigens presented in the groove of class II MHC molecules located on the surface of antigen-presenting cells. This dynamic leads to T-cell help and B-cell activation.
Note Lymphocyte homing is controlled by cell adhesion molecules (CAMs).
4. Once the activation has occurred, the lymphocytes stay trapped in peripheral lymphoid tissues, where they differentiate to mature effector cells in the case of T cells or plasma cells in the case of B cells. Lymph node swelling is thus a product both of lymphocyte trapping and division. S. Different populations oflymphocytes express different homing receptors. Naive and memory cells also express different receptors.
Note Cells of the immune system develop in the bone marrow.
Note Macrophages and neutrophils both phagocytose bacteria that are coated with antibody and/or complement. The C3b fragment of complement and/or IgG can bind to bacteria, then bind to receptors on phagocytic cells and signal them to engulf the organisms. The Fc receptors on macrophages are also useful for opsonization of bacteria by antibody. They react with the Fc region of IgG antibody molecules and hold the microbe close to the phagocytic cell membrane, thus facilitating the engulfment process.
CELLS OF THE IMMUNE SYSTEM Leukocytes include lymphocytes (B cells, T cells, and large granular lymphocytes or NK cells), mononuclear phagocytes (monocytes and macrophages), polymorphonuclear granulocytes (neutrophils, eosinophils, and basophils), mast cells, and dendritic cells. All leukocytes, as well as erythrocytes and platelets, initially develop in the adult bone marrow (described below); many complete maturation there, although T cells complete their maturation in the thymus. A. Monocytes and macrophages are derived as follows: stem cell-7 monoblast -7 promonocyte -7 circulating monocyte -7 tissue macrophage. Macrophages have a life span of months to years. 1. Function of monocytes and macrophages. These cells playa central role in cell-mediated immunity. They ingest particles via pinocytosis or phagocytosis and modulate inflammation via secretion of mediators. They also act as cells that process and present antigens to T lymphocytes (antigen-presenting cells).
a. Macrophages secrete over 100 mediators, including interleukins 1,8, and 12, collagenase, elastase, lipase, proteases, prostaglandins, leukotrienes, thromboxanes, lysozyme, and interferon. They also secrete complement component C2. b. Macrophages have Fc receptors and class II MHC molecules on the cell surface that mediate their biologic functions. The Fc receptors allow the uptake of immune complexes (Ig complexed to antigen), and class II molecules present antigenic pep tides to Th cells. c. Circulating monocytes differentiate into tissue macrophages with specific names. For example, tissue macrophages present within the sinusoids of the liver are called Kupffer cells. Macrophages found in the lung are termed alveolar macrophages; macrophages in the brain are called microglial cells. (1) Kupffer cells encounter antigens first from intestinal lumen absorption and serve
to clear particulate and soluble matter from portal circulation. They phagocytose bacterial endotoxin, soluble immune complexes, activated clotting factors, and microorganisms.
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(2) Alveolar macrophages destroy inhaled antigens and microbes. 2. Morphology of macrophages a. Epithelioid cells, usually found in granulomas, are derived from blood monocytes and are activated by an immune response to antigen. b. Multinucleated giant cells are formed by the fusion of macrophages. 3. Macrophage activation. Macrophages are stimulated by lymphokines (mostly IFN-y) to kill microorganisms and tumor cells. a. Activated macrophages have increased lysosomal hydrolytic enzymes and an increased chemotactic response. C5a and various cytokines from lymphocytes, neutrophils, and fibroblasts are chemoattractants for activated macrophages. b. Antigen coated with appropriate complement proteins (e.g., C3b) and/or antibody is more readily phagocytosed. This process is called opsonization. The C3b receptor is called CRI or CD35. c. Morphologic changes occur during activation and include increases in size, number of
pseudopods, and pinocytotic vesicles. The process takes several days. d. Macrophages experience a respiratory burst during phagocytosis via the hexosemonophosphate shunt pathway. This is a source of energy needed for cell membrane synthesis, and it also generates toxic oxygen metabolites such as singlet oxygen, superoxide anion, and hydrogen peroxide.
IgG Immunoglobulin
~ Macrophage
Antibody and complement C3b
Bacterial organism
In a Nutshell Figure VI-1-4. Antibody and complement C3b.
4. Macrophages present extracellular antigens to T helper cells. Antigen undergoes phagocytosis or pinocytosis. Once in the cytoplasm of the macrophage, the antigen is degraded into small peptides. The pep tides are then noncovalently bound to class II MHC molecules in an endosomal vesicle. The complex is then transported to the cell surface, where it stimulates class II-restricted antigen-specific Th cells (CD4). B. Dendritic cells are present in peripheral blood and lymphoid organs. Their primary func-
tion is to digest and process antigen for presentation to Th cells. Dendritic cells include Langerhans cells of skin, veiled cells in afferent lymphatics, and interdigitating reticulum cells in spleen and lymph nodes.
Macrophages process exogenous antigens and present the epitopes in a groove of the class II MHC molecules. CD4 T cells bearing receptors specific for the epitope will react with the epitope/MHC complex and be triggered to release cytokines. Endogenous antigens are similarly presented to CDS cells on class I MHC, also present on macro phages and other APCs.
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C. Granulocytes are also called polymorphonuclear leukocytes. There are three types of granulocytes: neutrophils, eosinophils, and basophils.
1. Neutrophils (polymorphonuclear leukocytes, PMNs) represent 60% of circulating blood leukocytes. Circulating neutrophils have a receptor for the Fc region of IgG (FcyR) and C3b (CD35) (Figure VI-I-4).
In a Nutshell PMNs kill microbes via: • Toxic oxygen metabolites • Digestive enzymes present in lysosomal granules
Clinical Correlate Eosinophilia is a hallmark of: • Atopic allergies • Worm infections It can also be seen in collagen vascular diseases, neoplastic disorders, and any skin rash.
a. Neutrophils have a multilobulated nucleus with dense chromatin and cytoplasmic lysosomes containing peroxidases and acid hydro lases. b. They reach the tissues by diapedesis, inserting pseudopodia with cell adhesion molecules between the endothelial cells so that movement through blood vessel walls occurs. c. Neutrophils are the first cells to arrive at acute inflammatory sites. They actively kill bacteria; their half-life is about 10 hours in the blood and 3 days in tissues. d. Cytoplasmic granules contain digestive enzymes. These include azurophilic granules that contain myeloperoxidase and specific granules that contain lactoferrin. e. Neutrophils phagocytize and then kill the organism by generation of H 2 0 2 and toxic oxygen radicals and via the action of granule-derived enzymes. 2. Eosinophils represent 1-3% of circulating leukocytes. a. Approximately 50% of circulating eosinophils have receptors for complement. b. Eosinophils have a bilobed nucleus and contain crystalloid granules staining red with Giemsa. c. Eosinophil chemotactic factors include histamine, C5a, LTB 4 , PAF, and ECF-A (eosinophil chemotactic factor of anaphylaxis).
Note Eosinophil granule contents that help control allergic reactions include histaminase and arylsulfatase.
d. They are functionally important in late inflammatory reactions, particularly in parasitic infections and allergy. e. Some important contents of eosinophils and their functions include: ( 1) Histaminase degrades histamine. (2) Pyrogen produces fever. (3) Peroxidase kills microorganisms. (4) Arylsulfatase degrades leukotrienes C4' D 4 , and E4 • (5) Major basic proteins are toxic to worms. 3. Basophils represent 1% of circulating leukocytes and are the smallest type of granulocytes. a. They contain abundant granules with RNA, mucopolysaccharides, and hypersensitivity mediators, such as histamine. b. They have receptors for the Fc portion of IgE. c. IgE binding promotes degranulation as it does in the tissue mast cells. Histamine and other mediators are released during degranulation and are responsible for the symptoms seen in atopic allergies. This is discussed further in the Clinical Immunology chapter. D. Lymphocytes, which include Band T lymphocytes, represent 30% of circulating leukocytes. Lymphocytes have a high nucleus-to-cytoplasm ratio and are distinguished by their antigen receptors and cell surface markers. 1. B lymphocytes. B lymphocytes terminally differentiate into plasma cells, which secrete large amounts of immunoglobulin (antibodies).
