Allan I. Basbaum PhD

• Professor and Chair, Department of Anatomy and W.M. Keck Foundation Center for integrative Neuroscience, University of California, San Francisco

https://bms.ucsf.edu/faculty/allan-basbaum-phd

The ability to form the maximum number of hydrogen bonds homeopathic remedy for erectile dysfunction causes generic 20mg tadacip overnight delivery, supplemented by van der Waals interactions in the core of this tightly packed structure erectile dysfunction most effective treatment tadacip 20 mg with visa, provides the thermodynamic driving force for the formation of an helix impotence at 30 years old tadacip 20mg low cost. Since the peptide bond nitrogen of proline lacks a hydrogen atom to contribute to a hydrogen bond erectile dysfunction medication reviews purchase tadacip 20mg, proline can only be stably accommodated within C C N C N C C N C C N C C 0 erectile dysfunction zyprexa purchase 20 mg tadacip mastercard. These amphipathic helices are well adapted to the formation of interfaces between polar and nonpolar regions such as the hydrophobic interior of a protein and its aqueous environment erectile dysfunction vitamin shoppe tadacip 20mg generic. The van der Waals radii of the atoms are larger than shown here; hence, there is almost no free space inside the helix. The second (hence "beta") recognizable regular secondary structure in proteins is the sheet. The amino acid residues of a sheet, when viewed edge-on, form a zigzag or pleated pattern in which the R groups of adjacent residues point in opposite directions. Unlike the compact backbone of the helix, the peptide backbone of the sheet is highly extended. Hydrogen bonds are indicated by dotted lines with the participating -nitrogen atoms (hydrogen donors) and oxygen atoms (hydrogen acceptors) shown in blue and red, respectively. Top: the enzyme triose phosphate isomerase complexed with the substrate analog 2-phosphoglycerate (red). Note the elegant and symmetrical arrangement of alternating sheets (light blue) and helices (green), with the sheets forming a -barrel core surrounded by the helices. The color of the polypeptide chain is graded along the visible spectrum from purple (N-terminal) to tan (C-terminal). Notice how the concave shape of the domain forms a binding pocket for the pentasaccharide, the lack of sheet, and the high proportion of loops and bends. Clusters of twisted strands of sheet form the core of many globular proteins (Figure 5­6). Loops & Bends Roughly half of the residues in a "typical" globular protein reside in helices and sheets and half in loops, turns, bends, and other extended conformational features. Loops are regions that contain residues beyond the minimum number necessary to connect adjacent regions of secondary structure. For many enzymes, the loops that bridge domains responsible for binding substrates often contain aminoacyl residues that participate in catalysis. Structural motifs such as the helix-loophelix motif that are intermediate between secondary and tertiary structures are often termed supersecondary structures. Proteins may contain "disordered" regions, often at the extreme amino or carboxyl terminal, characterized by high conformational flexibility. In many instances, these disordered regions assume an ordered conformation upon binding of a ligand. Tertiary & Quaternary Structure the term "tertiary structure" refers to the entire three-dimensional conformation of a polypeptide. It indicates, in threedimensional space, how secondary structural features-helices, sheets, bends, turns, and loops-assemble to form domains and how these domains relate spatially to one another. A domain is a section of protein structure sufficient to perform a particular chemical or physical task such as binding of a substrate or other ligand. Most domains are modular in nature, contiguous in both primary sequence and three-dimensional space (Figure 5­8). Simple proteins, particularly those that interact with a single substrate, such as lysozyme or triose phosphate isomerase (Figure 5­6) and the oxygen storage protein myoglobin (Chapter 6), often consist of a single domain. Examples include alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, quinone oxidoreductase, 6-phosphogluconate dehydrogenase, D-glycerate dehydrogenase, formate dehydrogenase, and 3, 20-hydroxysteroid dehydrogenase. Hydrophobic membrane domains anchor proteins to membranes or enable them to span membranes. Localization sequences target proteins to specific subcellular or extracellular locations such as the nucleus, mitochondria, secretory vesicles, etc. Regulatory domains trigger changes in protein function in response to the binding of allosteric effectors or covalent modifications (Chapter 9). Combining domain modules provides a facile route for generating proteins of great structural complexity and functional sophistication (Figure 5­9). Proteins containing multiple domains also can be assembled through the association of multiple polypeptides, or protomers. Quaternary structure defines the polypeptide composition of a protein and, for an oligomeric protein, the spatial relationships between its protomers or subunits. Ribbon diagrams (Figures 5­6 & 5­8) trace the conformation of the polypeptide backbone, with cylinders and arrows indicating regions of helix and sheet, respectively. In an even simpler representation, line segments that link the carbons indicate the path of the polypeptide backbone. The color of the polypeptide chain is graded along the visible spectrum from blue (N-terminal) to orange (C-terminal). Similarly, the C-terminal portion forms a contiguous domain responsible for binding pyruvate. Similarly, the C-terminal portion forms a contiguous, helix-rich domain responsible for binding the peptide substrate. Principal among these are hydrophobic interactions that drive most hydrophobic amino acid side chains into the interior of the pro- tein, shielding them from water. Other significant contributors include hydrogen bonds and salt bridges between the carboxylates of aspartic and glutamic acid and the oppositely charged side chains of protonated lysyl, argininyl, and histidyl residues. The kinase and bisphosphatase activities of 6-phosphofructo-2-kinase/fructose2,6-bisphosphatase are catalyzed by the N- and C-terminal proximate catalytic domains, respectively. Some proteins contain covalent disulfide (S-S) bonds that link the sulfhydryl groups of cysteinyl residues. Formation of disulfide bonds involves oxidation of the cysteinyl sulfhydryl groups and requires oxygen. For solution of its structure by x-ray crystallography, a protein is first precipitated under conditions that form large, well-ordered crystals. To establish appropriate conditions, crystallization trials use a few microliters of protein solution and a matrix of variables (temperature, pH, presence of salts or organic solutes such as polyethylene glycol) to establish optimal conditions for crystal formation. Crystals mounted in quartz capillaries are first irradiated with monochromatic xrays of approximate wavelength 0. Protein crystals may then be frozen in liquid nitrogen for subsequent collection of a high-resolution data set. The patterns formed by the x-rays that are diffracted by the atoms in their path are recorded on a photographic plate or its computer equivalent as a circular pattern of spots of varying intensity. The data inherent in these spots are then analyzed using a mathematical approach termed a Fourier synthesis, which summates wave functions. The wave amplitudes are related to spot intensity, but since the waves are not in phase, the relationship between their phases must next be determined. The traditional approach to solution of the "phase problem" employs isomorphous displacement. Prior to irradiation, an atom with a distinctive x-ray "signature" is introduced into a crystal at known positions in the primary structure of the protein. Heavy atom isomorphous displacement generally uses mercury