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3 Understanding Genetic, Molecular, Cellular, and Physiological Processes The biological well-being of humans is a composite of genetics, nutri- tion, and other environmental influences that may explain the renaissance of research in nutrition science. In this chapter, we present selected op- portunities in the nutrition sciences at the molecular and cellular levels. The development of the techniques of molecular genetics and of analyti- cal equipment and powerful computer-basecT techniques to analyze the resulting data has driven much of the progress and created many of the research opportunities in the basic sciences related to nutrition. Much of this technology is described in this chapter, but it is also relevant to many sections in Chapters 4 and 5. Research in nutrition, as in any field, evolves as a mosaic of informa- tion gathered from technological advances and conceptual breakthroughs. Two disciplines have made particularly important contributions: nuclear physics and molecular biology. During the first half of this century, devel- opments in nuclear physics led scientists to produce radioactive and stable isotopes. In the mid-1930s, stable isotopes were used to show that body lipids labeled with deuterium are in a dynamic state, with constant inter- action among the body pools and substantial influence by absorbed di- etary lipids. Experiments with i5N-labeled amino acids provided early in- sights into the dynamics of protein synthesis. The knowledge of nutrient metabolism gained during the last half century would not have been pos- sible without use of radioisotopes. Molecular biology made its impact on nutrition through advances in 47

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4S OPPORTUNITIES IN THE NUTRITION AND F0019 SCIENCES understanding the regulation of gene expression. In the early 1960s, stud- ies of the regulation of lactose metabolism in the bacterium, Escherichia cold began our understanding of nutrient metabolism at the molecular level. Lactose was found to stimulate the synthesis of enzymes involved in the conversion of lactose to galactose and glucose and in transporting galactose into the cell. As we discuss later, nutrients regulate the metabolic fate of mammalian cells and control the metabolism of other nutrients. Nutrition as a field of scientific inquiry poses important questions that can be investigated from many perspectives. Major contributions of basic biological research to the understanding of nutrient metabolism and func- tion at the cellular level include: (1) nutrient transport in the brain and intestine, (~) uptake and utilization of nutrients by cells, (3) control of gene expression by nutrients, and (4) hormonal regulation of nutrient metabolism. Results of research in these areas of biology provide the framework for understanding nutrient assessment, dietary recommenda- tions, and the interactions of nutrients in disease and health. In this chap- ter, we briefly review several examples of contemporary discoveries in nutrition where basic science was skillfully applied to illuminate a physi- ological process. We also describe several rapidly advancing technologies creating new opportunities for basic research in nutrition science. These examples illustrate several of the many opportunities that lie ahead. ACCOMPLISHMENTS AND RELATED POSSIBILITIES Brown and Goldstein and Lipid Metabolism Cholesterol, a small lipid molecule, is essential to membrane integrity and is the precursor of bile acids, steroid hormones, and vitamin D. Yet elevated cholesterol in blood plasma (hypercholesterolemia) is one of three major risk factors, along with smoking and hypertension, of atheroscle- rotic heart disease, the major cause of death in the United States. Athero- sclerosis is characterized by an accumulation of esterified cholesterol within the smooth muscle cells and macrophages of the artery wall, eventually leading to cell death and hardening of the arteries. The resulting obstruc- tion of the vessel can reduce or cut off blood flow, causing heart attack or stroke. Cholesterol is both synthesized in the body and contained in foods commonly found in most Western diets. A fundamental problem in cell biology is understanding how cells control their cholesterol content. Epi- demiological studies identified elevated concentrations of low-density li- poprotein (LDL), carrier of the major portion of cholesterol in human plasma, as the main factor causing atherosclerosis. Diets high in saturated fat and cholesterol elevate LDL levels in most individuals. A genetic dis

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GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 49 ease called familial hypercholesterolemia (FH) causes severe hyper- cholesterolemia in a few people that can be improved, but not normal- ized, through dietary treatment alone. By the early 1970s, it was known that FH exists in two clinical forms: a severe, homozygous form, in which LDL cholesterol is elevated 6 to 10 times normal and heart attacks may begin in childhood; and a less severe, hetero%ygous form, in which plasma LDL cholesterol is elevated ~ times normal at birth and heart attacks begin in the fourth to fifth decade of life. Heterozygous FH occurs in approximately 1 person in 500; the ho- mozygous form affects about 1 in 1 million persons. In 1972, Michael Brown and Joseph Goldstein hypothesized that FH might be caused by a failure of cells to repress cholesterol synthesis. The concept of feedback inhibition was well established, but genetic defects in end-product regulation had not previously been associated with human or animal disease. But how could cholesterol regulation, then thought to be the provenance of the liver and intestine, be studied in patients? Using an assay for the rate-limiting enzyme in cholesterol synthesis, Brown and Goldstein found that cholesterol synthesis increased in normal fibroblasts when lipoproteins were removed from the culture medium. Conversely, synthesis was rapidly suppressed when LDL was added back. Moreover, cells from patients with homozygous FEI had a high rate of cholesterol synthesis even when LDL was added. These studies identify LDL as a regulator of cellular cholesterol synthesis and FfI as a disease of impaired end-product regulation of cholesterol biosynthesis. Studies originally undertaken to explore how LDL cholesterol is de- livered to cells provided new insight into fundamental pathways of protein movement across cell membranes. Studies of Brown and Goldstein and others showed that the regulated uptake of LDL could be separated into distinct steps: (1) binding to the LDL receptor on the plasma membrane, (2) movement of LDL receptors into regions of the plasma membrane (coated pits) that invaginate to form endocytic vesicles that contain LDL, (3) dissociation of LDL from the LDL receptor in lysosomes at acid pH, (4) lysosomal degradation of the LDL protein and hydrolysis of LDL's esterified cholesterol, and (5) recycling of LDL receptors to the plasma membrane. The elucidation of the LDL receptor pathway was possible in large part because LDL uptake in FH fibroblasts contained mutations in the proteins that mediate most of these distinct steps. Eventually, the combination of genetic, biochemical, and molecular analyses revealed at least five separate classes of mutations, each of which causes the FEI phenotype (Figure 3.1~. The concepts of LDL receptor saturation, inter- nalization, and down-regulation of LDL receptor synthesis also helped to explain why diets high in fat stimulate production of LDL from its precur- sors and elevate circulating LDL levels. -