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a. There are two major subsets of B lymphocytes: CDS-positive cells produce 19M antibody to soluble polysaccharides and self-antigens. They are stimulated by nonspecific cytokines from Th cells. CDS-negative cells produce 19G, 19A, or 19E antibody specific to individual to protein antigens, cellular antigens, and bacteriallipopolysaccharides. These cells require direct physical interaction with specific Th cells. b. Memory B cells, generated after the primary exposure to an antigen, produce antibody with increased affinity for its antigen because of somatic mutation of 19 genes. c. Mature B cells have surface 19M and IgD that serve as the B-cell receptor and bind antigen. B cells can also present antigen to Th cells via class II MHC. d. B cells respond to two types of antigens. (1) T-cell-independent antigens activate B cells in the absence of CD4+ Th cells.
These antigens are usually composed of polysaccharides or carbohydrates with repeating structures. (2) T-cell-dependent antigens (most all protein antigens) require B- and T-cell interaction. The T cell then drives B cells to switch antibody classes (class switching) via direct contact and by cytokine secretion. 2. T lymphocytes. Two major types of T cells exist and are classified based on the expression of the cell surface proteins CD4 or CDS. Cellular differentiation (CD) proteins are present on many cells of the body. They reflect the function of the cell and are thus "markers" for particular cells. For example, CD3 is found in the membrane of all mature T cells, whereas CD4 and CDS proteins are found on certain subsets of these mature T cells. a. Th cells are CD4 positive. There are two distinct subsets of Th cells with different functions (Th1 and Th2). The Th2 cell's function is to stimulate B lymphocytes to proliferate and differentiate into antibody-producing cells. The Th1 cell's function is to promote cytotoxic T-cell (CDS+ cells) responses and the delayed-type hypersensitivity response. (1) Activation and proliferation of Th cells depends on corecognition of specific antigenic peptides and MHC class II molecules on antigen-presenting cells such as macrophages, dendritic cells, or B cells (APCs). (2) Activated Th cells produce cytokines, differentiation factors, and inflammatory cytokines. b. Cytotoxic T cells are CD8 positive. They lyse cells such as virus-infected cells and tumor cells. Cytotoxic T cells act by recognizing foreign antigen and MHC class I molecules with their T-cell receptor. 3. Natural killer (NK) cells represent lO-15% oflymphocytes in the peripheral circulation. a. NK cells kill certain tumor cells (without damaging normal tissues) and defend against viral infection. Unlike T cells, NK cells recognize foreign antigen that does not have to be presented on MHC molecules. b. NK cells mediate antibody-dependent cellular toxicity (ADCC). This function allows NK cells to kill antibody-coated target cells. e. NK cells are activated by cytokines such as 1FN-y and 1L-2.
Note T-celi-independent antigens induce IgM antibody only and do not cause immunologic memory (anamnesis). Much of this antibody is secreted by CDS B celis.
Note There are two subsets of helper T celis. Th 1 releases IL-2 and IFN-y, whereas Th2 releases other interleukins (e.g., 4, 5, 6, 10). Th1 celis stimulate proliferation and cytoxic responses of cytotoxic T celis and macrophages, whereas Th2 celis stimulate B-celi maturation, differentiation, and immunoglobulin class switching.
In a Nutshell • Helper T celis
--7
• Cytotoxic T celis
CD4+
--7
CD8+
Note ADCC may be mediated by NK celis, eosinophils, or neutrophils, ali of which have Fc receptors
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THE IMMUNE RESPONSE Immune cells and the responses they generate can be categorized into innate and acquired. The innate responses are necessary for immediate reponse to a pathogen and for establishing local inflammation to recruit circulating phagocytes and, later, activated T cells and B cells. The T and B lymphocytes make the acquired response and are responsible for many of the classic characteristics of immunity, including memory, antigen specificity, and tolerance to self. The characteristics of antigen presentation and recognition by T and B cells are discussed below. A. Innate versus acquired immunity. The immune system is composed of cells that defend against foreign invaders by both nonspecific mechanisms (termed innate or natural immunity), and antigen-specific mechanisms (known as acquired immunity). The nonspecific response is typically observed first because no prior exposure to antigen is necessary. This response may not be sufficient to clear the foreign antigen, and an antigen-specific mechanism produced by Band T cells may be required.
In a Nutshell
1. Innate immunity (also known as natural immunity) is present at birth in all individuals.
This response does not increase upon repeated exposure to a given antigen.
Innate Immunity (Also Called Natural Immunity)
a. The first line of defense is intact skin and mucous membranes.
• Present at birth
b. Innate immunity allows elimination of foreign substance (i.e., bacteria or virus) without previous exposure.
• Composed of skin, mucous membranes, secretions such as saliva and tears, phagocytic cells, and NK cells Acquired Immunity • Developed in response to a specific immunogen exposure • Composed of antibodies (lgG, IgA, etc.) and lymphocytes (B and T cells)
c. Innate immunity is enhanced by either natural antibodies or natural cytotoxic cells,
including macrophages, neutrophils, eosinophils, and NK cells. 2. Acquired immunity is established late in fetal life, and development continues during childhood. Continued exposure to foreign antigen will stimulate acquired immunity. a. Responses are specific to individual antigens because of antigen-specific recognition by surface antibody on B cells and by T-cell receptors (TCRs) on T cells. b. Individual lymphocytes recognize their appropriate antigens (discussed below) and are activated to proliferate into effector cells and memory cells with the same antigen specificity (clonal expansion). c. May have anamnestic response, in which subsequent response to previously recog-
nized antigen is more avid and often magnified (this is a result of the production of memory T and B cells and somatic mutation of Ig molecules). d. Antigen is eliminated due to specific antigen recognition and effector functions (discussed below). 3. Self-tolerance describes the absence of immune responses to one's own tissue antigens. It is necessary to prevent autoimmune responses. Mechanisms of self-tolerance include the following: a. Thymic deletion, the elimination of clones of developing T cells with receptors against self-antigens, occurs during cell maturation in the thymus. b. Anergy of B lymphocytes bearing receptors for self-antigens is maintained by the lack of costimulatory signals when naive cells encounter self-antigens. B. Antigens and antibodies. An antigen is any substance that can be specifically bound by an antibody or T-cell receptor, whereas an immunogen is an antigen that induces an immune response. Because antibodies and TCRs recognize different types of antigens, and antibody production requires helper Th-cell activation and assistance, immunogens typically possess both antibody and T-cell-reactive regions.
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1. An epitope or antigenic determinant is a specific site on the antigen that is recognized by
a B- or T-cell receptor, i.e., the part of the antigen to which an antibody binds. a. Most antigens express many epitopes. b. B-cell epitopes are short peptides of 10-25 amino acids that usually occur at the hydrophilic surface, whereas T-cell epitopes may be embedded within the protein. 2. Antibodies are able to bind to epitopes on a wide variety of molecules, including proteins, carbohydrates, nucleic acids, and small organic molecules. TCRs are only able to recognize peptides bound to major histocompatiblity (MHC) proteins on the surface of a cell (discussed below). 3. Many macromolecules can be antigenic. a. Proteins (glycoproteins, lipoproteins, or nucleoproteins) are the most common form of antigen and are usually very good immunogens. (1) Their antigenicity is based on amino acid composition, three-dimensional con-
formation, and/or biochemical properties (such as charge, etc.). (2) Peptides derived from processed proteins and bound to cell-surface MHC proteins (either class I molecules for CD8 T cells or class II molecules for CD4 cells) are the only type of antigen that T-cell receptors can recognize. b. Large, repetitive polysaccharides can activate B cells with little or no helper T aid (but cannot be recognized alone by TCRs) and so are considered T-cell-independent antigens. c. Nucleic acids can be recognized by antibodies but are poor immunogens because they cannot act as T-cell antigens unless they are bound to a specific protein carrier. Nucleic acids can stimulate antigen-presenting cells to make cytokines that enhance T-cell responses. d. Lipids are usually not immunogenic. When lipids are coupled to protein antigens, they tend to induce T-cell-mediated delayed hypersenstivity rather than antibody production. e. Haptens are small molecules that can act as an antibody epitope but will not induce immune responses because they are not recognized as T-cell antigens. When combined with a carrier protein, the hapten-carrier complex can produce hapten-specific and carrier-specific antibodies because the Th cell can recognize carrier peptides. Drugs that become metabolized in the body are common sources of haptens. Many allergens (e.g., penicillin) are haptens. C. Major histocompatibility complex (MHC). The MHC is a collection of highly polymorphic
Note Thymus-independent antigens do not induce memory cell production.