or uranium, which bind to cysteine residues. An alternative approach uses the expression of plasmid-encoded recombinant proteins in which selenium replaces the sulfur of methionine. Alternatively, if the unknown structure is similar to one that has already been solved, molecular replacement on an existing model provides an attractive way to phase the data without the use of heavy atoms. Finally, the results from the phasing and Fourier summations provide an electron density profile or three-dimensional map of how the atoms are connected or related to one another. Laue X-Ray Crystallography the ability of some crystallized enzymes to catalyze chemical reactions strongly suggests that structures determined by crystallography are indeed representative of the structures present in free solution. Classic crystallography provides, however, an essentially static picture of a protein that may undergo significant structural changes such as those that accompany enzymic catalysis. The time-consuming process of rotating the crystal in the x-ray beam is avoided, which permits the use of extremely short exposure times. Detection of the motions of residues or domains of an enzyme during catalysis uses crystals that contain an inactive or "caged" substrate analog. An intense flash of visible light cleaves the caged precursor to release free substrate and initiate catalysis in a precisely controlled manner. Using this approach, data can be collected over time periods as short as a few nanoseconds. When the three-dimensional structure is known, molecular dynamics programs can be used to simulate the conformational dynamics of a protein and the manner in which factors such as temperature, pH, ionic strength, or amino acid substitutions influence these motions. Molecular docking programs simulate the interactions that take place when a protein encounters a substrate, inhibitor, or other ligand. Virtual screening for molecules likely to interact with key sites on a protein of biomedical interest is extensively used to facilitate the discovery of new drugs. Secondary structure algorithms weigh the propensity of specific residues to become incorporated into helices or sheets in previously-studied proteins to predict the secondary structure of other polypeptides. In homology modeling, the known three-dimensional structure of a protein is used as a template upon which to erect a model of the probable structure of a related protein. Scientists are working to devise computer programs that will reliably predict the three-dimensional conformation of a protein directly from its primary sequence, thereby permitting the structures of the many unknown proteins for which templates are currently lacking to be determined. Folding into the native state does not involve a haphazard search of all possible structures. Native contacts are favored, and regions of native structure persist even in the denatured state. Discussed below are factors that facilitate folding and refolding, and the current concepts and proposed mechanisms based on more than 40 years of largely in vitro experimentation. Not only does this obviate the need to form crystals (a particular advantage when dealing with difficult to crystallize membrane proteins), it renders real-time observation of the changes in conformation that accompany ligand binding or catalysis possible. It also offers the possibility of perhaps one day being able to observe the structure and dynamics of proteins (and metabolites) within living cells. The Native Conformation of a Protein Is Thermodynamically Favored the number of distinct combinations of phi and psi angles specifying potential conformations of even a relatively small- 15-kDa-polypeptide is unbelievably vast. Clearly, protein folding in cells takes place in a more orderly and guided fashion. In the first stage, as the newly synthesized polypeptide emerges from the ribosome, short segments fold into secondary structural units that provide local regions of organized structure. In the second stage, the hydrophobic regions segregate into the interior of the protein away from solvent, forming a "molten globule," a partially folded polypeptide in which the modules of secondary structure rearrange until the mature conformation of the protein is attained. Considerable flexibility exists in the ways and in the order in which elements of secondary structure can be rearranged. In general, each element of secondary or super-secondary structure facilitates proper folding by directing the folding process toward the native conformation and away from unproductive alternatives. Proline-cis, trans-Isomerase All X-Pro peptide bonds-where X represents any residue- are synthesized in the trans configuration. Isomerization from trans to cis is catalyzed by the enzyme proline-cis, trans-isomerase (Figure 5­10). Auxiliary Proteins Assist Folding Under appropriate laboratory conditions, many proteins will spontaneously refold after being denatured (ie, unfolded) by treatment with acid or base, chaotropic agents, or detergents. Cells employ auxiliary proteins to speed the process of folding and to guide it toward a productive conclusion. Folding Is a Dynamic Process Proteins are conformationally dynamic molecules that can fold and unfold hundreds or thousands of times in their lifetime. First, unfolding rarely leads to the complete randomization of the polypeptide chain inside the cell. Unfolded proteins generally retain a number of contacts and regions of secondary structure that facilitate the refolding process. Second, chaperone proteins can "rescue" unfolded proteins that have become thermodynamically trapped in a misfolded dead end by unfolding hydrophobic regions and providing a second chance to fold productively. Glutathione can reduce inappropriate disulfide bonds that may be formed upon exposure to oxidizing agents such as O2, hydrogen peroxide, or superoxide (Chapter 52). The central cavity of the donut-shaped hsp60 chaperone provides a sheltered environment in which a polypeptide can fold until all hydrophobic regions are buried in its interior, eliminating aggregation. They include Creutzfeldt­Jakob disease in Protein Disulfide Isomerase Disulfide bonds between and within polypeptides stabilize tertiary and quaternary structure. Prion diseases may manifest themselves as infectious, genetic, or sporadic disorders. Because no viral or bacterial gene encoding the pathologic prion protein could be identified, the source and mechanism of transmission of prion disease long remained elusive. Today it is recognized that prion diseases are protein conformation diseases transmitted by altering the conformation, and hence the physical properties, of proteins endogenous to the host. Human prion-related protein, PrP, a glycoprotein encoded on the short arm of chromosome 20, normally is monomeric and rich in helix. Pathologic prion proteins serve as the templates for the conformational transformation of normal PrP, known as PrPc, into PrPsc. PrPsc is rich in sheet with many hydrophobic aminoacyl side chains exposed to solvent. As each new PrPsc molecule is formed, it triggers the production of yet more pathologic variants in a conformational chain reaction. Because PrPsc molecules associate strongly with one other through their exposed hydrophobic regions, the accumulating PrPsc units coalesce to form insoluble protease-resistant aggregates. Many polypeptides are initially synthesized as larger precursors called proproteins. The "extra" polypeptide segments in these proproteins often serve as leader sequences that target a polypeptide to a particular organelle or facilitate its passage through a membrane. Other segments ensure that the potentially harmful activity of a protein such as the proteases trypsin and chymotrypsin remains inhibited until these proteins reach their final destination. However, once these transient requirements are fulfilled, the now superfluous peptide regions are removed by selective proteolysis.