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so OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES Of ~ > ICC ~ ~- Mutation Class 1 2 3 4 Endosome Coated Pit Recycling Synthesis Transport Binding Clustering ~ LDL FIGURE 3.1 Five classes of mutations at the LDL receptor locus. These muta- tions disrupt the receptor's synthesis in the endoplasmic reticulum (ER), transport to the Golgi, binding of apolipoprotein ligands, clustering in coated pits, and recycling in en.dosomes. Each class is heterogenous at the DNA level. From. Hobbs et al. (19909. Reproduced, with permission, from the Annual Review of Genetics, Vol. 74, copyright 1990 by Annual Reviews Inc. As this example illustrates, elucidating the LDL pathway has had an impact far beyond that of understanding cholesterol homeostasis. Recep- tor-mediated endocytosis also applies to receptors for nutrients and hor- mones. The significance of these observations has been recognized fre- quently and ultimately led to the awarding of the Nobel Prize in Physiology or Medicine to Brown and Goldstein in 1985. The role of cholesterol and blood lipids in atherosclerosis is discussed further in Chapter 5. Retinoic Acid Shortly after "fat-soluble A" was described as a required dietary factor in the early l900s, investigators recognized that vitamin A must play es- sential roles in reproduction and in maintaining normally differentiated epithelial cells in many organs throughout the body. Epithelia of the ocu- lar conjunctive and respiratory and genitourinary tracts showed especially marked histopathological changes during vitamin A deficiency. By 1931, the chemical structure of vitamin A was identified as retinal, and in the 1950s, the retinal metabolite, 11-cis-retinaldehyde, was identified as the critical light-absorbing molecule of the visual pigment rhodopsin. Further chemical studies led to the synthesis of retinoic acid, the carboxylic acid derivative of retinal, and the demonstration that retinoic acid could substitute for retinal in growth assays and in maintaining nor

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GENETZC, MOLECULAR, CELLULAR, AND PHYSZOLOGZCAL PROCESSES 51 mat cellular differentiation. In the late 1970s, it was discovered that retinoic acid regulates growth and causes embryonic stem cells to differentiate- that is, to undergo a permanent change in the pattern of gene expression. At the same time, it was found that vitamin A inhibited the promotion phase of carcinogenesis. Together, these studies implied that retinoic acid functioned in the nucleus to regulate the transcription of specific genes. A major recent landmark in vitamin A research was the discovery of retinoic acid receptor (RAR) proteins in the nuclei of cells. These recep- tors function as transcription factors that regulate retinoid-responsive genes. Critical features of the nuclear receptors that mediate the actions of ste- roid hormones and thyroid hormones were already understood. Knowl- edge of these receptors served as a base for a search for additional ho- mologous proteins with unknown ligands. Investigators cloned a cDNA encoding a novel protein structurally related to the steroid/thyroid hor- mone receptors. In cultured cells expressing the new receptor, addition of retinoic acid activated specific genes (see box). Subsequent experiments identified more RARs. These RARs are now recognized as members of the steroid/thyroid hormone gene superfamily. This research has estab- lished retinoic acid as an important "hormonal" form of vitamin A that acts through a mechanism analogous to that of the steroid hormones. This basic discovery has had important consequences for nutrition, development, and cancer research. For example, previous studies of em- br,vonic development had identified retinoic acid as a morphogen that could control the form of body parts during embryonic development. Chemi- cal methods showed that retinoic acid is present in the embryo early in development. Researchers now hypothesize that retinoic acid provides a signal for normal cell migration. (, . Vitamin D Receptors and Metabolism Continuing interest in vitamin D derives from its importance in en- abling the body to make use of available calcium. Early in this century, vitamin D was shown to cure or prevent rickets, a bone disease common at the time and a major public health problem (see Chapter 1~. Our cur- rent interest in this nutrient centers on the influence that natural and synthetic analogues of vitamin D have on human diseases such as osteo- porosis, endocrine disorders, skin disease, and cancer. Sufficient calcium deposition in bone, as stimulated by vitamin D (particularly early in life), minimizes the risks and consequences of osteoporosis, a painful, debilitat- ing disorder that afflicts millions of older women.

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.52 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES CLONING THE FIRST NUCLEAR RETI N O l C AC I D REC E PTO R (RAR) The discovery of the first nuclear RAR, RAR-alpha, occurred nearly simultaneously in laboratories in France and the United States. The estro- gen and glucocorticoid receptors have modular"cassette" structures comprised of six main domains (see illustration below). The DNA-binding domain (domain C) mediates binding of the receptor to the promotor region of specific genes. The ligand-binding domain (domain E) mediates activation of the receptor complex by ligand. Investigators postulated that new genes with strong sequence similarity to the steroid/thyroid receptor genes might code for as-yet-unidentified nuclear receptors. They used cDNA probes specific for the highly conserved regions of known nuclear receptors to screen human cDNA libraries. This search identified a "can- didate receptor," one similar in size and domain structure to the known nuclear receptors. Domain A B C D Steroid receptor domain structure Bacterial chloramphenicol acetyltransferase E F DNA binding Ligand binding l Glucocorticoid Unknown receptor's receptor's domain C domain E 1 1 1 1 1 1 1 1 Transfect cells with cnimeric gene Add reporter (CAT)- gene with promoter activated by binding to domain C of the steroid receptor Add test ligand (retinoic acid, etc.) Examine for expression of CAT gene product

