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80 THE LIFE SCIENCES virus, SV40, and seven different human adenoviruses, continue to syn- thesize viral-specific mRNA, as demonstrated by the formation of DNA- RNA hybrids when their pooled mRNA is mixed with pure viral DNA. A surprisingly large amount of the total mRNA in the polyribosomes of acenov~rus-transtormea cells Is v~ral-specific, 2 to 5 percent, suggesting that a small amount of viral DNA present in the tumor cell is preferentially transcribed while most of the host RNA goes unexpressed. This selective transcription during viral carcinogenesis may represent only a specialized case of the more general phenomenon occurring during cell differentiation, or it may occur by an entirely different mechanism. It seems likely that these viral mRNA molecules are translated into proteins, some of which account for the altered growth and antigenic properties of the tumor cell. Host-cell DNA synthesis is inhibited during the cycle of infection with most DNA viruses. However, infection of nondividing cells with polyoma or SV40 virus induces the synthesis of host-cell DNA, a phenomenon thought to be of importance in viral transformation. The oncogenic RNA viruses, including the avian and murine leukemia and sarcoma viruses, are capable both of transforming cells and of replicat- ing within the same cell. Particularly intriguing are those viruses that are defective in the genes for synthesis of viral coat protein; they transform cells without the production of infectious virus. Others are defective only within certain host cells. Co-infection of tumor cells induced by such a defective virus in conjunction with a second virus ("helper virus") is re- quired for the synthesis of infectious virus; newly synthesized virus then contains the genome of the transforming virus and the coat protein of the helper virus. FORM AND FUNCTION For the isolated cell, structural form is correlated with its simplest needs- to remain alive in the face of adversity, to grow, and to reproduce by fission. To accomplish these simple ends, cells possess a variety of sub- structures, each specialized for the performance of a specific chemical task. Multicellular organisms man himself-are made possible by the collective structures, and the functions they permit, of organized groups of cells, organs that serve the entire organism much as cellular organelles serve the cell. One need consider only the brain, the gastrointestinal tract, the cardiovascular system, the kidney, the musculoskeletal system, and the genitalia to recognize the extent of this subdivision of labor. The goal of practitioners of the anatomical sciences and of physiologists has been to

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FRONTIERS OF BIOLOGY obtain detailed understanding of these correlations of form and function- to understand how nerves conduct, muscles contract, kidneys regulate the composition of the extracellular fluid, the gastrointestinal tract degrades foodstuffs into metabolizable nutrients; how joints permit motion; how gases are taken into the body or removed; and how the organism is inte- grated into a harmonious whole through the operation of the nervous and endocrine systems. The last two decades have witnessed an immense expansion of research in these areas, which has been made possible by new awareness of the nature of the problems, by new experimental tools such as the electron and phase-contrast microscopes, fast multichannel recorders, microspectrophotometers, the hying-spot microscope, x-ray analysis, and radioisotopes and techniques for their detection and measurement, as well as by the entire armamentarium of the biochemist. In no area of the life sciences is the melding of such classical disciplines as anatomy, physiology, pharmacology, anthropology, and biochemistry so clearly evident. Review here of the multitude of accomplishments at this level of biological con- sideration is impossible; accordingly, a few examples will be cited only as illustrations of current approaches to a few classical problems. Muscular Contraction For decades, muscular contraction has been an attractive object of study. Although it is easily amenable to experimental approach, no useful working concept of the fundamental mechanism was developed until two quite independent approaches revealed complementary information that, com- bined, led to a highly satisfying model of the nature of this process. As shown in Figure 22, phase-contrast and electron microscopy revealed the presence in muscle of two quite distinct types of fibers: In skeletal muscle, filaments of about 200 ~ are each surrounded by six filaments about 100 ~ in diameter. When muscle is stretched, the two sets of fila- ments pull away from each other, and when muscle is contracted, they telescope into each other. The individual filaments do not themselves shorten but appear to interact with each other by a "clawing" action that pulls the filaments past each other. The thick filaments are constructed of the protein, myosin; each molecule of myosin (mol. wt. 480,000) is an elongated multiple-stranded helix, about 1,800 ~ by about 20 A, with a "knob," oriented at right angles to the fiber axis at one end. The knob portion has the properties of an enzyme capable of catalyzing hydrolysis of ATP. The thin filaments are formed of a second protein, actin, the fundamental unit of which is a simple globular molecule (mol. wt. 60,000), which polymerizes into a filament analogous to a string of beads 81