Fatty acids that occur in natural fats usually contain an even number of carbon atoms erectile dysfunction medications online buy cheap tadacip 20mg on-line. Phospholipids: Lipids containing erectile dysfunction doctor in hyderabad buy 20 mg tadacip fast delivery, in addition to fatty acids and an alcohol erectile dysfunction at 55 discount tadacip 20 mg overnight delivery, a phosphoric acid residue impotence exercises for men buy tadacip 20mg fast delivery. They frequently have nitrogen containing bases and other substituents cheap erectile dysfunction pills online uk buy 20mg tadacip fast delivery, eg for erectile dysfunction which doctor to consult discount tadacip 20mg without prescription, in glycerophospholipids the alcohol is glycerol and in sphingophospholipids the alcohol is sphingosine. Fatty Acids Are Named after Corresponding Hydrocarbons the most frequently used systematic nomenclature names the fatty acid after the hydrocarbon with the same number and arrangement of carbon atoms, with -oic being substituted for the final -e (Genevan system). Various conventions use for indicating the number and position of the double bonds (Figure 15­1); eg, 9 indicates a double bond between carbons 9 and 10 of the fatty acid; 9 indicates a double bond on the ninth carbon counting from the -carbon. In animals, additional double bonds are introduced only between the existing double bond (eg, 9, 6, or 3) and the carboxyl carbon, leading to three series of fatty acids known as the 9, 6, and 3 families, respectively. A few branched-chain fatty acids have also been isolated from both plant and animal sources. Unsaturated Fatty Acids Contain One or More Double Bonds Unsaturated fatty acids (Table 15­2) may be further subdivided as follows: 1. Polyunsaturated (polyethenoid, polyenoic) acids, containing two or more double bonds. They are synthesized in vivo by cyclization of the center of the carbon chain of 20-carbon (eicosanoic) polyunsaturated fatty acids (eg, arachidonic acid) to form a cyclopentane ring (Figure 15­2). A related series of compounds, the thromboxanes, have the cyclopentane ring interrupted with an oxygen atom (oxane ring) (Figure 15­3). The leukotrienes and lipoxins (Figure 15­4) are a third group of eicosanoid derivatives formed via the lipoxygenase pathway (Figure 23­11). Leukotrienes cause bronchoconstriction as well as being potent proinflammatory agents, and play a part in asthma. Most Naturally Occurring Unsaturated Fatty Acids Have cis Double Bonds the carbon chains of saturated fatty acids form a zigzag pattern when extended at low temperatures. A type of geometric isomerism occurs in unsaturated fatty acids, depending on the orientation of atoms or groups around the axes of double bonds, which do not allow rotation. If the acyl chains are on the same side of the bond, it is cis-, as in oleic acid; if on opposite sides, it is trans-, as in elaidic acid, the trans isomer of oleic acid (Figure 15­5). Double bonds in naturally occurring unsaturated long-chain fatty acids are nearly all in the cis configuration, the molecules being "bent" 120 degrees at the double bond. This has profound significance for molecular packing in cell membranes and on the positions occupied by fatty acids in more complex molecules such as phospholipids. Trans fatty acids are present in certain foods, arising as a by-product of the saturation of fatty acids during hydrogenation, or "hardening," of natural oils in the manufacture of margarine. An additional small contribution comes from the ingestion of ruminant fat that contains trans fatty acids arising from the action of microorganisms in the rumen. Consumption of trans fatty acids is now known to be detrimental to health and is associated with increased risk of diseases including cardiovascular disease and diabetes mellitus. This has led to improved technology to produce soft margarine low in trans fatty acids or containing none at all. An end product of carbohydrate fermentation by rumen organisms1 Spermaceti, cinnamon, palm kernel, coconut oils, laurels, butter Nutmeg, palm kernel, coconut oils, myrtles, butter Common in all animal and plant fats Also formed in the cecum of herbivores and to a lesser extent in the colon of humans. Possibly the most common fatty acid in natural fats; particularly high in olive oil. Dienoic acids (two double bonds) 18:2;9,12 6 Linoleic all-cis-9,12-Octadecadienoic Corn, peanut, cottonseed, soy bean, and many plant oils. Trienoic acids (three double bonds) 18:3;6,9,12 18:3;9,12,15 6 3 -Linolenic -Linolenic all-cis-6,9,12-Octadecatrienoic all-cis-9,12,15-Octadecatrienoic Some plants, eg, oil of evening primrose, borage oil; minor fatty acid in animals. Tetraenoic acids (four double bonds) 20:4;5,8,11,14 6 Arachidonic all-cis-5,8,11,14-Eicosatetraenoic Found in animal fats; important component of phospholipids in animals. Pentaenoic acids (five double bonds) 20:5;5,8,11,14,17 3 Timnodonic all-cis-5,8,11,14,17-Eicosapentaenoic Important component of fish oils, eg, cod liver, mackerel, menhaden, salmon oils. Hexaenoic acids (six double bonds) 22:6;4,7,10,13,16,19 3 Cervonic all-cis-4,7,10,13,16,19-Docosahexaenoic Fish oils, phospholipids in brain. A triacylglycerol containing three saturated fatty acids of 12 carbons or more is solid at body temperature, whereas if the fatty acid residues are 18:2, it is liquid to below 0°C. In practice, natural acylglycerols contain a mixture of fatty acids tailored to suit their functional roles. The membrane lipids, which must be fluid at all environmental temperatures, are more unsaturated than storage lipids. Lipids in tissues that are subject to cooling, eg, in hibernators or in the extremities of animals, are more unsaturated. Mono- and diacylglycerols, wherein one or two fatty acids are esterified with glycerol, are also found in the tissues. These are of particular significance in the synthesis and hydrolysis of triacylglycerols. Dipalmitoyl lecithin is a very effective surface-active agent and a major constituent of the surfactant preventing adherence, due to surface tension, of the inner surfaces of the lungs. Most phospholipids have a saturated acyl radical in the sn-1 position but an unsaturated radical in the sn-2 position of glycerol. Phosphatidylethanolamine (cephalin) and phosphatidylserine (found in most tissues) are also found in cell membranes and differ from phosphatidylcholine only in that ethanolamine or serine, respectively, replaces choline (Figure 15­8). Carbons 1 & 3 of Glycerol Are Not Identical To number the carbon atoms of glycerol unambiguously, the -sn (stereochemical numbering) system is used. It is important to realize that carbons 1 and 3 of glycerol are not identical when viewed in three dimensions (shown as a projection formula in Figure 15­7). Enzymes readily distinguish between them and are nearly always specific for one or the other carbon; eg, glycerol is always phosphorylated on sn-3 by glycerol kinase to give glycerol 3-phosphate and not glycerol 1-phosphate. Phosphatidic acid is important Phosphatidylinositol Is a Precursor of Second Messengers the inositol is present in phosphatidylinositol as the stereoisomer, myoinositol (Figure 15­8). Phosphatidylinositol 4,5-bisphosphate is an important constituent of cell membrane phospholipids; upon stimulation by a suitable hormone agonist, it is cleaved into diacylglycerol and inositol trisphosphate, both of which act as internal signals or second messengers. It is also found in oxidized lipoproteins and has been implicated in some of their effects in promoting atherosclerosis. On hydrolysis, the sphingomyelins yield a fatty acid, phosphoric acid, choline, and a complex amino alcohol, sphingosine (Figure 15­11). Cardiolipin Is a Major Lipid of Mitochondrial Membranes Phosphatidic acid is a precursor of phosphatidylglycerol which, in turn, gives rise to cardiolipin (Figure 15­8). This phospholipid is found only in mitochondria and is essential for mitochondrial function. Decreased cardiolipin levels or alterations in its structure or metabolism cause mitochondrial dysfunction in aging and in pathological conditions including heart failure, hypothyroidism and Barth syndrome (cardioskeletal myopathy). Galactosylceramide is a major glycosphingolipid of brain and other nervous tissue, found in relatively low amounts elsewhere. Galactosylceramide (Figure 15­12) can be converted to sulfogalactosylceramide (sulfatide), present in high amounts in