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GENETIC, MOLECULAR, CELLULAR, AND PHYSZOLOGICAL PROCESSES 53 How could the ligand for this new receptor be identified when its target genes were unknown' An ingenious strategy, the swap experiment, provided the key. Molecular cloning techniques were used to construct a new gene. Region C of the candidate receptor was replaced with region C of the glucocorticoid receptor. When the new hybrid (or chimeric) receptor was expressed, it interacted with its novel ligand through its unique domain E and bound to known glucocorticoid response elements through domain C. When cells were cotransfected with plasmid DNA that directed synthesis of the chimeric receptor and with DNA that contained a glucocorticoid-sensitive promotor linked to a reporter gene, candidate ligands could be tested for their ability to activate the reporter gene. Several natural and synthetic molecules were added as putative ligands for the chimeric receptor. Only retinoic acid was a strong activa- tor. Based on the sequence similarity between the nuclear RAR and steroid/thyroid receptors and on the ability of RAR to activate gene transcription, the RARs are new members of the steroid/thyroid super- family of ligand-dependent nuclear receptors. Metabolism from Vitamin to Steroid Hormone In the late 1960s, researchers demonstrated that vitamin D is hy- droxylated to a biologically active form in the liver and kidneys. This research demonstrates the application of multiple approaches, a charac- teristic of nutrition research. Chromatographic methods were developed and used to show that vitamin D3 (cholecalciferol) undergoes hydroxyla- tion to biologically active polar metabolites, mainly 1,25 dihydroxyvitamin D3 (calcitriol). Specialized chemical syntheses and analytical mass spec- trometry were used to identify these structures. The side-chain conforma- tions of these vitamin D derivatives were identified using high-resolution proton nuclear magnetic resonance. The structure and relationships of vitamin D and its key metabolites are shown in Figure 3.2. Over 30 metabolites of vitamin D have been described. Calcitriol, the active form of vitamin D, is generated in the kidneys from the 25-hycTroxy- lated metabolite produced in the liver. The enzyme responsible for hy- droxylation in liver, caTciferol-25-hydroxylase, is regulated by a feedback mechanism that protects against vitamin D toxicity (which would cause abnormal calcification) ant! helps to conserve vitamin D when dietary intake is Tow or formation of vitamin D in the skin is decreased. Physiological Actions via Genomic anti Nongenomic Pathways Calcitrio! regulates metabolism of calcium and phosphorus and the expression of many genes of known anti unknown function. This contem

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54 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES HOW 7-Dehydrocholesterol | DIET | SKIN / HO'v a' Vitamin D3 LIVER | | KIDNEY| '~` I' OH a' HOW OH 1,25-Dihydroxyvitamin D3 HOW - ~ I OH HOW 25-Hydroxyvitamin D3 /\ \ HOW> Vitamin D2 | KIDNEY | >. I other tissues | OH ~ 1>H 24,25-Dihydroxyvitamin D3 FIGURE 3.2 Vitamin D metabolism in the skin, liver, and kidney. From Henry, ILL., et al. 1992. The cellular and molecular regulation of 1,25~0H):D3 produc- tion. [. Steroid Biochem. Molec. Biol. 41:401-407. Copyright 1992, reprinted with kind permission from Pergamon Press Ltd. porary biomedical research is at the interfaces of physiology, biochemis- try, nutrition, and molecular biology. Our understanding of the calcium- related functions of vitamin D was enhanced with the discovery of the calcium-binding protein calbindin. Vitamin D stimulates the production of this protein by increasing transcription of the calbindin gene. Calbindin, by a mechanism not yet defined, transports calcium across intestinal cells and delivers it to a calcium pump on the basolateral membrane that is regulated by calcitriol. The pump transports calcium across the mem- brane into the blood and then to bone and soft tissues. Calcium metabo- lism is controlled by parathyroid hormone (PTH) and calcitriol (Figure

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GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 55 HORMONAL LOOP DERIVED FROM VITAMIN D '~ 1,25 (OH)eDs 7 ( <_PTH it_ `~1,25(0H)2D5 ~ ~Co++HPO Ca++HPO~ ~CHpo,,- \~ A lomg/looml ~ INTESTINE ~35- 1} ~ ' c~circa'. // BLOOD CALCIUM / ~ y~ 1 ~: I (s I ~ ( 45 1 1,25 (OH)2Ds - ~*~' ~ 1 ~ PARATHYRO'D GLANDS ~ _ _ ~ OTI 1ER FACTORS K D^OHD;~-Elydrox . 25 OH D' 425T~O LIVER \ ~ \_/ VITAMI N D FIGURE 3.3 Diagrammatic representation of the regulation of plasma calcium (ECF) concentration by the vitamin D endocrine system and the parathyroid glands. Low plasma calcium is detected by parathyroid glands. Parathyroid hormone stimulates production of 1,25~0H)~D3. The two hormones act either indepen- dently or in concert to mobilize calcium from bone, kidney tubules, and small intestine, bringing about an elevation of plasma calcium concentration that in turn suppresses parathyroid hormone secretion. H.F. DeLuca, from Nutrition: An In- tegrated Approach, third edition (p. 149), by Ruth L. Pike and Myrtle L. Brown, [ohn Wiley & Sons, copyright 1984. Reprinted with permission. 3.31. A subnormal concentration of calcium in the blood stimulates secre- tion of PTlI, which in turn increases the synthesis of calcitriol in the kidneys. Increased expression of the calbindin gene stimulates intestinal transport of dietary calcium, which increases calcium concentrations in the blood. In addition, calcitriol, in conjunction with PTH, increases mo- bilization of calcium and phosphorus from bone to the blood. Through these mechanisms, calcium in the blood is maintained at concentrations sufficient to calcify bones and perform important intracellular functions such as signal transduction. In addition to regulating calcium metabolism, vitamin D increases or decreases expression of 50 other genes. The vitamin D receptor is a mem