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82 THE LIFE SCIENCES Skeletal Muscle Muscle Fasciculus -- " , , ~ -,,# Am_ _ ~ ,_ ~ Hi_ ,,' Z Sarcomere ; I"` Myofibril "` G-Actin Molecu le 11 1 11 1 ,' 'I I I / 1 / I / I ! / ! O~Booo ~ ~ K ~~ ooo~ooo~ooo F-Actin Filament __~ L Myosin Fi lament \ \ Myosin Molecule \ H ~ I ~ -~-~-. v - ~ ~Li 9 htHeavy Meromyosi n Meromyosin ~\ \ \ ~\ \ \ FIGURE 22 Structure and function of skeletal muscle. F. G. H. and I are cross sections at the levels indicated. (From W. Bloom and D. W. Fawcett, A Textbook of Histology, 9th ea., 1968. Copyright (I) 1968 W. B. Saunders Company. Drawing by Sylvia Collard Keene.)

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FRONTIERS OF BIOLOGY with two such strands wrapped around each other in a double helix. In solution, purified actin and myosin react rapidly with each other to form a tightly bonded complex, bond formation occurring between the knobs of the myosin and some aspect of the actin molecule. Addition of ATP permits transient separation of the actin-myosin complex, which is re- formed as the myosin hydrolyzes the ATP. Presumably, some equivalent interaction is responsible for the ratchet-like action of actin and myosin in contracting or relaxing muscle, although the details of this process remain obscure. How then does an intact muscle contract? The process begins with the arrival of a nerve impulse, itself the consequence of a rapid change in the surface properties of the nerve such that sodium ions leave and potassium ions enter. At the muscle-nerve junction a similar wave of excitation commences and rapidly sweeps over the muscle surface. This wave is essen- tially of similar character- a change in the distribution of sodium and potassium ions near the muscle cell surface. A few milliseconds later the entire cell begins to contract. In a brilliant series of experiments, microelectrodes with tips less than a micron in diameter were touched to the muscle surface, applying shocks too small to cause a general excitation. Local contraction responses were obtained, but only at particular spots on the cell surface. Electron micros- copy revealed these activating spots to be narrow indentations of the surface membrane, which penetrate deep into the cell. These structures, "transverse tubules," carry the change in surface charge into the cell in proximity with the contractile elements- the actin and myosin filaments. Contraction itself therefore occurs in consequence of a change in the local ionic environment of these proteins. To some degree, it is the change in sodium and potassium concentration that is meaningful, but more im- portantly, these changes act to release calcium ions from some bound form, and it is the increase in calcium ions, specifically, that stimulates the ATP- hydrolyzing activity of the myosin and makes contraction possible. Unless the ionic changes were reversed, the cell would continue to remain con- tracted until all available ATP had been utilized. However, in close rela- tion to the transverse tubules is a network of extremely tiny tubes, the "sarcoplasmic reticulum," a system that, utilizing the energy of ATP, sequesters the calcium inside the tubules, thereby bringing contraction to a halt and permitting the muscle to relax until the next wave of excitation arrives. Numerous details of this fundamental life process remain to be unraveled. But, in the main, the totality is a satisfying concept, consistent with all known evidence, and represents the ultimate convergence of physiological, biochemical, and anatomical studies. 83