myelin. Glucosylceramide is the predominant simple glycosphingolipid of extraneural tissues, also occurring in the brain in small amounts. Neuraminic acid (NeuAc; see Chapter 14) is the principal sialic acid found in human tissues. In the shorthand nomenclature used, G represents ganglioside; M is a monosialo-containing species; and the subscript 3 is a number assigned on the basis of chromatographic migration. However, biochemically it is also of significance because it is the precursor of a large number of equally important steroids that include the bile acids, adrenocortical hormones, sex hormones, D vitamins, cardiac glycosides, sitosterols of the plant kingdom, and some alkaloids. All steroids have a similar cyclic nucleus resembling phenanthrene (rings A, B, and C) to which a cyclopentane ring (D) is attached. The carbon positions on the steroid nucleus are numbered as shown in Figure 15­14. If the compound has one or more hydroxyl groups and no carbonyl or carboxyl groups, it is a sterol, and the name terminates in -ol. Because of Asymmetry in the Steroid Molecule, Many Stereoisomers Are Possible Each of the six-carbon rings of the steroid nucleus is capable of existing in the three-dimensional conformation either of a "chair" or a "boat" (Figure 15­15). In naturally occurring steroids, virtually all the rings are in the "chair" form, which is the more stable conformation. The junction between the A and B rings can be cis or trans in naturally occurring steroids. The A ring of a 5 steroid is always trans to the B ring, whereas it is cis in a 5 steroid. A H 10 9 13 Cholesterol Is a Significant Constituent of Many Tissues Cholesterol (Figure 15­17) is widely distributed in all cells of the body but particularly in nervous tissue. It is often found as cholesteryl ester, where the hydroxyl group on position 3 is esterified with a long-chain fatty acid. Ergosterol Is a Precursor of Vitamin D Ergosterol occurs in plants and yeast and is important as a precursor of vitamin D (Figure 15­18). When irradiated with ultraviolet light, ring B is opened to form vitamin D2 in a process similar to that which forms vitamin D3 from 7-dehydrocholesterol in the skin (Figure 44­3). Lipid peroxidation is a chain reaction providing a continuous supply of free radicals that initiate further peroxidation and thus has potentially devastating effects. They include ubiquinone (Chapter 13), which participates in the respiratory chain in mitochondria, and the long-chain alcohol dolichol (Figure 15­20), which takes part in glycoprotein synthesis by transferring carbohydrate residues to asparagine residues of the polypeptide (Chapter 47). Plant-derived isoprenoid compounds include rubber, camphor, the fat-soluble vitamins A, D, E, and K, and -carotene (provitamin A). Antioxidants fall into two classes: (1) preventive antioxidants, which reduce the rate of chain initiation and (2) chainbreaking antioxidants, which interfere with chain propagation. The reaction is initiated by an existing free radical (X·), by light, or by metal ions. Malondialdehyde is only formed by fatty acids with three or more double bonds and is used as a measure of lipid peroxidation together with ethane from the terminal two carbons of 3 fatty acids and pentane from the terminal five carbons of 6 fatty acids. Peroxidation is also catalyzed in vivo by heme compounds and by lipoxygenases found in platelets and leukocytes. Other products of auto-oxidation or enzymic oxidation of physiologic significance include oxysterols (formed from cholesterol) and isoprostanes (formed from the peroxidation of polyunsaturated fatty acids such as arachidonic acid). However, fatty acids, phospholipids, sphingolipids, bile salts, and, to a lesser extent, cholesterol contain polar groups. Therefore, part of the molecule is hydrophobic, or water-insoluble; and part is hydrophilic, or water-soluble. They become oriented at oil:water interfaces with the polar group in the water phase and the nonpolar group in the oil phase. A bilayer of such amphipathic lipids is the basic structure in biologic membranes (Chapter 40). Liposomes are of potential clinical use-particularly when combined with tissue-specific antibodies-as carriers of drugs in the circulation, targeted to specific organs, eg, in cancer therapy. In addition, they are used for gene transfer into vascular cells and as carriers for topical and transdermal delivery of drugs and cosmetics. Emulsions are much larger particles, formed usually by nonpolar lipids in an aqueous medium. These are stabilized by emulsifying agents such as amphipathic lipids (eg, lecithin), which form a surface layer separating the main bulk of the nonpolar material from the aqueous phase (Figure 15­22). Amphipathic lipids also contain one or more polar groups, making them suitable as constituents of membranes at lipidwater interfaces. The lipids of major physiologic significance are fatty acids and their esters, together with cholesterol and other steroids. Long-chain fatty acids may be saturated, monounsaturated, or polyunsaturated, according to the number of double bonds present. Eicosanoids are formed from 20-carbon polyunsaturated fatty acids and make up an important group of physiologically and pharmacologically active compounds known as prostaglandins, thromboxanes, leukotrienes, and lipoxins. The esters of glycerol are quantitatively the most significant lipids, represented by triacylglycerol ("fat"), a major constituent of lipoproteins and the storage form of lipid in adipose tissue. Phosphoacylglycerols are amphipathic lipids and have important roles-as major constituents of membranes and the outer layer of lipoproteins, as surfactant in the lung, as precursors of second messengers, and as constituents of nervous tissue. Peroxidation of lipids containing polyunsaturated fatty acids leads to generation of free radicals that damage tissues and cause disease. Metabolic pathways fall into three categories: (1) Anabolic pathways, which are those involved in the synthesis of larger and more complex compounds from smaller precursors-eg, the synthesis of protein from amino acids and the synthesis of reserves of triacylglycerol and glycogen. Knowledge of normal metabolism is essential for an understanding of abnormalities underlying disease. Normal metabolism includes adaptation to periods of starvation, exercise, pregnancy, and lactation. Abnormal metabolism may result from nutritional deficiency, enzyme deficiency, abnormal secretion of hormones, or the actions of drugs and toxins. Larger animals require less, and smaller animals more, per kg body weight, and growing children and animals have a proportionally higher requirement to allow for the energy cost of growth. For human beings this requirement is met from carbohydrates (40­60%), lipids (mainly triacylglycerol, 30­40%), and protein (10­15%), as well as alcohol. The mix of carbohydrate, lipid, and protein being oxidized varies, depending on whether the subject is in the fed or fasting state, and on the duration and intensity of physical work. The requirement for metabolic fuels is relatively constant throughout the day, since average physical activity increases metabolic rate only by about 40­50% over the basal metabolic rate. However, most people consume their daily intake of metabolic fuels in two or three meals, so there is a need to form reserves of carbohydrate (glycogen in liver and muscle) and lipid (triacylglycerol in adipose tissue) in the period following 16 C H A P T E R a meal, for use during the intervening time when there is no intake of food. If the intake of metabolic fuels is consistently greater than energy expenditure, the surplus is stored, largely as triacylglycerol in adipose tissue, leading to the development of obesity and its associated health hazards. By contrast, if the intake of metabolic fuels is consistently lower than energy expenditure, there are negligible reserves of fat and carbohydrate, and amino acids arising from protein turnover are used for energy-yielding metabolism rather than replacement protein synthesis, leading to emaciation, wasting, and, eventually, death (see Chapter 43). In the fed state, after a meal, there is an ample supply of carbohydrate, and the metabolic fuel for most tissues is glucose. In the fasting state glucose must be spared for use by the central nervous system (which is largely dependent on glucose) and the red blood cells (which are wholly reliant on glucose).