Note Carrier Effect Poorly immunogenic or nonimmunogenic molecules acquire immunogenicity when they are chemically linked to proteins that serve as carriers and impart the diversity and T-cell reactivity needed.
genes encoding the proteins that regulate immune responses. These genes include, most notably, the class I and class II cell surface proteins and the class III genes that encode complement proteins. In humans, the MHC genes are termed human leukocyte antigens (HLAs) and are found on the short arm of chromosome 6. The HLA proteins are glycoproteins present on cell surfaces that enable T cells to recognize and bind antigenic peptides, i.e., they function in immune recognition. 1. HLA class I antigen (Figure VI-1-5). Class I proteins are membrane glycoproteins on the
surface of all nucleated cells and platelets. They bind endogenous peptides processed from protein synthesized in the cell's cytosol and are necessary for antigen recognition by CD8+ cytotoxic T lymphocytes (CTLs). In humans, the three types of class I genes are referred to as HLA-A, HLA-B, and HLA-C antigens.
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Figure VI-1-5. HLA class I antigen.
In a Nutshell Class I MHC
a. The class I proteins share common structures, including three globular domains (with a molecular weight of 45 kD) in noncovalent association with ~2 microglobulin (not an HLA-coded protein). (1) The carboxy terminus lies within the cytoplasm, and the hydrophobic surface
traverses the membrane.
• Ali nucleated cells
(2) The outer two globular domains form the peptide-binding groove.
• Human class I genes = HLA-A, HLA-8, and HLA-C
(3) Each allele of each HLA class I protein has a different range of peptides that can bind to it.
• Cytotoxic T cells (CDS) recognize class I MHC on infected celis (viruses, intracellular bacteria, parasites, tumor antigens) Class II MHC • Class II is found only on celis that interact with helper T cells = antigenpresenting cells (8 celis, macrophages, dendritic cells) • Human class II genes = HLA-DR, HLA-DQ, and HLA-DP • Helper T cells (CD4) bind to class II MHC on antigenpresenting cells
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b. Proteins synthesized in the cell cytosol are routinely degraded by proteases; peptides from these proteins are transported, through a peptide transporter known as the TAP complex, into the endoplasmic reticulum, where they have an opportunity to bind to freshly synthesized HLA class I proteins. (1) Most commonly, peptides of normal intracellular proteins are bound to HLA
class I proteins and displayed on the cell surface. (2) Proteins made by intracellular viruses, bacteria, parasites, or neoantigens made by transformed tumor cells will also be displayed on HLA class I proteins. c. Cytotoxic T cells recognize viral, intracellular bacterial, parasitic, or tumor antigens in association with class I molecules. CDS on the surface of these cells recognizes a nonpolymorphic region of the class I MHC molecule.
2. HLA class II antigen (Figure VI-1-6). HLA class II proteins are expressed on a more restricted set of cells, including antigen-presenting cells (dendritic cells, Langerhans cells, activated macrophages), B cells, and thymic epithelial cells involved in T-cell maturation. These proteins bind exogenous peptide epitopes processed from endocytosed molecules and are necessary for antigen recognition by Th cells. Class II genes encode for cell surface glycoproteins with two polypeptide components called a and ~ (Figure VI-1-6). In humans, the class II genes include HLA-DR, HLA-DQ, and HLA-DP.
Basic Immunology
Peptide groove
I
Figure VI-1-S. HLA class II antigen.
a. The class II proteins share a common structure, with each ex and ~ chain containing two globular domains. (1) The outer domains of the ex and groove.
~
chains combine to form the peptide-binding
(2) The ex and ~ chains are strongly associated in the cell membrane but are not linked by covalent bonds. (3) Each allele of each HLA class II protein has a different range of peptides that can bind to it. b. Class II proteins are found on cells that interact directly with Th cells, including B cells, monocyte-macrophages, Langerhans cells, dendritic cells, and thymic epithelium. c. Proteins endocytosed, pinocytosed, or phagocytosed by the cell are routinely degraded
by proteases; peptides from these proteins are able to bind to freshly synthesized HLA class II proteins. (1) Most commonly, peptides of normal extracellular proteins are bound to HLA class II proteins and displayed on the cell surface. (2) Proteins made by extracellular bacteria or parasites or injected proteins (e.g., vaccines) will also be displayed on HLA class II proteins. d. CD4 cells recognize bacterial, parasite, or injected proteins in association with class II. The CD4 molecule binds a nonpolymorphic region of class II. 3. HLA class III antigen. Class III genes encode for complement components or regulators of serum complement component levels. C2 and factor B are encoded by class III MHC genes.
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4. HLA disease associations. Many diseases are associated with increased frequency of certain HLA antigens. Ankylosing spondylitis is typically associated with HLA-B27; other diseases are also associated with specific HLA antigens: rheumatoid arthritis (DR4), Sjogren syndrome (DR3), and insulin-dependent diabetes mellitus (DR3 and DR4). a. Most of the HLA-associated diseases are of unknown etiology and have associated immunologic abnormalities. b. Tissue typing for organ transplantation involves matching the HLA class I and class II antigens. D. Antibodies. Antibodies act as antigen-specific receptors on B cells, and, when secreted by plasma cells, mediate humoral responses. Antibodies comprise approximately 20% of plasma proteins. They are produced by B cells in response to the introduction of foreign substances (i.e., antigens) into the body. Antibodies are bifunctional: They bind epitopes on antigens, thereby directly attacking the antigen, and they stimulate other biologic phenomena such as activating complement and binding Fe receptors on other lymphoid cells (opsonization, ADCC). 1. Structure. Antibodies consist of four polypeptide chains-two identical light chains and
two identical heavy chains-all bound together by disulfide bonds (Figure VI -1-7). Heavy and light chains have hypervariable regions at the amino terminus (responsible for antigen-binding specificity) and constant regions at the carboxy terminus. There are intrachain disulfide links that divide each chain into subunits of 110 amino acids; thus light chains have two domains referred to as the variable and constant domains, whereas heavy chains have four or five domains-one variable domain and three or four constant ones.
Hypervariable regions
iI
I I
",.Ught chain
It. t.
Disulfide bonds
" . Heavy chain
"'*" ~
....
........ ..... --:::~~, ~
Hypervariable regions .. - - Papain site
~
T pFc'
Fc
....
, , ' ..... Hinge region ........ Pepsin site
J
Figure VI-1-7. Schematic structure of immunoglobulin molecule.
a. Light chains. The molecular weight of each light chain is approximately 23 kD and is composed of 220 amino acids. There are two subtypes of light chains: kappa and lambda. (1) Kappa chains differ from lambda chains on the basis of structural differences in the constant region. The genes coding for each are not on the same chromosome (2 and 22, respectively).