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84 THE LIFE SCIENCES THE CONSTANCY OF THE Milieu Interieur The free and independent life of vertebrate animals became possible when their ancestral forms first developed closed circulations, which assured that all cells of the body, no matter how remote from the external environ- ment, had a nutrient supply, removal of waste products, and an environ- mental composition compatible with life. In turn, such an arrangement renders imperative some device for assuring the constancy of that internal environment, despite the vicissitudes of life. On some days, one may drink little or nothing; on others, rather prodigious quantities. Salt intake may vary equally widely. Some diets are effectively alkaline; some are acid. Changes in the rate of respiration may affect not only the oxygen supply but also the rate of removal of carbon dioxide and hence the acidity of the blood. That we survive and scarcely notice such variation is a tribute to a remarkable organ, the kidney. Until relatively recently, thoughts concerning renal function rested on speculations based largely on the appearance of the anatomical structure of the kidney. More than a century ago it was recognized that the funda- mental operating unit of the kidney is the "nephron," of which millions are arrayed, in parallel, in the kidney cortex (Figure 23~. Each commences with a small arteriole that is branched rapidly from the main renal artery and becomes a tuft of capillaries (the glomerulus) that coalesce in a venule. Surrounding the tuft of capillaries is a structure made of connective tissue that leads into a miniature tubule. The latter goes straight down toward the renal medulla, makes a hairpin turn, returns toward the outer surface, and then descends again into a thicker channel, the collecting duct, which drains into the hilum of the kidney. Particularly noteworthy is the fact that the blood in the venule formed by coalescence of the glomerular capillaries also surrounds the ascending tubule and the collecting duct before entering into the larger veins for exit from the kidney. From the appearance of this structure it was deduced that the glomerulus must be a filter through which passes an "ultrafiltrate" containing all the plasma constituents except the relatively large plasma proteins. It was further assumed that, as the glomer- ular filtrate traverses the tubules, materials are removed from it, ultimately leaving the presumptive urine. Slowly, over the first two thirds of this century, evidence accumulated suggesting the essential validity of this concept. Indirect techniques that permitted measurement of the magnitude of these operations were devised. In a 160-lb man, the overall rate of glomerular filtration is about 125 milliliters per minute, or about 180 liters per day, a volume 65 times that of the entire volume of plasma. Almost all (99.5 percent) the water and

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FRONTIERS OF BIOLOGY 85 Art . it. - Glomerulus Proximal ~_ Proximal Distal Thin Segment ~ Collecting Duct FIGURE 23 The nephron fundamental unit of the kidney. (Adapted from H. W. Smith, The Kidney, Oxford University Press, New York, 1935.) its solutes are reabsorbed as this filtrate passes along the tubules, for urine output is only about one liter per day. The experimental challenge has been to ascertain the mechanisms by which this versatile organ so alters its behavior as to assure excretion of very dilute urine when water intake has been copious or extremely con- centrated urine when water intake has been meager or salt intake excessive, to excrete alkaline or acid urine as may be appropriate to physiological circumstance, and to assure that none of the material in the glomerular filtrate that is valuable to the body, e.g., glucose, is lost. Although indirect evidence in these regards was accumulated over a long period, the detailed picture now available has been the consequence of recent development of techniques for micropuncture, originally used in frogs and mud puppies