Several metabolic defects result in vitamin B6-responsive or -unresponsive homocystinurias diabetic erectile dysfunction icd 9 code cheap tadacip 20mg without prescription. Defective carrier-mediated transport of cystine results in cystinosis (cystine storage disease) with deposition of cystine crystals in tissues and early mortality from acute renal failure causes of erectile dysfunction in 40 year old buy tadacip 20 mg with amex. Interconversion of serine and glycine catalyzed by serine hydroxymethyltransferase impotence quoad hoc purchase tadacip 20 mg with mastercard. Catabolism of L-cysteine via the cysteine sulfinate pathway (top) and by the 3-mercaptopyruvate pathway (bottom) impotence kegel exercises discount tadacip 20mg. Oxidation of acetaldehyde to acetate is followed by formation of acetyl-CoA (Figure 30­10) erectile dysfunction ed treatment order 20mg tadacip free shipping. Catabolism of 4-hydroxy-L-proline forms impotence brochures quality 20 mg tadacip, successively, L-1-pyrroline-3-hydroxy-5-carboxylate, -hydroxy-L-glutamate-semialdehyde, erythro-hydroxy-L-glutamate, and -keto-hydroxyglutarate. Since ascorbate is the reductant for conversion of p-hydroxyphenylpyruvate to homogentisate, scorbutic patients excrete incompletely oxidized products of tyrosine catabolism. Subsequent catabolism forms maleylacetoacetate, fumarylacetoacetate, fumarate, acetoacetate, and ultimately acetyl-CoA. The probable metabolic defect in type I tyrosinemia (tyrosinosis) is at fumarylacetoacetate hydrolase (reaction 4, Figure 30­12). Late in the disease, there is arthritis and connective tissue pigmentation (ochronosis) due to oxidation of homogentisate to benzoquinone acetate, which polymerizes and binds to connective tissue. Conversion of the tryptophan metabolite 3-hydroxykynurenine to 3-hydroxyanthranilate is impaired (see Figure 30­15). False-positives in premature infants may reflect delayed maturation of enzymes of phenylalanine catabolism. Lysine first forms a Schiff base with -ketoglutarate, which is reduced to saccharopine. In one form of periodic hyperlysinemia, elevated lysine competitively inhibits liver arginase (see Figure 29­9), causing hyperammonemia. Restricting dietary lysine relieves the ammonemia, whereas ingestion of a lysine load precipitates severe crises and coma. In a different periodic hyperlysinemia, lysine catabolites accumulate, but even a lysine load does not trigger hyperammonemia. In addition to impaired synthesis of saccharopine, some patients cannot cleave saccharopine. Tryptophan is degraded to amphibolic intermediates via the kynurenine-anthranilate pathway (Figure 30­15). Tryptophan oxygenase (tryptophan pyrrolase) opens the indole ring, incorporates molecular oxygen, and forms N-formylkynurenine. Since kynureninase requires pyridoxal phosphate, excretion of xanthurenate (Figure 30­16) in response to a tryptophan load is diagnostic of vitamin B6 deficiency. Hartnup disease reflects impaired intestinal and renal transport of tryptophan and other neutral amino acids. The defect limits tryptophan availability for niacin biosynthesis and accounts for the pellagralike signs and symptoms. Subsequent reactions form propionyl-CoA (Figure 30­18) and ultimately succinyl-CoA (see Figure 19­2). This multimeric enzyme complex of a decarboxylase, a transacylase, and a dihydrolipoyl dehydrogenase closely resembles pyruvate dehydrogenase (see Figure 17­5). Its regulation also parallels that of pyruvate dehydrogenase, being inactivated by phosphorylation and reactivated by dephosphorylation (see Figure 17­6). In isovaleric acidemia, ingestion of protein-rich foods elevates isovalerate, the deacylation product of isovalerylCoA. Figures 30­20, 30­21, and 30­22 illustrate the subsequent reactions unique to each amino acid skeleton. The biochemical defect involves the -keto acid decarboxylase complex (reaction 2, Figure 30­19). Plasma and urinary levels of leucine, isoleucine, valine, -keto acids, and -hydroxy acids (reduced -keto acids) are elevated. Prompt replacement of dietary protein by an amino acid mixture that lacks leucine, isoleucine, and valine averts brain damage and early mortality. Mutation of the dihydrolipoate reductase component impairs decarboxylation of branched-chain keto acids, of pyruvate, and of -ketoglutarate. The impaired enzyme in isovaleric acidemia is isovaleryl-CoA dehydrogenase (reaction 3, Figure 30­19). The analogous first three reactions in the catabolism of leucine, valine, and isoleucine. Subsequent reactions remove any additional nitrogen and restructure the hydrocarbon skeleton for conversion to oxaloacetate, -ketoglutarate, pyruvate, and acetyl-CoA. Metabolic disorders of cysteine catabolism include cystine-lysinuria, cystine storage disease, and the homocystinurias. Metabolic diseases of lysine catabolism include periodic and persistent forms of hyperlysinemiaammonemia. Metabolic disorders of branched-chain amino acid catabolism include hypervalinemia, maple syrup urine disease, intermittent branched-chain ketonuria, isovaleric acidemia, and methylmalonic aciduria. Subsequent catabolism of the methacrylyl-CoA formed from L-valine (see Figure 30­19). In addition, many proteins contain amino acids that have been modified for a specific function such as binding calcium or as intermediates that serve to stabilize proteins-generally structural proteins-by subsequent covalent cross-linking. The amino acid residues in those proteins serve as precursors for these modified residues. Small peptides or peptide-like molecules not synthesized on ribosomes fulfill specific functions in cells. Neurotransmitters derived from amino acids include -aminobutyrate, 5-hydroxytryptamine (serotonin), dopamine, norepinephrine, and epinephrine. Many drugs used to treat neurologic and psychiatric conditions affect the metabolism of these neurotransmitters. Biosynthesis of carnosine is catalyzed by carnosine synthetase in a two-stage reaction that involves initial formation of an enzyme-bound acyl-adenylate of -alanine and subsequent transfer of the -alanyl moiety to L-histidine. Many drugs, drug metabolites, and other compounds with carboxyl groups are excreted in the urine as glycine conjugates. Glycine is incorporated into creatine (see Figure 31­6), the nitrogen and -carbon of glycine are incorporated into the pyrrole rings and the methylene bridge carbons of heme (Chapter 32), and the entire glycine molecule becomes atoms 4, 5, and 7 of purines (Figure 34­1). Homocarnosine (Figure 31­2), present in human brain at higher levels than carnosine, is synthesized in brain tissue by carnosine synthetase. Phosphorylated Serine, Threonine, & Tyrosine the phosphorylation and dephosphorylation of seryl, threonyl, and tyrosyl residues regulate the activity of certain enzymes of lipid and carbohydrate metabolism and the properties of proteins that participate in signal transduction cascades. Mammalian tissues form -alanine from cytosine (Figure 34­9), carnosine, and anserine (Figure 31­2). Body fluid and tissue levels of -alanine, taurine, and 264 Methionine S-Adenosylmethionine, the principal source of methyl groups in the