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(2) Each antibody molecule has either two kappa or two lambda chains. (3) A specific immunoglobulin always has identical kappa or lambda chains. (4) The ratio of chain subtypes is constant within a species. b. Heavy chains. The molecular weight of each heavy chain is 50-75 kD. Each is composed of 450-580 amino acids. (1) There are five different isotypes corresponding to the individual heavy chain gene utilized. Structural differences in the constant regions of the various types of heavy chains account for their different biologic properties. (a) 19G has gamma (y) heavy chains. (b) 19A has alpha (ex) heavy chains. (c) 19M has mu (f.l) heavy chains. (d) 19E has epsilon (£) heavy chains. (e) 19D has delta (8) heavy chains. (2) Heavy chains are further divided into subclasses based on the number of interchain disulfide bridges. For example, 19G 1 and 19G4 have two disulfide bonds, 19G2 has four disulfide bonds, and 19G3 has 15 disulfide bonds. (3) Genes coding for heavy chains are on the same chromosome (14). c. Disulfide bonds link together the different components of the immunoglobulins by
binding between cysteine residues. The bonds confer the three-dimensional structure of immunoglobulins. (1) 1nterchain bonds. There is usually one interchain disulfide bond between the
cysteine residue of the heavy chain and the penultimate or ultimate carboxy terminal cysteine residue of the light chain. 1nterchain disulfide bonds between heavy chains are variable in number; this helps to differentiate subclasses of immunoglobulins. (2) 1ntrachain disulfide bonds also confer structural properties by linking cysteine residues located on the same polypeptide chain. d. The hinge region is located between the second and third 220-amino-acid segments of the heavy chains. This region allows flexibility in the F(ab)2 portion of the molecule and consists of flexible glycine residues flanked by proline-rich inflexible segments. 2. Immunoglobulin fragments generated by enzymatic digestion allow the functional regions of 19 to be characterized. a. Papain digestion of an immunoglobulin yields three fragments, two Fab fragments Cab" stands for "antigen-binding") and one Fc fragment ("c" stands for "crystalline"; the form this region takes in low ionic strength buffers). (1) The Fab fragment contains the amino terminal half of the heavy chain and the
entire light chain. Its molecular weight is 50 kD. (2) The Fc fragment contains the two carboxy terminal halves of the heavy chain held together by disulfide bonds. This region is responsible for binding to Fc receptors on phagocytic cells and for activating complement components. b. Pepsin digestion of an immunoglobulin yields one F(ab')2 fragment and two pFc fragments.
Mnemonic The "e" in Fe could stand for: • Crystallizable • Carboxy terminus of the molecule • Complement-binding region (CHR) • Cell tropism (e.g., FcyR on phagocytes, Fc£R on basophils, FcyR on syncytiotrophoblasts)
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(1) The F(ab')2 fragment is composed of the two light chains, each of which is disul-
fide bonded to the amino terminal portion of a heavy chain (see Figure VI-l-7). The F(ab')2 fragment maintains bivalent antigen-binding ability and is 100 kD in size. (2) The pFc' fragment is composed of the two free carboxy terminal heavy chains.
Note that the pepsin cleavage site is below the disulfide bonds, thus generating the two free heavy chain fragments that individually have no biologic functions. 3. Antigen-binding sites are found in the variable region located in the 1l0-amino-acid segment of the amino terminal end of the heavy and light chains. a. The variability of amino acid sequence confers antigen-binding specificity to individual immunoglobulin molecules. This site has both framework and hypervariable or complimentarity determining subregions. Framework subregions are relatively conserved amino acid sequences that preserve the three-dimensional structure of the variable region and stabilize the hypervariable regions. Hypervariable regions are folded to form the antigen-binding site (also called the paratope). b. The affinity (or binding strength) of an antibody is defined as the concentration of antigen at which half of the antibodies are in a complex with antigen and half are free. The relative affinity of an antibody to antigen is measured in moles per liter and is referred to as the dissociation constant, Kd • Higher numbers mean greater affinity. c. Different structurally related antigens may be cross-reactive and bound by the same antigen-combining site on different antibodies. This is important in diseases such as rheumatic fever, where streptococcal antigens are structurally related and cross-reactive with heart tissue (cardiac myosin). d. Biologic effects of antigen binding result primarily from cross-linking of immunoglobulin molecules resulting from multivalent antigen binding. 4. Idiotypes, allotypes, and isotypes a. Idiotype is the term used to describe the area of the variable region responsible for antigen specificity. These are the unique epitopes that are found in the paratope (see above) of the antibody. b. Isotype is the term used to describe the subclasses of immunoglobulins ("G-A-M -E-D") that are distinguished by unique constant regions encoded by the heavy chain gene. The individual isotypes have unique effector mechanisms, such as the ability to bind complement or mediate hypersensitivity responses (described below).
Note Monoclonal Antibody Plasma cell + myeloma cell = hybridoma. The hybridoma secretes antibody identical to the one produced by the plasma cell. The myeloma cell gives the hybridoma its immortality.
c. Allotype is the protein product of an allele that may be detected as an antigen by another member of the same species. It involves different alleles at a specific site in the constant region of the heavy chain. S. Immune serum contains a heterogeneous population of antibodies with varying affinity for antigens; the average affinity increases as the period of time after immunization lengthens due to somatic mutations. 6. Monoclonal antibodies are antibodies that are specific for a single epitope determinant. They are produced in vitro by hybridomas, created from the fusion of plasma cells to myeloma cells that are grown in tissue culture. E. Immunoglobulin genetics. The multiple genes encoding immunoglobulin molecules undergo a structured rearrangement process leading to the expression of functional protein. It is through this rearrangement that the diversity of immunoglobulin molecules is acquired. This section reviews the recombination events that are required for immunoglobulin synthesis.
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1. Genes coding for light and heavy chain variable regions
a. Kappa light chain is encoded by a single locus on chromosome 2. It has a constant domain and a variable domain. Lambda light chain is encoded by a gene complex on chromosome 22. (1) The variable domain is encoded by two separate gene segments. The variable segment (VK or VA) codes the first 95 amino acids, and the joining segment OK or Til,) codes the final 12 amino acids. (2) There are an estimated 350 variable genes and six joining gene segments encoded
in the human K chain gene. b. Heavy chains (VDJ complex) are encoded by a single region on chromosome 14. (1) The constant region (CH ) sequences for each of the heavy chain isotypes are in
tandem on the chromosome. There is a single copy of each gene. (2) There are four joining segments OH)' several hundred variable segments (VH)' and a dozen diversity segments (DH ). (3) The heavy chain is assembled via VDJ recombination. 2. Antibody diversity is generated by random combinations between individual heavy and light chains, as well as by random combinations of variable, joining, and diversity genes. These intrachain recombinational events occur in the following manner (Figure VI-1-8) for light chains. a. The DNA loops around, bringing a variable gene allele close to a joining gene segment. b. The introns interact in a specific manner (Figure VI-1-9), as detailed in sections c through e below.
G~rm line DNA
_fill[][][F!=f= V~ Vvx2 V vxn
j
~ Gene
I
J1 J2 J3 J4 J5
C x
V-J joining
In a Nutshell Antibody diversity is due to: • Recombination of immunoglobulin genes for:
rearrangement
- Variable region
Plasma cell DNA Vx1
J1 J2 J3 J4 J5
!
- Joining region Cx
Transcription
Plasma cell RNA
Nucleoplasm Cytoplasm
- Diversity region (in H chains only) • Recombination of heavy chains with light chains • Addition of nucleotides to DNA during genetic recombination by terminal deoxynucleotidyl transferase enzyme
Figure VI-1-8.lntrachain recombinational events in light chain synthesis.
• Somatic mutations
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c. The recognition signals adjacent to the V and J segments hold the two strands together so that the recombinase enzyme products of the RAG-l and RAG-2 genes can affect gene fusion, creating a bifunctional codon (a DNA segment coding for both the V- and J-region polypeptides). The recognition signals (recombinant signal sequences [RSS]) are composed of a nine-nucleotide segment (nonamer) and a seven-nucleotide segment (heptamer) separated by 12 or 23 base pairs. d. The spacer of nucleotides in the intron region of the DNA allows the strand to loop around and bring the bases into proximity for optimal interaction. This is called the 1223 spacer because there are 12 nucleotides in one DNA segment and 23 in the other. e. The variable and joining gene segments are fused by the action of two recombinase enzymes. An endonuclease cleaves the DNA strand, splitting out the intron. The two gene segments are then united by DNA ligase.