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86 THE LIFE SCIENCES but since extended to mammals, permitting direct sampling from precise locales within the minute renal tubules under a variety of conditions. Ten years were then expended in perfecting adequate microanalytical techniques for the examination of these tiny specimens (0.01-0.1 r11. Once the tech- niques were developed, thousands of such determinations were undertaken, and from them has emerged a detailed blueprint of the modus operandi of this organ. A few aspects of this operation warrant summary. First it was established that the pressure in the glomerular capillaries is unusually high (about 60 mm Hg) and thus sufficient to ensure filtration through the capillaries. As the fluid passes through the descending proximal tubule, a large fraction of the total is removed; with it most of the desirable organic compounds, e.g., glucose, are removed by processes of active trans- port similar to those operating in most living cells. Only about 10 percent of fluid remains at the level of the hairpin turn. The fluid in this region is decidedly more concentrated than earlier in the proximal tubule, largely due to removal of water by simple osmotic forces because of the high concentration of salt in the surrounding region. As the fluid rises in the ascending limb, specific facultative adjustments are made in the sodium, potassium, and hydrogen content. In large measure, sodium ions removed in this region are exchanged either for hydrogen or potassium ions. It is in this region also that the tubular cells manufacture ammonia (NH3), which is secreted into the duct fluid, to combine with hydrogen ions that the same cells have secreted into that fluid. By this process a considerable amount of acid can be secreted without unduly acidifying the presumptive urine. Final adjustments are made in the early portion of the collecting duct, where the final salt concentration is achieved. A series of controls assures that the composition of the final urine is commensurate with physiological requirements of the moment. Foremost among these is the ingenious mechanism that makes salt concentration pos- sible. It was long known that man can excrete urine about four times as concentrated in salt as his own blood plasma. Other animals, particularly desert-dwellers, are considerably more adept at this task than are we; for example, the kangaroo rat need never drink water and survives by excreting urine 14 times as concentrated as his own plasma! The principal feature that distinguishes the kidney of the kangaroo rat from that of man is that in the kangaroo rat the descending tubule dips much farther down into the cortical and medullary tissue of the kidney; i.e., the tubule is decidedly longer relative to kidney size than is that of man. Inspection of the kidneys of a variety of species indicated that, in general, concentrating ability is a function of tubular length. It was this observation that suggested that the entire apparatus is patterned after the principle used by engineers in the design of heat-exchanging apparatus i.e., as a countercurrent multiplier.

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FRONTIERS OF BIOLOGY This notion has been subject to a variety of tests and has indeed proved to be valid, operating as shown in Figure 24. Since then, over examples of the same process have been sought, and it was learned that the circulation of large pelagic fish utilizes the same principle to conserve heat, a feature particularly evident in the tuna. \ ~ mu] 3003~ 300 ~ _ Corte:\(~\~; ~) j /~ 300 300 Inner Medulla 300 300 /: /300 l r ~[~ kI [it- ~ 400 ~00 400 200 Outer H2O Exchange of Na for K,H, N H4 - FIGURE 24 Salt concentrations in various regions of the nephron, basis for the countercurrent concentration device. Summary of passive and active exchanges of water and ions in the nephron in the course of elaboration of hypertonic urine. Con- centrations of tubular urine and peritubular fluid in mOsm/L; large boxed numerals show estimated percent of glomerular filtrate remaining within the tubule at each level. (From Physiology of the Kidney and Body Fluids, Second Edition, by Robert F. Pitts. Copyright (I) 1968 Year Book Medical Publishers, Inc. Used by permission.) 87

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88 THE LIFE SCIENCES Several hormones also control renal function. Individuals with Addison's disease were long known to become dehydrated due to excessive urinary excretion of sodium and chloride, with retention of potassium and acid. Such individuals fail to secrete the normal hormones of the adrenal cortical gland. The specific hormone in question has been identified as aldosterone, a steroid that is necessary to the process whereby sodium is reabsorbed in the distal tubule. Aldosterone secretion, in turn, is sensitive to the sodium concentration of blood passing through the adrenal cortex and through the pituitary gland. A second hormone was also recognized by virtue of a disorder of man- diabetes insipidus a syndrome characterized by excretion of as much as six gallons of urine per day. Individuals suffering from this disorder fail to elaborate in their posterior pituitary glands antidiuretic hormone, a relatively small polypeptide or protein, which determines the ease with which water may penetrate the walls of the collecting duct. The amount of hormone in contact with that duct determines the volume and final salt concentration . of urine. A third hormone, that of the parathyroid gland, governs the fate of phosphate as it traverses the tubule. This ion is removed in the descending tubule by active transport; the hormone blocks this process and phosphate continues into the urine. Secretion of this hormone, however, is sensitive not to the phosphate concentration but to the calcium concentration of blood plasma. When the latter is below the physiologically desirable level, hormone is secreted. It stimulates resorption of bone mineral; both calcium and phosphate concentrations rise in blood plasma, and the phosphate is then discarded in the urine under the action of the same hormone. Thus the intrinsic architecture of the kidney, the special adaptation of ordinary mechanisms for cellular active transport, the countercurrent appa- ratus, and the superimposed hormonal control all combine to permit sur- vival under a wide variety of environmental circumstances. For many years, hypertension has been associated with renal disease. Forty years of research addressed to the nature of this relationship culmi- nated in the demonstration of the following set of events. When circulation to the kidney is impaired, for whatever reason, e.g., a failing heart or an occluded renal artery, kidney cells release into the circulation a protein, renin, which has proved to be a proteolytic enzyme. The latter, in turn, digests from a normal plasma protein a medium-sized polypeptide that is somewhat further digested by a second enzyme, already present in plasma, to form angiotensin, the most powerful of known vasoconstrictive agents. The presence of this material in blood accounts for the disease of a large group of patients with hypertension. Moreover, angiotensin has been found not only to raise arterial blood pressure, but also directly to stimulate the