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56 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES ber of the gene family for steroid/thyroid receptors and functions accord- ingly. The hormone-receptor complex binds to DNA and regulates tran- scription of specific genes. Learning more about the cellular distribution of this receptor may shed light on other important roles of vitamin D. Despite the advances described above, knowledge of calcium's role in intracellular processes and for maintaining the skeletal system remains incomplete. We do not know (1) how dietary calcium homeostasis, regu- lated by vitamin D metabolites, contributes to bone calcium turnover, (2) the significance of the trans-acting vitamin D receptor system in differen- tiating tissues, or (3) how this receptor system interacts with cytokines and growth factors during development or nutritional deprivation or in diseases such as osteoporosis. These questions represent important oppor- tunities in nutrition research. Neurotransmitters Regulation and Action Molecular and immunocytochemical techniques and newly developed drugs have increased our understanding of the development and regula- tion of neurotransmission. We have achieved a new understanding of the relationships between neurotransmitter function, behavior and cognition, and the molecular and biophysical basis of the dominant excitatory and inhibitory receptor systems in the brain. During brain development, these systems are regulated at the level of the gene, and their functions may be influenced by dietary factors such as low protein intake. At least one class of excitatory receptors is involved in neurotoxic damage, some of which may occur early in development and be detectable only with careful cog- nitive testing over an extended period of time. Infinences on Eating Behaviors Monoamine neuropeptides and hormones appear to affect food intake anti aspects of feeding behavior in animals as well as humans. Concentra- tions of amines and neuropeptides in the brains of animals are highly responsive to circulating nutrients and hormones and to environmental variables that may contribute to eating disorders in humans. Injecting specific transmitters into the hypothalamus causes satiated animals to over- eat or hungry animals to stop eating. Understanding the brain pathways involved in eating motivation and satiety is vitally important to improving health. For example, altering the concentrations of serotonin (5lIT) and norepinephrine (NE) in the medial hypothalamus may modulate the tem- poral pattern of carbohydrate and protein intake by activating or inhibit- ing satiety mechanisms. Activation of serotonin receptors may directly antagonize the action of alpha-2-adrenergic receptors that normally func

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GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 57 tion to increase carbohydrate intake. In vivo microdialysis enables scien- tists to measure the actual concentrations of small molecules such as sero- tonin, dopamine, and cyclic-AMP in small, defined regions of the brain and has advanced this field considerably. Despite these new findings, much more needs to be learned about the interrelationships among the areas in the brain where particular neurotransmitters act as well as how these actions are coupled to dietary and environmental variables that influence food intake. Excitatory and Inhibitory Receptor Systems in the Brain The amino acids glutamate and aspartate and the amino acids glycine and gamma amino butyric acid (GABA) are the transmitters in the domi- nant excitatory and inhibitory pathways, respectively, of the vertebrate brain. Molecular cloning techniques have been used to learn the primary structures of many of the subunits that make up the complex receptors for these transmitters. Subunit composition varies dramatically from region to region in the brain, providing the receptors with different kinetics, bincI- ing affinities for the transmitters, and susceptibilities to cytoplasmic or extracellular modulators. Receptor composition also changes during de- velopment; thus the receptors on neurons in the young brain may produce markedly different responses than the same receptors on the same neu- rons in the adult brain. . There are two types of receptors in eukaryotic cell membranes. Ion- passing receptor complexes are ion channels, and at least three types are regulated by the excitatory amino acid glutamate. In contrast, metabotropic cell surface receptors transmit their signals to intracellular modifying en- zymes which, in turn, activate signaling pathways that lead to specific end effects. One receptor subtype for glutamate, the N-methyl-D-aspartic acid (NMDA) receptor, deserves particular mention because it has been impli- cated in a wide range of normal and pathological processes in the central nervous system. The NMDA receptor has a high affinity for the synthetic glutamate analog, N-methyl-D-aspartate. The complexity of the ligand- binding and voTtage-gating properties of this receptor make it unique among the known ion-passing receptors. Certain brain traumas cause glutamate to be released into the extracellular fluict and cause neuron death; the NMDA receptor has been implicated in this brain cell death. In addition, overactive NMDA receptors have been implicated in various forms of epilepsy. The other major class of ion-passing "glutamate" receptors- the alpha-amino-3-hyc3roxy-5-methyl-4-isoxazole propionic acicl/kainic acid (AMPA/KA) receptors have much lower binding affinities for NMDA, thus allowing the properties of the NMDA receptor channel to be studied selectively.