body, also contributes its carbon skeleton for the biosynthesis of the 3-diaminopropane portions of the polyamines spermine and spermidine (Figure 31­4). Homocarnosine Histidine Decarboxylation of histidine to histamine is catalyzed by a broad-specificity aromatic L-amino acid decarboxylase that also catalyzes the decarboxylation of dopa, 5-hydroxytryptophan, phenylalanine, tyrosine, and tryptophan. Histidine compounds present in the human body include ergothioneine, carnosine, and dietary anserine (Figure 31­2). Polyamines Ornithine & Arginine Arginine is the formamidine donor for creatine synthesis (Figure 31­6) and via ornithine to putrescine, spermine, and spermidine (Figure 31­3) Arginine is also the precursor of the intercellular signaling molecule niThe polyamines spermidine and spermine (Figure 31­4) function in cell proliferation and growth, are growth factors for cultured mammalian cells, and stabilize intact cells, subcellular organelles, and membranes. Arginine phosphate of invertebrate muscle functions as a phosphagen analogous to creatine phosphate of mammalian muscle (see Figure 31­6). Tryptophan Following hydroxylation of tryptophan to 5-hydroxytryptophan by liver tyrosine hydroxylase, subsequent decarboxylation forms serotonin (5-hydroxytrypta- mine), a potent vasoconstrictor and stimulator of smooth muscle contraction. Catabolism of serotonin is initiated by monoamine oxidase-catalyzed oxidative deamination to 5-hydroxyindoleacetate. Spermidine formed from putrescine (decarboxylated L-ornithine) by transfer of a propylamine moiety from decarboxylated S-adenosylmethionine accepts a second propylamine moiety to form spermidine. Serotonin and 5-methoxytryptamine are metabolized to the corresponding acids by monoamine oxidase. N-Acetylation of serotonin, followed by O-methylation in the pineal body, forms melatonin. The principal normal urinary catabolites of tryptophan are 5-hydroxyindoleacetate and indole 3-acetate. Tyrosine Neural cells convert tyrosine to epinephrine and norepinephrine (Figure 31­5). In the adrenal medulla, phenylethanolamine-N-methyltransferase utilizes S-adenosylmethionine to methylate the primary amine of norepinephrine, forming epinephrine (Figure 31­5). Synthesis of creatine is completed by methylation of guanidoacetate by S-adenosylmethionine (Figure 31­6). It is formed by decarboxylation of L-glutamate, a reaction catalyzed by L-glutamate decarboxylase (Figure 31­7). Conversion of tyrosine to epinephrine and norepinephrine in neuronal and adrenal cells. The two starting materials are succinyl-CoA, derived from the citric acid cycle in mitochondria, and the amino acid glycine. These four molecules condense in a head-to-tail manner to form a linear tetrapyrrole, hy270 Natural Porphyrins Have Substituent Side Chains on the Porphin Nucleus the porphyrins found in nature are compounds in which various side chains are substituted for the eight hydrogen atoms numbered in the porphin nucleus shown in Figure 32­1. Note that both of these uroporphyrinogens have the pyrrole rings connected by methylene bridges Table 32­1. The reaction is catalyzed by uroporphyrinogen decarboxylase, which is also capable of converting uroporphyrinogen I to coproporphyrinogen I (Figure 32­7). The mitochondrial enzyme coproporphyrinogen oxidase catalyzes the decarboxylation and oxidation of two propionic side chains to form protoporphyrinogen. Formation of Heme Involves Incorporation of Iron Into Protoporphyrin the final step in heme synthesis involves the incorporation of ferrous iron into protoporphyrin in a reaction catalyzed by ferrochelatase (heme synthase), another mitochondrial enzyme (Figure 32­4). Both erythroid and nonerythroid ("housekeeping") forms of the first four enzymes are found. Heme biosynthesis occurs in most mammalian cells with the exception of mature erythrocytes, which do not contain mitochondria. However, 1 the functions of the above proteins are described in various chapters of this text. This repression-derepression mechanism is depicted diagrammatically in Figure 32­9. Heme also affects translation of the enzyme and its transfer from the cytosol to the mitochondrion. Most of these drugs are metabolized by a system in the liver that utilizes a specific hemoprotein, cytochrome P450 (see Chapter 53). During their metabolism, the utilization of heme by cytochrome P450 is greatly increased, which in turn diminishes the intracellular heme concentration. The importance of some of these regulatory mechanisms is further discussed below when the porphyrias are described. This band is termed the Soret band after its discoverer, the French physicist Charles Soret. The double bonds joining the pyrrole rings in the porphyrins are responsible for the characteristic absorption and fluorescence of these compounds; these double bonds are absent in the porphyrinogens. An example is the absorption curve for a solution of porphyrin in 5% hydrochloric acid (Figure 32­10). Also, the photosensitivity (favoring nocturnal activities) and severe disfigurement exhibited by some victims of congenital erythropoietic porphyria have led to the suggestion that these individuals may have been the prototypes of so-called werewolves. Assay of the activity of one or more of these enzymes using an appropriate source (eg, red blood cells) is thus important in making a definitive diagnosis in a suspected case of porphyria. Thus, the use of appropriate gene probes has made possible the prenatal diagnosis of some of the porphyrias. As is true of most inborn errors, the signs and symptoms of porphyria result from either a deficiency of metabolic products beyond the enzymatic block or from an accumulation of metabolites behind the block. These compounds, when present in urine or feces, can be separated from each other by extraction with appropriate solvent mixtures. Enzyme 3 is also called porphobilinogen deaminase or hydroxymethylbilane synthase. Spontaneous oxidation of porphyrinogens to porphyrins Photosensitivity Figure 32­11. Patients exhibiting photosensitivity may benefit from administration of -carotene; this compound appears to lessen production of free radicals, thus diminishing photosensitivity. Damaged lysosomes release their degradative enzymes, causing variable degrees of skin damage, including scarring. These are generally organs or cells in which synthesis of heme is particularly active. The bone marrow synthesizes considerable hemoglobin, and the liver is active in the synthesis of another hemoprotein, cytochrome P450. Thus, one classification of the porphyrias is to designate them as predominantly either erythropoietic or hepatic; the types of porphyrias that fall into these two classes are so characterized in Table 32­2. Porphyrias can also be classified as acute or cutaneous on the basis of their clinical features. Thus, taking drugs that cause induction of cytochrome P450 (so-called microsomal inducers) can precipitate attacks of porphyria. The major findings in the six principal types of porphyria are listed in Table 32­2. When hemoglobin is destroyed in the body, globin is degraded to its constituent amino acids, which are reused, and the iron of heme enters the iron pool, also for reuse.