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FRONTIERS OF BIOLOGY cortex of the adrenal gland to secrete aldosterone, which, as we have seen, stimulates the mechanism for tubular reabsorption of sodium. In conse- quence, the kidney reabsorbs an inappropriately large amount of sodium, sodium excretion falls behind daily intake, and the body content of sodium, and with it water, increases. It is for this reason that the patient with con- gestive heart failure becomes waterlogged with edema fluid, usually evident as swelling of the extremities. Elucidation of this sequence of events was possible only after sophisticated techniques had been developed for the purification of proteins and of polypeptides. The actual molecular basis for the influence of aldosterone, antidiuretic hormone, parathormone, and angiotensin on the structures of the kidney and of the adrenal gland remains totally unknown. Each is secreted in response to changes in the internal ionic environment. The kidney is a versatile machine whose activities, therefore, are programmed by these hormones. The result is the remarkable constancy of the milieu interieur. Endocrines By 1945, students of endocrinology thought that the science of endocri- nology was well developed. The list of endocrine glands was thought to be essentially complete, and the gross consequences of under- or over-secretion of each were thought to be known, particularly as manifest in diseases of human beings. Yet the list of endocrines continues to proliferate, under- standing of the mode of action of these regulatory secretions becomes increasingly detailed, and the subtle manner in which they integrate the life of the organism becomes increasingly evident. The most surprising discovery of recent years was that certain nerve cells respond to neural signals by secreting hormones into the bloodstream, and in this way the two major integrative systems of the body are themselves functionally interlocked. The best demonstration of this is the picture that has emerged of the regulation of the female reproductive system. Sug- gestions of such interlocking came from old observations of the influence of the environment on the success of mating and pregnancy in domestic animals. Exciting stimuli related to photoperiodicity, ambient temperature, visual perception, odors, and even sounds were recognized. Among wild birds, the female generally requires the attention of the male before she will engage in nest building and egg laying. The female pigeon must perceive the visual image of her mate in order to produce crop milk for the young. Crowding can completely disrupt the reproductive processes of rats and mice. A female mouse who has mated with a male of her own strain does not become pregnant if, within 24 hours, she merely senses the odor of a 89