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GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGZCAl, PROCESSES S7 enable us to develop dietary interventions that improve function. Obesity is an important example of this type of disease. It is characterized by excessive deposits of fat in adipose tissue and is caused by ingesting more calories than needed. It is imperative that we understand more about the genetic, physiological, and biochemical bases of weight control. Research on the development and function of adipose tissue is crucial to under- standing the etiology of this common and potentially devastating problem. Obesity has a significant genetic basis, although in some cases interactions with the environment may be necessary before the genetic propensity manifests itself. The complexity of the metabolic and neurological changes that accompany the onset and maintenance of obesity has made it difficult to determine which of the observed changes are causing the problem. Interestingly, there are a number of mouse and rat models of obesity that are unequivocally the result of changes in single genes. In keeping with 1 l 1 . 1 . r 1 . . ~ . rr - - 1~ = ~ one mu~gen~c character or obesity Brent ~ene.s tinner to he involved in the different animal models. Obesity in these animal models is inher- ited in a simple Mendelian fashion. Despite the obvious importance of this debilitating disease, these genes have not been identified. Identification of these genes must be a priority for nutritional re- search. Positional cloning, or reverse genetics, has already identified some genes that cause diseases in humans, even though the molecular bases of these gene defects were unknown. Using approaches outlined earlier in this chapter, it should be possible to identify the genes that cause obesity in various animal models. This will have two important consequences. First, we will be able to examine the structure of those same genes in humans to see if any forms of human obesity have the same causes. Sec- ond, we can analyze the effects of early diet, stress, and other environ- mental factors on the expression of those genes in normal animals to determine the roles of those genes in nongenetically determined obesity. Other approaches to identifying obesity genes in humans have been out- lined earlier in this chapter. _)7 ~ t~ ~ark Structural Biology Techniques such as two-dimensional NMR and X-ray crystallography, described earlier in this chapter, have created unprecedented opportuni- ties to determine, at the atomic level, the three-dimensional structures of enzymes, enzyme-substrate complexes, and other proteins (with and with- out ligands such as hormones, vitamins, drugs, and metal ions). These techniques will enable us to also determine RNA and DNA structures and those of protein-DNA and protein-RNA complexes. These opportunities are related to nutrition. Determination of the atomic structure of a receptor protein with and without its ligand will

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88 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES enable investigators to identify the optimal chemical structure of a ligand for interaction with its protein partner. This type of interaction initiates virtually all regulatory activity in the body. Knowing the optimum struc- ture of the ligand will be useful in synthesizing effective agonists and antagonists for use in conventional drug therapy and therapy with modi- fied nutrients. Some of these therapeutics will be modified micronutrients such as vitamins or antioxidants. Others may be modified versions of ma- cronutrients, because it is clear that macronutrients or their metabolites regulate the activity of some enzymes by binding directly to them and altering their enzymatic activities. Alternatively, macronutrients or their metabolites may bind to and regulate the activity of proteins that control production of the enzyme; for example, by binding to proteins that regu- late transcription of the gene for that enzyme. Understanding the struc- ture and function of proteins that interact with regulatory ligands will facilitate the synthesis and effective use of therapeutic agents. The same principle can be applied to the synthesis of substrate analogs to be used as inhibitors of specific enzymes involved in intermediary metabolism. Understanding the three-dimensional structure of proteins will also lead to the design of more effective enzyme catalysts. These improved catalysts will be useful to the food industry (see Chapter 4~. To get im- provec! catalytic properties, we can apply the same principles described above. In this case, however, the goal will be to modify a protein's struc- ture to give it optimal binding or catalytic properties with respect to a specific ligand or substrate. Alternatively, the structure can be mollified to make it more stable or to function in a non-aqueous environment. Enzymes with improved catalytic properties may also be useful in gene therapy. If only a small amount of an enzyme can be introduced by gene therapy, a very efficient one would be very useful. A large number of enzymes and proteins are involved in nutritional processes, nutritional regulation of metabolic function, and food process- ing. Determining their structures, characterizing their functions, and de- signing therapies for improving their functions in specific situations pro- vide important opportunities in basic science with clear relevance to nutrition and food science. Stem Cell Biology Stem cells are critical for the development and maintenance of the organism. Their properties include the ability to self-renew, divide asym- metrically, and generate one or more irreversibly differentiated progeny cell types (Figure 3.5~. In the adult organism, continuously proliferating tissues such as slain, bone marrow, and intestinal epithelium depend on their respective stem cell populations to replace obligatory cell losses. The

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GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 89 Stem cell FIGURE 3.5 Stem cell asymmetric division, producing one daughter that is committed to ter- minal differentiation and another that remains as a stem cell. ,i, Terminally differentiated cell magnitude of the replacement is considerable; approximately 10~: epithe- lial cells are shed by the human small intestine each clay and must be replaced by new cells originating from the division of stem cells located deep within the intestinal crypts. Because of their high synthetic rates, these same tissues are particularly sensitive to nutritional inadequacies. Despite the importance of stem cells, our understanding of how their differentiation is regulated and how they function is limited. Until very recently, it was not possible to recognize and purify stem cells in suffi- cient numbers to perform meaningful experiments. In addition, we lacked in vitro systems appropriate for the culture and analysis of stem cells. Finally, our understanding of the factors regulating cell division, even in the less complicated case of non-stem cells, was limited. Recent basic science developments in all these areas, particularly with stem cells from the bone marrow, offer great opportunity and promise for improved un- clerstancling of the biology of stem cells. The quintessential characteristic of stem cells is their ability to un- dergo asymmetric cell divisions that is, one daughter retains its stem cell- ness while the other undergoes terminal differentiation. flow this is ac- complished is a fascinating, unanswered question and research opportunity. How nutritional state regulates this process is even more obscure and an equally important area for future research. These problems are made all the more difficult by our limited knowledge of the factors that control the normal mitotic cell cycle. The asymmetric cell division of stem cells prob- ably represents a special case of the normal mitotic cell cycle modified by internal or external signals that ensure the different fates of the two daughters. Recent work in a variety of organisms, including yeast, flies, amphibians, ant! mammals, has led to a general model of cell cycle control. It should now be possible to determine the factors responsible for the special char- acteristics of stem cells. A second area of great progress in basic research relative to stem cell