Schematic representation of the amplification of chorion protein genes s36 and s38 impotence treatment reviews cheap 20 mg tadacip with visa. This results in a different carboxyl terminal region of the encoded proteins such that the µm protein remains attached to the membrane of the B lymphocyte and the µs immunoglobulin is secreted erectile dysfunction medicine in dubai cheap 20mg tadacip with amex. It is not clear how these processing-splicing decisions are made or whether these steps can be regulated erectile dysfunction vitamins purchase tadacip 20mg mastercard. Lemon B erectile dysfunction unani medicine order tadacip 20 mg on line, Tjian R: Orchestrated response: a symphony of transcription factors for gene control erectile dysfunction doctor toronto quality 20 mg tadacip. Much has been learned about human genetic disease from pedigree analysis and study of affected proteins impotence under 30 tadacip 20mg for sale, but in many cases where the specific genetic defect is unknown, these approaches cannot be used. Understanding this technology is important for several reasons: (1) It offers a rational approach to understanding the molecular basis of a number of diseases (eg, familial hypercholesterolemia, sickle cell disease, the thalassemias, cystic fibrosis, muscular dystrophy). These bases are attached to the C-1 position of the sugar deoxyribose, and the bases are linked together through joining of the sugar moieties at their 3 and 5 positions via a phosphodiester bond (Figure 35­1). The alternating deoxyribose and phosphate groups form the backbone of the double helix (Figure 35­2). Indeed, the degree of base-pair matching (or mismatching) can be estimated from the temperature re396 * See glossary of terms at the end of this chapter. If an average gene length is 3 Ч 103 bp (3 kilobases [kb]), the genome could consist of 106 genes, assuming that there is no overlap and that transcription proceeds in only one direction. The exact function of the remaining ~98% of the human genome has not yet been defined. This condensation may serve a regulatory role and certainly has a practical purpose. Occasionally, such sequences are found within the gene itself or in the region that flanks the 3 end of the gene. Many eukaryotic genes (and some viruses that replicate in mammalian cells) have special regions, called enhancers, that increase the rate of transcription. This is a rigidly controlled process involving a number of complex steps, each of which no doubt is regulated by one or more enzymes or factors; faulty function at any of these steps can cause disease. The variation in size and complexity of some human genes is illustrated in Table 40­1. These enzymes were called restriction enzymes because their presence in a given bacterium restricted the growth of certain bacterial viruses called bacteriophages. Organization of a eukaryotic transcription unit and the pathway of eukaryotic gene expression. The structural region is bounded at its 5 end by the transcription initiation site and at its 3 end by the polyadenylate addition or termination site. The primary transcript has a special structure, a cap, at the 5 end and a stretch of As at the 3 end. The sizes of the genes include some proximal promoter and regulatory region sequences; these are generally about the same size for all genes. The -adrenergic receptor gene is intronless, and the thyroglobulin gene has 36 introns. The fragments produced can be isolated by electrophoresis on agarose or polyacrylamide gels (see the discussion of blot transfer, below); this is an essential step in cloning and a major use of these enzymes. Sticky ends of fragments can also anneal, so that tandem heterogeneous inserts form. To circumvent these problems, an enzyme that generates blunt ends is used, and new ends are added using the enzyme terminal transferase. By convention, these are written in the 5 or 3 direction for the upper strand of each recognition sequence, and the lower strand is shown with the opposite (ie, 3 or 5) polarity. This technique, though less efficient than sticky-end ligation, has the advantage of joining together any pairs of ends. The disadvantages are that there is no control over the orientation of insertion or the number of molecules annealed together, and there is no easy way to retrieve the insert. Plasmids have several properties that make them extremely useful as cloning vectors. These vectors are specially constructed to contain very active inducible promoters, proper in-phase translation initiation codons, both transcription and translation termination signals, and appropriate protein processing signals, if needed. Some expression vectors even contain genes that code for protease inhibitors, so that the final yield of product is enhanced. Thus, the parental plasmid, which provides resistance to both antibiotics, can be readily separated from the chimeric plasmid, which is resistant only to tetracycline. For this reason, various mammalian viral vectors are being investigated for use in gene therapy experiments. Importantly, neither modification (32P or fluorescent-label) affects the hybridization properties of the resulting labeled nucleic acid probes. A Library Is a Collection of Recombinant Clones the combination of restriction enzymes and various cloning vectors allows the entire genome of an organism to be packed into a vector. Hence, the chimeric plasmid will no longer survive when plated on a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin can therefore be used to distinguish clones of plasmid that contain an insert. Bacteria are grown on colonies on an agar plate and overlaid with nitrocellulose filter paper. A radioactive probe is added to the filter, and (after washing) the hybrid complex is localized by exposing the filter to x-ray film. By matching the spot on the autoradiograph to a colony, the latter can be picked from the plate. Gene families, in which there is some degree of homology, can be detected by varying the stringency of the hybridization and washing steps. Hybridization conditions capable of detecting just a single base pair mismatch between probe and target have been devised. This requirement can be satisfied by cloning the fragment of interest, using the techniques described above. By having a radioactive label incorporated at the end opposite the termination site, one can separate the fragments according to size using polyacrylamide gel electrophoresis. An autoradiograph is made, and each of the fragments produces an image (band) on an x-ray film. Most commonly employed is an automated procedure in which four different fluorescent labels-one representing each nucleotide-are used. Each emits a specific signal upon excitation by a laser beam, and this can be recorded by a computer. In the protein, or Western, blot, proteins are electrophoresed and transferred to nitrocellulose and then probed with a specific antibody or other probe molecule. Each synthetic cycle takes but a few minutes, so an entire molecule can be made by synthesizing relatively short segments that can