C C Nonamer
Sp~'
12 bases (one turn)
A A A A A
T T T T T
C
G
A
T lIE
23 bases (two turns)
[
G
T Heptamer
G G
C A
G A
C T
C
G
Site of V/J recombinase action
G
/
A
c
T
INS
Figure VI-1-9. Recognition Signal interactions.
f. Once gene recombination has occurred, the DNA is transcribed into nuclear RNA and the intervening sequence between the bifunctional codon and the constant region gene is spliced out by an RNAse. g. The RNA then moves to the cytoplasm, where it can function as a messenger RNA molecule. h. A similar process is involved in synthesis of the heavy-chain mRNA with the exception that there are two recombinational events: A diversity gene combination with a joining gene precedes the joining of the variable gene with this bifunctional codon to form, in this case, a trifunctional codon.
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3. General features of class (isotype) switching and gene recombination a. Mature B lymphocytes expressing IgM and IgD may switch antibody classes after contact with antigen to produce IgA, IgG, or IgE. b. The immunoglobulin produced has the same variable region of light and heavy chain as IgM and IgD on the cell surface, except for occasional mutational changes in the V region genes (somatic hypermutation). c. The constant region genes are maintained, allowing the synthesis of the different iso-
types all with the same antigen specificity. After VDJ recombination, a B cell may receive signals through cytokines or other cascades that a different isotype of immunoglobulin may be more appropriate. The cells will class switch to anyone of the downstream constant regions (y, ex, or £ chain genes). 4. Allotypes are antigens on immunoglobulin molecules encoded at one genetic locus and inherited as alleles. Not all members of the species have the same particular allotype in their genome. a. Km allotypes are a derivation of K light chains in humans from one of three alleles at a single locus, km. b. Gm allotypes are a derivation of the human IgG heavy-chain isotypes Gl, G2, G3, and G4. c. a2m allotypes are a derivation of the human a2 heavy-chain isotype from one of two alleles at a single locus, a2m (1 or 2).
d. Allelic exclusion is a term used to describe the phenotypic expression of a single allele in cells containing two different alleles for that genetic locus (one from each parent). The synthesis of a functional gene product from a light-chain gene suppresses recombination on all other light-chain genes; the same is true for heavy chains. Therefore, recombination events create an active H -chain or L-chain gene on only one of the two chromosomes. F. Properties of immunoglobulin subclasses. As previously noted, there are five immunoglobulin subclasses: IgG, IgA, IgM, IgE, and IgD (remember them by"G-A-M-ED"). They can be distinguished by the type of heavy chain they possess and by their distinct biologic functions. 1. IgG provides the major defense against bacteria and toxins. There are four subclasses,
varying by heavy-chain isotype (Gl, G2, G3, G4).
In a Nutshell
a. The relative amounts of serum IgG are IgGl, 60-70%; IgG2, 14-20%; IgG3, 4-8%; IgG4,2-6%.
Major Functions of IgG
b. IgG (molecular weight of 150 kD) comprises 85% of the total immunoglobulin, with serum concentration 800-1,700 mg/dL. It has a half-life of approximately 21 days, except IgG3, which has a half-life of 7 days.
• Opsonization
c. IgG is important in the secondary immune response to antigen and provides long-
lasting immunity.
• Passive immunity in fetus
• Complement activation • ADCC
d. IgG is the only class of immunoglobulin that crosses the human placenta into fetal circulation; it is responsible for protecting the newborn during the first 4-6 months of life. e. Three subclasses of IgG fix complement (IgG3 > IgG 1 > IgG2). f. The Fc region binds to Fcy receptors on polymorphonuclear leukocytes, monocytes, protein A of staphylococcal strains, and some B lymphocytes.
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2. IgM is important in the primary immune response to antigen it is usually the first antibody detected in the serum after exposure to a specific antigen.
In a Nutshell Major Features of IgM • First antibody synthesized • Found in B-cell membrane • Pentamer with J chain in serum • Very efficient activator of complement
a. Monomers serve as cell-surface receptors on mature, naive B lymphocytes. (1) The cell-surface form contains a hydrophobic segment of 25 amino acids in the
carboxy terminal of the heavy chains that serves to anchor the immunoglobulin to the membrane. (2) The secreted form contains an alternative hydrophilic chain terminus. b. Circulating IgM is a pentamer of five immunoglobulin molecules (10 heavy 11 chains and 10 light chains) and one disulfide-linked J chain, with a total molecular weight of approximately 900,000 daltons. The cysteine-rich J chain is linked by a disulfide bond to the penultimate cysteine residues of two 11 chains from separate monomers. c. The half-life of IgM in serum averages 5 days. The serum concentration, 50-190
mgldL, represents 10% of immunoglobulin in plasma. d. IgM fixes complement very efficiently because each circulating molecule has 10 Fc sites. e. Agglutination of antigens via the pentameric structure of IgM allows the cross-linking of antigens. f. Isohemagglutinins, rheumatoid factors, and heterophile antibodies are all IgM. 3. IgA has an important barrier function on mucosal surfaces and functions in the secretory immune response. a. 19A represents 15% of the total serum immunoglobulin. The serum half-life of 19A is 6 days with a concentration of 140-420 mg/dL.
In a Nutshell
b. The serum form is usually monomeric, consisting of two heavy chains (aI, (2) and two light chains with a molecular weight of 170 kD.
Major Features of IgA
c. The secretory form (sIgA) is found in tears, colostrum, saliva, milk, and other secretions.
• Secretory immunity
(1) It is a dimer joined by a polypeptide J chain.
• In secretions found as a dimer with a J chain and secretory piece (slgA)
(2) sIgA contains a 70,000-dalton secretory component added to polymeric 19A by epithelial cells as it passes through to the luminal side. (3) The secretory component confers stability to the molecule, making it less susceptible to proteolysis in the gastrointestinal tract. (4) It is produced by plasma cells in the lamina propria of the gastrointestinal and respiratory tracts. d. Subclasses of 19A are based on two heavy chain isotypes.
In a Nutshell Major Features of IgO • Very low concentration in plasma • High levels in membrane of mature B cell • Functions in antigen recognition by B cell
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(1) 19A1, 90% of serum 19A, is susceptible to bacterial proteases.
(2) 19A2, 60% of secretory 19A, is resistant to proteases. 4. IgD functions as a cell surface antigen receptor in naive B lymphocytes. IgD is very susceptible to proteolysis. a. Its serum concentration is very low, ranging between 0.3-4.0 mgldL with a serum half-life of 3 days and a molecular weight of 170 kD. b. IgD functions primarily as a receptor for antigen on B cells and stimulates B-cell proliferation. There are no subclasses of IgD. It disappears after antigenic stimulation.
Basic Immunology
5. IgE, also called the "reaginic antibody:' is associated with allergic response and immediate hypersensitivity. a. IgE has a serum half-life of 2 days in plasma and a serum concentration of ObBb(P) = 0 convertase
C4b, is the classical pathway C3 convertase, also known as C4b2a. This component can cleave many molecules of C3; this step is, therefore, the site of amplification of the complement pathway. C4b2a binds to cell membranes covalently. d. C3 is a heat -stable glycoprotein with two polypeptide chains, alpha and beta. C3 is responsible for the distinction between self and non-self. "Self" surfaces limit C3 deposition, whereas "non-self" surfaces allow rapid C3 deposition. C3 is activated by the classical pathway, the alternative pathway, thrombin, plasmin, and tissue proteases. (1) C3a is an anaphylatoxin, causes mast cell degranulation, and mediates vascular leakage.