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go THE LIFE SCIENCES male mouse of a different strain; her pregnancy is blocked by suppression of pituitary support of the corpora lutea. In all these situations the environ- mental stimuli perceived by sensory receptors in the brain are translated into chemical signals secreted by nerve cells in a small region at the base of the brain the hypothalamus. From this small group of cells, neuroendocrine secretions, simply termed "releasing factors," are transmitted to the nearby pituitary gland via the very short hypophyseal portal vein. At least two specific releasing factors are involved, causing the pituitary to release, respectively, its follicle-stimulating hormone and its luteinizing hormone. The former stimulates the development of ovarian follicles, while the latter effects the rupture of mature follicles and the release of eggs and causes the ruptured follicle to develop into the corpus luteum. Earlier, it had been supposed that the pituitary gland and the ovary were linked in a closed feedback regulatory mechanism. It was presumed that ovarian hormones act on the pituitary to suppress the secretion of its gonadotropins. But it has recently been demonstrated that the ovarian hormones act not on the pituitary but on the hypothalamus, suppressing the production of releasing factors. The latter, of unknown structure, are of enormous potency and are made and secreted only in the most minute quantities. Other releasing factors that serve to stimulate the pituitary to release its thyrotrophic, growth, and adrenocorticotrophic hormones are thought to be elaborated in this same small region of the brain. Thus, the notion is implied that the cells of this area are equipped with a set of chemical sensors exquisitely sensitive to many aspects of their environment, because it is here, also, that one experiences thirst, satiety, hunger, and external temperature changes, and responds accordingly. Another new addition to the family of hormones is thyrocalcitonin, a hormone secreted by the parafollicular cells of the thyroid gland and which, together with the long-known hormone of the parathyroid gland, serves to regulate calcium metabolism. Preliminary observations suggest that this new hormone may be precisely what is required to prevent the postmeno- pausal osteoporosis responsible for bone fractures in a large number of women of advanced years. Surprisingly late in the history of endocrinology was the discovery of a compound that appears to deserve the appellation "intracellular hormone" and may be, in the evolutionary sense, the first hormone. This compound, 3',5'-cyclic adenosine monophosphate (cyclic adenylate), is formed from ATP under the influence of the enzyme phosphoadenosine cyclase. Re- markably, this substance has been independently discovered in several connections. It was first noted as the material formed when the hormone epinephrine, from the adrenal gland, stimulates the release of glucose from

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FRONTIERS OF BIOLOGY storage as liver glycogen. Epinephrine operates by triggering the action of the liver cyclase, and the resultant cyclic adenylate then trips off a series of events that lead to the activation of the enzyme responsible for the degrada- tion of glycogen. But cyclic adenylate formation was also discovered as the primary event in the activation of the cells of the adrenal cortex by the trophic hormone of the pituitary; after arrival of the latter hormone, cyclic adenylate is formed, which somehow makes possible the formation and secretion of adrenocortical hormones. The same compound is made when pituitary antidiuretic hormone acts upon the collecting duct of the renal tubule; somehow its presence permits movement of water across that struc ture. The effects of cyclic adenylate are not confined to animal cells. It has been found to stimulate synthesis of enzymes in E. coli' normally controlled by catabolite repression, to induce aggregation in starved slime molds and to derepress the synthesis of several enzymes of the mold Neurospora- enzymes that usually appear in the course of spore foundation. It seems remarkable that the same compound can serve so many functions. The manner in which it does so is obscure in each instance, but from this brief account it is clear why this newly discovered material has been dubbed an "intracellular hormone." PLANT AND INSECT HORMONES At least five major hormones or groups of hormones have now been dis- cerned in plants, including the classical auxins, gibberellins, cytokinins, abscisin, and even the simple gas ethylene. Unlike the highly specific effects of animal hormones, plant hormones appear to lack single clearly defined functions. Each is involved in a variety of different processes of growth or development. The gibberellins stimulate stem and leaf growth, seed determination, growth of some fruits, and in some cases, the formation of flowers and even the sex of flowers. Cytokinins affect root and bud formation; like the gibberellins, they are capable of overcoming dormancy in some plants, and they have the interesting property of rendering plants less sensitive to all kinds of unfavorable conditions, such as shortage of water, extreme temperatures, and even the effects of weed killers. Abscisin generally seems to reduce the growth activities of plants and to induce dormancy, as observed in a variety of plant parts. Accordingly, it would appear that, at any time, the behavior of a given portion of a plant is deter- mined not by the absolute amount of any one of these hormones, but by the ratio of one to another. Insects elaborate at least three and probably several more hormones. The two that have commanded attention are large steroid-like molecules, 91