then be ligated to one another. Knowing which specific dideoxynucleotide reaction was conducted to produce each mixture of fragments, one can determine the sequence of nucleotides from the labeled end (asterisk) toward the unlabeled end by reading up the gel. Automated sequencing involves the reading of chemically modified deoxynucleotides. The exact area of hybridization is localized by layering photographic emulsion over the slide and, after exposure, lining up the grains with some histologic identification of the chromosome. Some of the human genes localized using these techniques are listed in Table 40­5. These bind two distinct primers that are directed at specific sequences on opposite strands and that define the segment to be amplified. Antibodies directed against this peptide can be used to assess whether this peptide is expressed in normal persons and whether it is absent in those with the genetic syndrome. An example of the latter is the attempt to engineer plants that are more resistant to drought or temperature extremes, more efficient at fixing nitrogen, or that produce seeds containing the complete complement of essential amino acids (rice, wheat, corn, etc). This may still be true, but if on reading the initial sections of this chapter one predicted that genetic disease could result from derangement of any of the steps illustrated in Figure 40­1, one would have made a proper assessment. This gene is located in a cluster on chromosome 11 (Figure 40­8), and an expanded version of the gene is illustrated in Figure 40­9. Deletions (solid bar) of the locus cause -thalassemia (deficiency or absence [0] of -globin). Each type of thalassemia tends to be found in a certain group of people, eg, the (A)0 deletion inversion occurs in persons from India. Schematic representation of the -globin gene cluster and of the lesions in some ge- different lesions in and around the -globin gene (Table 40­6). The altered codon specifies a different amino acid (valine rather than glutamic acid), and this causes a structural abnormality of the -globin molecule. Other point mutations in and around the -globin gene result in decreased production or, in some instances, no produc- 5 I1 I2 3 Figure 40­9. Reading from the 5 to 3 direction, the shaded areas are exons 1­3 and the clear spaces are introns 1 (I1) and 2 (I2). Again, a molecular analysis of -thalassemia produces numerous examples of these processes-particularly deletions-as causes of disease (Figure 40­8). Pedigree analysis has been applied to a number of genetic diseases and is most useful in those caused by deletions and insertions or the rarer instances in which a restriction endonuclease cleavage site is affected, as in the example cited in this paragraph. This approach allows for prenatal diagnosis of sickle cell disease (dash-sided square). This is proving useful in the human genome sequencing project and is an important component of the effort to understand various single-gene and multigenic diseases. The direction of extension is determined by restriction mapping, and the procedure is repeated sequentially until the desired sequence is obtained. The X chromosome-linked disorders are particularly amenable to this approach, since only a single allele is expressed. The strategy is to clone a gene (eg, the gene that codes for adenosine deaminase) into a vector that will readily be taken up and incorporated into the genome of a host cell. This procedure is repeated until fragment 4 hybridizes with fragment 5, which contains the entire sequence of gene X. Complementing this highthroughput, transcript-profiling method is the recent development of high-sensitivity, high-throughput mass spectrometry of complex protein samples. Newer mass spectrometry methods allow one to identify hundreds to thousands of proteins in proteins extracted from very small numbers of cells (< 1 g). Microarray techniques and mass spectrometric protein identification experiments both lead to the generation of huge amounts of data. Future work at the intersection of bioinformatics and transcript-protein profiling will revolutionize our understanding of biology and medicine. The transgenic approach has recently been used to correct a genetic deficiency in mice. This gene was expressed and regulated normally in the hypothalamus of a certain number of the resultant mice, and these animals were in all respects normal. Targeted Gene Disruption or Knockout In transgenic animals, one is adding one or more copies of a gene to the genome, and there is no way to control where that gene eventually resides. A complementary- and much more difficult-approach involves the selective removal of a gene from the genome. Gene knockout animals (usually mice) are made by creating a mutation that totally disrupts the function of a gene. This is then used to replace one of the two genes in an embryonic stem cell that can be used to create a heterozygous transgenic animal. Several hundred strains of mice with knockouts of specific genes have been developed. Clone: A large number of organisms, cells or molecules that are identical with a single parental organism cell or molecule. Intron: the sequence of a gene that is transcribed but excised before translation. Microsatellite repeat sequences: Dispersed or group repeat sequences of 2­5 bp repeated up to 50 times. Western blot: A method for transferring protein to a nitrocellulose filter, on which the protein can be detected by a suitable probe (eg, an antibody). Hybridization with a complementary radioactive polynucleotide (eg, by Southern or Northern blotting) is commonly used to generate the signal. Plasma membranes form closed compartments around cellular protoplasm to separate one cell from another and thus permit cellular individuality. The selective permeabilities are provided mainly by channels and pumps for ions and substrates. The plasma membrane also exchanges material with the extracellular environment by exocytosis and endocytosis, and there are special areas of membrane structure-the gap junctions- through which adjacent cells exchange material. In addition, the plasma membrane plays key roles in cellcell interactions and in transmembrane signaling. Changes in membrane structure (eg caused by ischemia) can affect water balance and ion flux and therefore every process within the cell. Specific deficiencies or alterations of certain membrane components lead to a variety of diseases (see Table 41­5). It brings to the cells nutrients (eg, glucose, fatty acids, amino acids), oxygen, various ions and trace minerals, and a variety of regulatory molecules (hormones) that coordinate the functions of widely separated cells. Extracellular fluid is characterized by high Na+ and Ca2+ content, and Cl- is the major anion. Vast changes would have been required for evolution of a completely new set of biochemical and physiologic machinery; instead, as it happened, cells developed barriers-membranes with associated "pumps"-to maintain the internal microenvironment. Plasma membrane Mouse liver cells Retinal rods (bovine) Human erythrocyte Ameba 0.

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