(2) C3b causes opsonization and immunologic adherence via specific cell-surface receptors for this component. C3b receptors are found on granulocytes, monocytes, lymphocytes, and erythrocytes. C3b combines with the classical pathway C3 convertase (C4b2a) to form the classical pathway CS convertase (C4b2a3b). 3. Alternative pathway. There are several key components of the alternative complement pathway. a. Factor B is a protein similar to C2. b. Factor D is an enzyme that cleaves factor B. (The active product is termed Bb.) c. Properdin is a group of proteins involved in stabilizing C3bBb of the alternative pathway. d. There are several initiators of the alternative pathway. (1) C3 spontaneously degrades at a low rate to form C3a and C3b. (2) Lipopolysaccharides, the cell wall components of gram-negative bacteria (3) Bacterial and plant polysaccharides (4) Cell-membrane constituents
• ObBb3b(P) = (S convertase
(5) Aggregated 19A, IgG, IgE, and IgM
Final common Pathway
(6) Cobra venom factor
(S convertase converts (S to (Sa and (sb (Sb combines with (6 and G; (sb67 complex inserts into plasma membrane (8 and (9 join the complex to form the final membrane attack complex
(7) Endotoxins that complex with factor B to form C3 convertase e. The alternative pathway starts by the binding of C3b to factor B. The binding of C3b makes Factor B susceptible to proteolysis by factor D, and a complex of C3b and Bb (the proteolytic product) is formed. Properdin then forms a stable complex with C3bBb. The alternative pathway then continues as does the classical pathway. 4. The membrane attack complex (MAC) is formed from the reactions among C5, C6, C7, C8, and C9. a. CS is a heat-stable protein that consists of two polypeptide chains, alpha and beta. The alpha chain is cleaved by C5 convertase, resulting in C5b and soluble C5a. C5a is an anaphylatoxin and chemotactic factor. It also causes mast cell degranulation and mediates vascular leakage. b. C6 and C7. C5b, C6, and C7 combine to form the CSb,6,7 complex. The C5b,6,7 complex is lipophilic and inserts itself into the hydrophobic plasma membrane. c. C8 and C9 are the terminal components in cell lysis. C8 combines with C5b,6,7 to form CSb,6,7,8 and becomes stably attached to the cell membrane. C9 then polymerizes in the membrane, leading to osmotic lysis of the cell.
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5. The products of the complement cascades have several biologic roles, including viral neutralization, lysis of infected cells, and direct pathogen lysis. Complement activation can also mediate immune adherence (the focusing of antigen on macrophage or lymphocyte surfaces) and promote phagocytosis. Finally, biologic responses such as anaphylaxis, kinin activity, smooth muscle contraction, vasodilatation, and chemotaxis are mediated by individual complement components.
Clinical Correlate Deficiencies in complement components predispose patients to certain diseases: G deficiency
->
Increased susceptibility to pyogenic infections
Q deficiency
->
Increased incidence of connective tissue disorders
B. Inflammation is a pathologic state initially characterized by pain, redness, heat, and swelling. These features of inflammation are caused by increased vascular permeability, leading to an infiltration of leukocytes (primarily neutrophils and macrophages, although small numbers of eosinophils and basophils can also be found). Lymphocytes can also enter sites of inflammation, where the release of cytokines can enhance the inflammatory response. A variety of compounds or chemicals can elicit inflammatory responses, and a variety of chemotactic factors are responsible for the migration of cells to sites of inflammation.
(5-8 deficiency ->
infections (meningococcal, gonococcal)
1. Vasoactive and smooth muscle constrictors a. Histamine (1) Histamine is stored as granules in mast cells, basophils, and platelets, with higher concentrations found in the intestine, lung, and skin. (2) Histamine is released from mast cells when antigen contacts IgE bound on the mast cell. It may also be released by nonimmunologic mechanisms, e.g., trauma or cold. (3) Histamine interacts with target cell receptors HI, H2, and H3. HI causes contraction of smooth muscle, increases vascular permeability, and elevates intracellular cyclic GMP. H2 increases gastric acid secretion, respiratory mucus production, and intracellular cyclic AMP. H3 is found in the central nervous system and functions in the negative feedback inhibition of histamine release and synthesis. (4) Histamine can be isolated from inflammatory sites in early inflammation, but its concentration dwindles within 1 hour. b. Arachidonic acid products. Arachidonic acid is derived from cell membrane phospholipids after conversion from linoleic acid. It is degraded via two pathways: the cyclooxygenase pathway and the lip oxygenase pathway. (1) The cyclo-oxygenase pathway converts arachidonic acid to prostaglandin G2 (PGG 2 ). PGG 2 is converted to PGH2 , which is ultimately converted to thromboxane ~ (T~), other more stable prostaglandins (PGF2 cx, PGE2 , PGD 2 ), and prostacyclin (CPGI 2 ).
Recurrent
Neisseria
esterase inhibitor deficiency
(1
->
Hereditary angioneurotic edema
In a Nutshell The inflammatory response can be triggered by local tissue damage that results in enzyme activation or by mast cell degranulation caused by anaphylatoxin interaction with specific receptors (e.g., OaR, C5aR) on the cytoplasmic membrane. Allergen reacting with cell-bound IgE can also trigger degranulation.
(2) The lipoxygenase pathway produces the leukotrienes, including LTC 4 , LTD 4 , and LTE 4 , which are collectively known as the slow-reacting substance of anaphylaxis. When administered by aerosol, these leukotrienes are more potent constrictors of human airway smooth muscle than is histamine. LTB4 is chemotactic for neutrophils, eosinophils, and macrophages. c. Platelet-activating factor (PAF) is derived from cell-membrane lipid and is synthesized by basophils, neutrophils, monocytes, and epithelium. (1) PAF activates platelets by initiating the release of platelet granule constituents,
causing platelets to clump. (2) PAF stimulates the synthesis of prostaglandins and leukotrienes. (3) PAF also increases the adhesiveness of neutrophils for endothelial cells.
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Immunology
d. Adenosine is an inflammatory agent derived from mast cells after ATP breakdown. It interacts with Al and A2 receptors on the cell membrane. The A2 receptor is associated with increased levels of intracellular cyclic AMP, an effect that is blocked by methylxanthine drugs.
Clinical Correlate 2. Chemotactic factors
Eosinophils are important cells of the innate immune system; they augment acquired immunity to metazoans. They contain basic proteins in their granules that are toxic to worms.
a. Eosinophil chemotactic factors (ECF). There are six major eosinophil chemotactic factors: histamine, ECF-A; eosinophil stimulation promoter; soluble immune complexes, C5a; and hydroxyeicosatetraenoic acid (HETE), a lipoxygenase pathway product. b. Neutrophil chemotactic factors. There are six neutrophil chemotactic factors. Their primary role is to attract neutrophils to sites of inflammation. (1) High-molecular-weight neutrophil chemotactic factor (HMW-NCF)
(2) LTB 4 , which is derived from arachidonic acid metabolism, via the lip oxygenase pathway (3) PAF (4) IL-l
(5) IL-8 (6) C5a Phospholipid
t
Phospholipase
Arachidonic Acid ~ 5-Lipoxygenase
Cyclo·oxygenase / ' Thromboxane A2 ~ Prostaglandin (PG) 12 ~ PGF . . PGE:
¥'
PGG 2
5-HPETE
... ...
...
GH P 2
LTA4
... PGD 2
12-Lipoxygenase
12-HPETE
t
-+ LTB4
~+:}
SRS-A
LTE4
12-HETE Eicosanoids are derived from arachidonic acid and mediate inflammation.
Figure VI-1-12. Eicosanoid formation pathways.
3. Enzyme mediators a. Neutral proteases are products of mast cells. (1) Tryptic protease has trypsin-like potency and is capable of cleaving C3 to form C3a. It also alters blood-clotting proteins. (2) Neutral protease has a specificity similar to that of chymotrypsin. One of its substrates may be angiotensinogen.