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So OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES biology has been the identification of several cytokines and growth fac- tors. The interleukins, granulocyte macrophage colony-stimulating factor, erythropoietin, insulin-like growth factors, basic fibroblast growth factor, and many others have been described and made available for experimen- tal use in culture systems. Supplementation of culture medium with vari- ous combinations of these growth factors allows culture of stem cells with preservation of their characteristics. Available evidence suggests that these potent molecules are a necessary part of the microenvironment of stem cells. In fact, one hypothesis for the mechanism of asynchronous cell divi- sion suggests that each stem cell has an optimal microenvironment, or niche, and that when the cell divides, only one daughter can remain in the niche the other is physically forced out. Away from the optimal microen- vironment, the clisplaced cell commits to differentiation. EIow nutritional state regulates concentrations of the growth factors and composition of the microenvironment is a largely unexplored research opportunity. Great progress has been macle in the identification and isolation of stem cells, particularly of bone marrow. The search began with the recog- nition that lethally irradiated mice suffered bone marrow failure and that this could be reversed by injection of unirradiated bone marrow cells. This assay for the ability of cells to reconstitute the hematopoietic system made it possible to sort through the complex mixture of cells found in the marrow to identify stem cells. Work in several laboratories led to the development of monoclonal antibodies that identify cell surface differen- tiation antigens characteristic of various stages in the lineage of hemato- poietic cells. Availability of these reagents, coupled with advances in puri- fication of cells by fluorescence-activated cell sorting (FACS) and other antibody-based cell-separation techniques, allowed mouse hematopoietic stem cells to be purified to virtual homogeneity. As few as 30 of these cells are sufficient to reconstitute all blood cell types in a lethally irradi- ated mouse. Similar cells have now been identified in human marrow. An even more primitive cell gives rise both to hematopoietic stem cells and the marrow stromal cells that produce cytokines essential for the hemato- poietic microenvironment. Hematopoiesis is one of several processes es- sential to maintenance of the immune system. The importance of main- taining good immune function is discussed in Chapter 5. These advances in basic research set the stage for studies that will analyze the role of nutrition in regulating the function of stem cells. Nu- trition scientists will be able to study stem cells of the intestinal mucosa to learn how these cells function in health and disease. We can anticipate learning how stem cells accomplish asynchronous cell division, what sig- nals are responsible for a commitment to terminal differentiation, and what factors determine how often stem cells divide. The answers to each of these questions will be important landmarks in nutrition research.

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GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 9 Nutrient Transport Systems of the Bloocl-Brain Barrier The blood-brain barrier (BBB) refers to the selective permeability of the vasculature in brain capillaries in the central nervous system. The blood-cerebrospinal fluid (CSF) barrier represents the permeability of the capillaries of the dense vascular beds of the third ant! fourth brain ven- tricles, known as the choroid plexus. These vessels separate the blood from the CSF. Thus, the interstitial fluids that bathe nerve cells and the CSF of the ventricles and subarachnoid space are different from each other and significantly different from the fluid that bathes other cells of the body. The term "barrier" is something of a misnomer derived from early studies in which lipid-insoluble dyes were perfused intravenously and shown to be excluded from brain tissue. In fact, the permeability characteristics of the BBB and the blood-CSF barrier are complex, as is the relationship between the two. Endothelial cells act as molecular sieves, passing hydrophilic mol- ecules of up to 40,000 molecular weight. In contrast to other capillary beds of the body, the endothelium of the brain capillaries is essentially impermeable, in either direction, to proteins and ions in the blood. Mor- phologically, these reticuloendothelial cells show tight junctions completely blocking the intracellular spaces, and they are not interrupted by gap junctions. If free flow between cell junctions is ruled out as a common route of passage for molecules in the normal adult brain, what are the alternative routes? For small molecules, these include a variety of pumps and active transport systems. There is general agreement among workers in the field that the brain enclothelium itself is the major component of the BBB. However, there is another structural difference between brain capillaries and the capillary beds of other body organs that could play an important role in BBB per- meability, either by acting as another filter or by modulating endothelial cell permeability. Specifically, brain capillaries are completely enclosed by the endfeet of astrocytes, separating them from direct contact with neurons. There appear to be no gaps in the junctions between these endfeet. Thus, the sequence of potentially specialized surfaces separating the blood plasma from brain cells is as follows: the luminal wall of the brain endo- thelium, the basal wall of the epithelium, an unusually thick basement membrane, and the two surfaces of the endfeet of astrocytes. In short, most of the molecules that enter the brain have to travel through special- ized transport systems or, if they are lipid-soluble, to navigate successfully two types of cells. On the other hancl, plasma-borne molecules of a wide range of sizes can enter the brain when there are disruptions of the physi- cal endothelial barrier. Such disruptions can be brought about by disrup- tions in osmolarity or by physical damage.

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99 OPPORTUNITIES IN TlIE NUTRITION AND FOOD SCIENCES Basic Research in Nutrition and the Blood-Brain Barrier Recent work in the private sector has concentrated on developing molecules that will cross the BBB. In addition, there has been consider- able basic research on extracellular matrix proteins in basement mem- branes and the way these molecules interact with neuronal and glial cell surface receptors. Molecular microbiology has created a wealth of new knowledge on specialized glial pumping systems, "transmitter" receptors in glia, and complex peptide and growth-factor-mediated interactions be- tween neurons and glia. In contrast, there is a paucity of basic research on interactions between various types of endothelial cells. Whether particular molecules entering the bloodstream after ingestion and intestinal absorp- tion are harmful to neurons or brain function and whether nutrients are adequate to support healthy brain function are complicated issues. An- swers will have to be determined not only on the basis of epithelial per- meability, as measured under some controlled conditions, but also on how long the molecules in question remain in brain fluid and on the final concentrations they reach in the brain. The latter depends on how effec- tively they are cleared into CSF and on the efficacy of active uptake systems for them in particular brain regions, in the neurons themselves, and in the endothelia and glia that surround brain capillaries. Moreover, like other aspects of nutrition, these factors will undoubtedly vary in indi- viduals of differing age and genotype. The specialized properties of brain endothelial cells apparently de- rive from their contact with brain cells. Long-term cultures would provide the easiest approach to studying the selective permeability or transport systems of brain endothelia. However, such cultures apparently do not retain the selective permeability characteristics of brain endothelia in the intact animal. Thus, in the area of nutrition in the brain, many of the required basic research models are simply not available. This is a particu- lar concern with respect to the effects of food additives and nutrition on the developing brain. There is considerable controversy over differences between fetal and adult BBBs and over when the fetal BBB is established for molecules of various sizes. The CSF-brain barrier of the young fetus is less permeable to large proteins than is that of older fetuses or adults. Many receptors and ionic pumps present in the adult brain differ in their subunit composition and probably, therefore, in their function in the fetal brain. Consequently, the young differentiating neurons in the fetal brain may be relatively unprotected from potentially dangerous molecules in the blood. Basic aspects of the nutrition of the brain thus provide the molecular bases for the cliet-related cognition studies described in later chapters. The topic of what passes into the developing brain has become con J J