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b. Acid hydrolases are found in the lysosomes of many cell types. They degrade membrane components such as chondroitin sulfate. 4. ProteoglY3,000 IUjmL • ~ Antibody responses
· i
Respiratory infections, especially s. aureus
• Eosinophilia • Dermatitis • Growth retardation
B. Acquired immunodeficiency. Acquired immunodeficiency syndrome is the defining infec-
tious disease of our generation. 1. Causative agent. AIDS is caused by the human immunodeficiency virus (HIV; see Figure VI -2-1 for an illustration of the HIV replication cycle). a. HIV was first suspected as the etiologic agent of AIDS in 1981. In some communities in central African countries, the rate of infectivity is as high as 30-40% among the adult population. Two types of HIV, termed HIV-l and HIV-2, are responsible for most infections worldwide. b. HIV is a C-type retrovirus belonging to the Lentivirus family. Its genome encodes nine proteins, the most important of which are gag (encoding viral core proteins), pol (reverse transcriptase), and env (envelope proteins). c. The RNA core is surrounded by a lipid envelope that is derived from the host plasma membrane. The viral membrane contains the transmembrane protein gp160. This protein is commonly detected in Western blot analysis as two fragments-gp41, which promotes the fusion of viral envelope with the host cell membrane, and gp120, an adhesion molecule that binds to CD4 on Th cells. d. The core proteins include reverse transcriptase and two nonglycosylated proteins designated p18 and p24. 2. Risk groups for HIV infection include homosexual males, intravenous drug users, children born to infected mothers, sexual partners of people in high-risk groups, and recipients of infected blood products and secretions. The latter risk is now minimal, given the excellent methods of screening blood products for potential contamination.
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Adhesion
Il-=--'%--llr---
RNA genome
gag
HIV fusion and entry
A.
Viral assembly and budding
B.
D. CD4 T-cell activation induces HIV replication and assembly
c. Figure VI-2-1. HIV replication cycle.
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3. Mechanism of transmission. HIV is primarily spread by contact with infectious bodily fluids such as semen and blood. Sexual intercourse, either homosexual or heterosexual, is the principle route of transmission, although vertical transmission of virus from mother to newborn can also occur. The sharing of needles by intravenous drug users continues to be an important source of infection. 4. Mechanism of infection. AIDS is characterized by a profound loss of CD4+ T cells. The virus binds to the CD4 molecule on the T cell via gp120. The chemokine receptor CXCR4 is required as a coreceptor on the target cell. Some HIV also infects macrophages and monocytes and requires another chemokine coreceptor, termed CCRS. Once the virus enters into the cell, the RNA is reverse transcribed and integrated within the host DNA. Exposure of the T cell to activating stimuli such as cytokines or antigens results in activation of the virus by stimulating transcription of virally encoded genes. The long terminal repeat (LTR) sequences of the viral genome contain binding sites for a mammalian transcription factor. Once activated, the virus replicates within the cell. Extensive viral budding can lead to death of the T cell. In contrast to the T cell, the macrophage is more resistant to death from viral infection and appears to serve as an important viral reservoir. This cytolysis leads to profound immune suppression. A number of opportunistic infections, as noted below, overwhelm the patient's weakened immune response. 5. Cellular consequences of HIV infection a. T cells. A central consequence of infection with HIV is a loss of CD4+ Th cells. However, in addition to a loss of CD4+ T cells, there is also a decrease in the response of T cells to antigen, impaired production of cytokines and such as IL-2 and IFN-y, decreased intracellular signaling. The hypermetabolic wasting syndrome seen in HIVinfected individuals may be caused by increased levels of cytokines such as TNF-a (cachectin).
Note The loss of CD4+ cells leads to defects in cell-mediated immunity, with an increased incidence of infection due to fungi, viruses, and mycobacteria.
Clinical Correlate HIV patients can present with a wide range of problems across organ systems. If you encounter an HIV (or high-risk group) patient on the exam, you should know that there is a high incidence of the following disorders in HIV patients: • Oral thrush (Candida)
b. B cells. Although patients with early HIV infection appear to have polyclonal activation of B cells with circulating immune complexes in their plasma, they steadily lose the ability to mount an effective antibody response to new antigens.
• Esophagitis (Candida, CMV,
c. Macrophages. HIV can enter the macrophage through binding of gp120 to CD4 and a second membrane receptor, CCRS (a chemokine receptor). Both circulating monocytes and macrophages serve as a reservoir for the virus. Although more resistant to death from viral infection, infected macrophages have decreased IL-l secretion, as well as an impaired ability to present antigen to T cells.
- Bacterial (Sa/monella,
6. Natural history of HIV infection. The Centers for Disease Control (CDC) has proposed three clinical subgroups for AIDS-infected individuals: a. Category A. This group of individuals is characterized by an acute infection. After the initial infection, the patient may develop a syndrome resembling infectious mononucleosis, characterized by rash, sore throat, fever, or even aseptic meningitis. The illness typically resolves after 1-2 weeks, upon which seroconversion to HIV proteins arise. This group of individuals may also be characterized by asymptomatic infection. The disease becomes clinically latent and can remain so for 7-10 years. c. Category B. This group is characterized by persistent generalized lymphadenopathy together with fever, rash, and fatigue. AIDS-related complex (ARC) represents a nonspecific cluster of signs and symptoms of AIDS that is not accompanied by a decrease in CD4+ cells. A diagnosis of early ARC is made if the individual has one or two of the following symptoms: fatigue, fever, weight loss, persistent skin rash, oral hairy leukoplakia, herpes simplex, and oral thrush. Advanced ARC is determined if the individual has two or more of these symptoms.
HSV) • Diarrhea Shigella, Yersinia, Campy/obader, Mycobaderium aviumintracellu/are, C diffici/e) - Parasitic (Cryptosporidium, Isospora, Giardia, Entamoeba) - Viral (CMV colitis) - Fungal (Candida) • Intestinal neoplasms: lymphoma, Kaposi sarcoma • Pneumonias (especially Pneumocystis carinit) (Continued)
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Clinical Correlate (conrd) • Tuberculosis and atypical mycobacterial infection • Fungal respiratory disorders: histoplasmosis, coccidioidomycosis • Pulmonary neoplasms: Kaposi sarcoma, lymphoma • Hematologic problems: anemia, leukopenia, thrombocytopenia • Neurologic disorders - Cryptococcal meningitis - Toxoplasmosis - Progressive multifocal leukoencephalopathy - CMV encephalopathy - CMV retinitis - AIDS dementia - CNS lymphoma • Sexually transmitted diseases • Skin - Shingles - Kaposi sarcoma - Seborrheic dermatitis - Herpes simplex • Disseminated infections - Cytomegalovirus - M. avium-intracellulare
- Histoplasmosis • Papillomavirus-induced tumors - Carcinoma of the cervix, penis - Anal condylomas
d. Category C. This group is characterized by generalized disease, including neurologic opportunistic infections and secondary neoplasms. Ultimately, the disease progresses to the breakdown of the immune system; full-blown AIDS is characterized by the development of secondary tumors and numerous opportunistic infections. (1) Among the tumors common to AIDS is Kaposi sarcoma, which can be found in individuals even before breakdown of the immune system, caused by HSVS. (2) Non-Hodgkin lymphomas are increasingly common tumors that are found in severely immunocompromised patients. (3) HIV-infected individuals are also susceptible to opportunistic infection by protozoa (e.g., Cryptosporidia and Toxoplasma gondii) , fungi (e.g., Cryptococcosis, Candidiasis, and Pneumocystis carinii), bacterial (e.g., Mycobacterium aviumintracellulare and M. tuberculosis), and viruses (e.g., Cytomegalovirus [CMV], herpes simplex, and varicella-zoster). (4) Central nervous system (CNS) involvement is a common feature of AIDS (60%). It can result either from direct infection of the CNS by the virus or from opportunistic infections (e.g., toxoplasmosis). 