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GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 93 tentious in some areas, such as that of glutamate and other excitatory amino acids used as additives in many foods, largely because of the in- creasing number of research reports suggesting an important role for glutamate receptors in neural development and increasing evidence that amino acids can function as excitotoxic agents. For fetal development, the issue be- comes the degree of permeability of the blood-placenta barrier. One study in primates during late gestation indicated very little transfer of even high concentrations of infused glutamate through the placenta. In addition, several studies suggest that ingestion of large amounts of glutamate has relatively little effect on glutamate concentration in circulating blood or in the milk of nursing mothers. However, some scientists studying brain development or excitotoxicity feel that the existing studies are inadequate. We lack sensitive longitudinal behavioral assessments of the development of cognitive and motor skills in children exposed to large amounts of glutamate in their diet and good basic research studies on when and how the developing BBB is established and maintained. Thus, the issue of the high concentrations of potentially toxic additives in food regularly ingested by children and women of childbearing age should not be dismissed. Be- havioral biologists have begun to develop and employ in humans and non- human primates the kinds of analyses that might identify effects of long- term exposures to low levels of excitotoxins in the diet. Prevention and Repair of Oxidative Damage Structural changes in lipids, proteins, or nucleic acids caused by chemical or photochemical oxidation have been linked to aging, cancer, and other degenerative diseases. Because a host of oxidative processes are essential for life, it is critical to distinguish pathophysiological changes from physi- ological ones and to determine how the pathological ones can be pre- vented or reversed. Dietary factors, especially the antioxidant vitamins C and E, beta-carotene, the sulfur-containing amino acids, and proteins with redox functions, may prevent or control oxidative damage. Various metals have either pro-oxidant or antioxidant properties, and some may have both, depending on their chemistry, concentration, and environment. Thus, both pro- and antioxidant activities exist together in cells, and a major challenge is to elucidate the mechanisms of each and the factors that balance their actions in physiological situations. The results of clinical investigations support the preventive or thera- peutic value of antioxidant vitamins in certain diseases. For instance, vita- min E may prevent retinal damage (retrolental fibroplasia) in infants un- dergoing oxygen therapy, and high levels of beta-carotene may be efficacious in the genetic photosensitivity disorder erythropoietic protoporphyria. In addition, beta-carotene or other dietary antioxidants in fruits and veg -

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94 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES etables may be beneficial in reducing the risk of some cancers, .1 . . a. including lung cancer, a tumor that is often associated with oxidative damage from smoking. The dietary components that mediate the protective effects of fruits and vegetables need to be identified; advances in chromatography and spectroscopy are likely to facilitate this research. Another area in need of research is the interaction of antioxidants with intestinal bacteria. Such studies should increase our understancling of the role of antioxidants in reducing the production of metabolites with the potential for pro-carci- nogenic activity in the colon. The effects of antioxidants on tumor cell biology, including the ability of cells to elaborate growth factors, proteases, and cell-surface antigens associated with metastasis, also may be explored by new methods of mo- lecular biology, electron spin resonance spectroscopy, and immunocytochem- istry. Little is known about the effects of antioxidants on the activation of cellular oncogenes, another promising area for future research. Likewise, the relationship of oxidants to DNA damage and the ability of antioxidants to promote repair deserve further study. Some recent intervention trials were designed to examine the associa- tion of antioxidant vitamin intake and cancer. Unexpectedly, the antioxi- dant vitamins also benefited sufferers of other diseases, such as heart disease. In cells in culture, oxidative damage to lipids and sterols has been linked to changes in lipoprotein metabolism reminiscent of those that occur in atherosclerosis. Oxidation of lipoprotein fatty acids can stimulate cells of the artery to release mediators that attract monocytes from the blood. Oxidation of apoproteins can trigger lipoprotein uptake by the scav- enger receptor pathway instead of the LDL receptor pathway. There is some evidence that vitamin E and possibly carotenoids in plasma LDL can prevent such oxidation. Antioxidants also may play important roles in tissue repair following injury. Studies of the effect. of ~iet~rv ~ntioxi3nntc on time Renoir in_ ~A. _ _ _ ~ _ . ~ ~ ~ ~ v ~ ~ ~ -~.r ~^ ~ ~ ~ ~ ~ clucking new vessel formation (an~io~enesis) may he airled by n~v~n~?c in microscopy (e.g., confocal), use of molecular probes (e.g., for cytokines and growth factors), and identification of cell surface changes related to cell adhesion or migration (e.g., cell adhesion molecules). The "oxidative burst" of polymorphonuclear leukocytes and macro- phages is a normal process critical to antimicrobial activity. Little is known of the effects of dietary antioxidants on the normal function of these cells. The relationship between nutrients with antioxidant activity and cytokine production also requires investigation in a variety of systems. Recent evi- clence supports synergistic interactions between retinal, formed in cells from beta-carotene, and various cytokines on cell functions, including oxi- dative metabolism and proliferation. The cellular levels at which these ~,, , ~, ~ r 1 1 ~/

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GENETIC, MOLEC ULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 95 effects are exerted (e.g., transcription, translation, and secretion) remain to be learnecl. There are also new opportunities to study the functions and require- ments for sulfur-containing amino acids, seleno-proteins, and metalloproteins as antioxidants. With molecular techniques it should be possible to ex- press specific proteins in bacteria that depend on sulfur or selenium for growth. Incorporation of isotopic sulfur or selenium into newly expressed proteins will facilitate molecular and structural studies. Our future ability to follow metabolism in intact cells using NMR ancl other methods may lead to new understanding of the influence of oxidation state on growth and cell function. Genetic manipulations and transfection of cells to main- tain more oxidized or reducer! states may leas! to new insight into the role of oxidation status on cell proliferation, function, and survival. Clearly, the functions of dietary oxidants and antioxidants pose a great variety of intriguing basic science questions. Research in this area, capital- izing on new techniques for manipulating cells and monitoring their me- tabolism, has great potential for improving our understanding of how dis- ease can be prevented or its onset delayed. Some additional discussion of oxidative damage and its control by diet can be found in Chapters 2 and 5. Retinoic-Acid-Regulatecl Nuclear Receptors The recent discovery of the retinoic-acid-regulated nuclear receptors RAR and RXR and their expression during early embryonic development has opened new opportunities for learning how retinoids regulate differ- entiation and development in many tissues. Retinoid receptors have been identified in neural tissues not previously recognized as targets of retinoid action. A family of genes known to regulate the pattern of development of body parts in lower organisms (e.g., the HOX genes in Drosophila) also play an important role in mammalian development. The discovery that certain genes, first iclentifiec3 by virtue of their rapid response to retinoic acid, are in fact homologous to genes in the HOX family opens the way to a new understancling of when and how retinoicis function in development and how they act as morphogens. Similarly, the discovery of BAR and RXR proteins in embryonic neural tissue has opened the way for cletailec3 studies on retinoid-directect development throughout the nervous system. Information concerning the RAR can also be applied to a better un- derstancting of the mutations related to certain cancers. A hallmark of acute promyelocytic leukemia (APL) is an abnormal chromosomal pattern in which part of human chromosome 17 is translocated to chromosome 15. After the gene for RAR-alpha was identified, it was localized to chro- mosome 17. Investigators found that the transIocation resulted in fusion of a portion of RAR-alpha with another, uncharacterized gene (now termed

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96 i OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES PML) in a number of APL patients. This new information implies that disruption of RAR-alpha has profound consequences on the differentia- tion of blood cells. The future holds great promise for understanding the relationship between abnormalities of retinoid metabolism and certain cancers such as APL. Understanding the roles of RAR and PML in the formation of blood cells is now a high priority in cancer research. Both molecular and metabolic studies may lead to important insights into spe- cific cellular requirements for retinoic acid or other newly identified retinoids during differentiation. Knowing that the actions of the nuclear RAR and RXR are controlled n a concentration-dependent manner by retinoic acid or other retinoids, investigators have refocused attention on understanding the enzyme path- ways through which bioactive retinoids are formed from their nutrient precursors. These enzymic transformations take place in the cell's cyto- piasm, where certain retinoid-binding proteins are also known to exist. New studies have pointed to the importance of these proteins in control- ling the metabolism of vitamin A. They control ligand concentration and direct retinal, retinaldehyde, or retinoic acid to specific enzymes that catalyze important esterification, hydrolytic, and oxidation reactions. Future re- search must address how cells take up retinal or retinoic acid from plasma, how cells regulate the conversion of retinal to retinoic acid, and what types of catabolic reactions prevent the buildup of bioactive retinoids. The relationship of vitamin A nutrition, including the consumption of carotenoids and preformed vitamin A, to cellular retinoid metabolism is not well understood. There are many opportunities for nutritional bio- chemists and cell or cancer biologists to work together to understand how vitamin A and the carotenoids exert their effects on cell differentiation. The demonstration of a link between an abnormal RAR-alpha and APL suggests that other abnormalities of retinoid metabolism might also be linked to cancer susceptibility. Such basic science discoveries may lead to tests to identify individuals with a genetic predisposition to cancer. As noted earlier, providing vitamin A to children at risk of vitamin A defi- ciency decreases their rate of death. There are clearly opportunities for basic studies on the relationship of vitamin A deficiency to immune de- fenses and cellular growth; such studies would contribute to improved strategies for nutritional supplementation for children in vulnerable popu- lations and to our knowledge of the underlying effects of retinoids on the immune system. CONCLUDING REMARKS We have learned much, but there is still much more to be learned! In this chapter, we have tried to convey a sense of some recent accomplish

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GENETIC, MOLECULAR, CELLULAR, AND PHYSZOLOGICAL PROCESSES 97 meets and future opportunities in basic biology related to nutrition sci- ence. One reason for doing so is to demonstrate how new technologies are resulting in more powerful approaches to the resolution of long-standing research problems in nutrition science. These technologies have made possible increased understanding of basic biological phenomena at the cellular, molecular, and physiological levels. Our increased understanding, in turn, provides the intellectual foundation for pursuing future opportu . . notes. Continued technological advances give us the ability to meet our in- tellectual challenges. Two areas of technology have been particularly im- portant in creating the new opportunities. The first of these is the transgenic technology that has grown out of the revolution wrought by the develop- ment of recombinant DNA procedures. The second is the evolution of instrumentation that, with the ever-growing power of computers, permits us to measure the amount and identity of almost any small molecule with precision and exquisite sensitivity and to analyze the structure of large molecules at the atomic level of resolution. We have tried to convey how these two great technologies will contribute, separately and jointly, to the solution of a number of research problems in basic nutritional science. A growing number of these techniques are being applied to important re- search concerns in clinical nutrition, food science, and public health. Sub- sequent chapters provide numerous examples of this sort. - .