7. Diagnosis a. Seroconversion against HIV proteins usually occurs within 2 months after infection or, in rare cases, later in the illness. Serum antibodies against surface envelope proteins gp41, gp120, and/or gp160 and at least one gag protein and one pol protein can be detected by ELISA and by Western blot. The latter assay is typically used to confirm seroconversion first identified by ELISA. b. Peripheral CD4 counts can additionally be used as a marker of disease progression and as a predictor of the risks for opportunistic infection. c. Polymerase chain reaction (PCR) can identify HIV RNA from blood, CSF, and tissues of infected individuals even before seroconversion. Additionally, PCR can differentiate HIV-l from HIV-2 infection. S. Prognosis and treatment a. Only a small fraction of patients (5%) develop overt clinical symptoms within the first 2 years of HIV infection. However, upon clinical expression of disease and opportunistic infections, the median survival is approximately 2 years. Treatment is directed at reducing the viral load and at treating the symptoms of opportunistic infection. The principal therapy of choice is nucleoside analogs that prevent reverse transcriptase activity of HIV and newly defined protease inhibitors. The most studied dideoxynucleoside analog is azidothymidine (AZT) , which is typically used in combination therapy with other analogs and with protease inhibitors, including indinavir, saquinavir, and ritonavir. Other commonly used nucleoside analogs are dideoxycytidine (ddC) , dideoxyinosine (ddI), didehydrodeoxythymidine (d4T), dideoxythiacytidine (3TC), and nevirapine. b. Treatment of opportunistic infection can take many directions depending on the presentation of individual patients. For example, trimethoprim-sulfamethoxazole (TMP-SMX) is now effective treatment for P. carinii pneumonia, as well as other opportunistic infections such as Listeria, Nocardia, and Toxoplasma. C. Phagocytic cell disorders are characterized by a heightened incidence and severity of
infections caused by bacteria, particularly pyogenic organisms such as staphylococci and streptococci. They may be classified into syndromes defective in the production and/or
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maturation of cells (production defects) or syndromes where cells are present but functionallyaberrent (functional defects). 1. Cyclic neutropenia is defined as a periodic neutropenia, usually occurring every 21 days, with corresponding intermittent bone marrow maturation arrest at the promyelocyte stage. Splenectomy may improve clinical symptoms, and GM-CSF will stimulate neutrophil production and reduce symptoms and infections. Patients are typically sick for 1 week with fever, malaise, ulceration of skin and mucous membranes, abdominal pain, sore throat, lymphadenitis, and arthritis. 2. Hereditary neutropenia is usually fatal by 1 year of age. It is characterized by the absence of peripheral blood neutrophils from birth and by the arrested maturation of bone marrow precursors. 3. Acquired neutropenia has several known causes, including: a. Hyposplenism (as seen in sickle cell disease) b. Neoplasms, particulary myelocytic leukemia and metastatic carcinoma c. Overwhelming infection from tuberculosis, typhoid fever, measles, infectious mononucleosis, and leishmaniasis d. Drug toxicity from many cancer chemotherapeutic agents, antibiotics, and diuretics e. Irradiation f. Hemodialysis and cardiopulmonary bypass patients 4. Opsonic defects are functional defects of phagocytic cells commonly caused by the lack of serum factors, primarily complement products. Opsonization refers to the clearance of organisms (or other target cells) via antibody or complement attaching to the target organism and by the engulfment of this complex after attachment to complement or Fc receptors. In newborns, opsonic defects can arise due to C3 deficiency or from IgM deficiency. Other complement "deficiencies" are primarily from a lack of receptors for C3b that normally reside on phagocytic cells. 5. Chemotactic defects refer to the impaired ability of cells to respond or move toward a chemical stimulus. As described previously, cells of the lymphoid system can normally migrate toward a variety of stimuli, such as cytokines and mediators of inflammation. Defects in chemotaxis can be due to serum inhibitors, possibly associated with alcoholic liver disease, recurrent staphylococcal infections, Hodgkin disease, or agammaglobulinemia. In addition, drugs (steroids, colchicine) or C5 defects can affect normal chemotaxis. Several syndromes are associated with impaired cellular chemotactic responses, including lazy leukocyte syndrome and leukocyte adhesion deficiency. a. Lazy leukocyte syndrome refers to inadequate chemotactic responses and reduced random mobility of leukocytes. Once again, bacteria are the major threat to these patients. b. Leukocyte adhesion deficiency is an impairment of leukocyte interaction caused by defective synthesis of the ~-chain component of the CD18 integrin (adhesion) molecule. The loss of the chain results in failure of diapedesis of neutrophils and impaired cell interactions among leukocytes. 6. Functional deficits of phagocytic cells included deficiencies in which the phagocytes are unable to kill ingested pathogens. a. Chediak-Higashi syndrome is an autosomal recessive disease in which neutrophils have decreased chemotactic response. Gigantic lysosomal granules form; the basic cell defect involves microtubule dysfunction. This disease may present as peripheral
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neuropathy, and patients are susceptible to viral and bacterial enteric infections. Partial albinism is also present. b. Chronic granulomatous disease is characterized by the inability of phagocytes to displaya typical respiratory burst during phagocytosis. The defect is in the NADPH oxidase system. Infection with catalase-positive microorganisms such as Staphylococcus au reus, Salmonella, Nocardia, Aspergillus, and Serratia are common. In the laboratory, the disease is marked by the failure of granulocytes to reduce nitroblue tetrazolium (NBT) dye to formazan. D. Complement deficiencies are caused by defects (either genetic or functional) in individual complement components.
In a Nutshell
1. C2 deficiency is the most common complement component deficiency. Collagen vascular disease is commonly observed in C2-deficient patients. Encapsulated bacterial septicemia and CNS infections are also found in these patients.
Immune Deficiency
Predominating Opportunist
Bcell
Pyogenic bacteria
Tcell
Viruses and fungi; also mycobacteria
3. C5 deficiency clinically presents with infection by gram-negative rods and Neisseria species. C5 dysfunction is known as Leiner syndrome (eczema, diarrhea, recurrent gramnegative sepsis).
C3
Pyogenic bacteria
4. Late complement component deficiencies (C5-C8) can result in disseminated Neisseria meningitidis or N. gonorrhoeae infections.
(5-(8
Neisseria
5. CI inhibitor deficiency results in hereditary angiodema (HAE).
Phagocytes
Pyogenic bacteria
Note These reactions are called "immediate" because symptoms of the allergy will be seen in a patient 10-20 minutes after exposure to the allergen. There must have been a previous antigenic encounter to induce the sensitivity. Always think of type I (atopic, IgE-mediated) hypersensitivity when the symptoms (rash, wheezing, cramps) occur rapidly after antigen injection/ingestion.
2. C3 deficiency causes recurrent infections with encapsulated bacteria. It may also be associated with cirrhosis, immune complex disease, and SLE.
HYPERSENSITIVITIES Hyperimmune reactions cause disease syndromes when the normal protective functions of immunity become imbalanced. Excessive immunoglobulin E (lgE) can lead to allergy; antigen-antibody (Ag-Ab) complexes can provoke arteritis, arthritis, and glomerulonephritis; and cytotoxic antibodies can destroy red blood cells (RBCs), white blood cells (WBCs), platelets, or, less frequently, other cells in the body. Inappropriate or undesirable cell-mediated immunity can lead to graft rejection or demyelinating disease. There are four distinct types of hypersensitivity reactions characterized by the time course required for the induction of the response, the immune cells and soluble factors involved, and the types of antigen involved. A. Type I hypersensitivity (or immediate, atopic, or anaphylactic hypersensitivity) requires an initial exposure to antigen in order to sensitize the person. Re-exposure to the same antigen causes cross-linking of IgE receptors on the surface of basophils and mast cells (via FCE receptors). The mast cells then release a variety of pharmacologic mediators. The systemic reactions occur within minutes of secondary exposure to the allergen. Smooth muscle contraction leads to constriction of bronchi and bronchioles, and vasodilation and increased vascular permeability result in peripheral edema. The significant cutaneous response is a wheal and flare reaction ("hives" or urticaria). The typical clinical syndromes associated with type I hypersensitivity include asthma, atopic dermatitis, eczema and allergic rhinitis (hay fever). 1. The important preformed mediators (and their primary biologic functions) released from basophils and mast cells upon the cross-linking of IgE are: a. Histamine (smooth muscle contraction and vasodilatation) b. Heparin (anticoagulant)
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Bridge to Respiratory System IgE-Fc receptor on mast cell surface
I
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