Opportunities in the Nutrition and Food Sciences: Research Challenges and the Next Generation of Investigators (1994)

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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

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

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. -

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

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.

.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

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

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

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

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

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.

ss OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES The NMDA receptor also may be involved in the actions of some toxins in food. Several toxins found in food cause the death of brain cells. Overactivation of NMDA receptors may mediate some of these effects. In the Pacific Islands, an endemic neurological disease producing symptoms of amyotrophic lateral sclerosis (ALS), parkinsonism, and dementia has been linked to ingestion of seeds from the cycad plant; these seeds con- tain large quantities of beta-N-methylamino-L-alanine. In vitro, this toxin has excitotoxic properties that are blocked by NMDA receptor antago- nists. ALS has been linked to loss of neurons expressing high numbers of NMDA receptors. Although no history of unusually high short- or long- term exposures to excitatory amino acids has been found in victims of ALS, parkinsonism, or dementia, the NMDA receptor complex may play a role in the etiology of these conditions. A familial form of ALS is caused by mutations in the gene encoding Zn++/Cu++ superoxide dismutase. This suggests that free radicals may modulate the NMDA receptor, disrupt cytoplasmic Ca++ homeostasis, and lead to degeneration of motor neurons. Dietary factors may play a role in the onset of N MDA-linked neurodegenerative diseases. Wernicke-Korsakoff syndrome, for example, is associated with thiamin deficiency and frequently results from alcohol- ism in humans. The impairment of memory and cognitive function charac- teristic of this syndrome is associated with damage to specific sites in the central nervous system. In thiamin-deficient rats, homologous sites in the brain are damaged. The effect of thiamin deficiency can be attenuated with a NMDA receptor antagonist. Among the elderly, altered amino acid receptor function may be caused by altered subunit expression, accumulated dietary deficiencies, altered transport functions for amino acids, or altered dietary intake of amino acicls. One or more of these may be important in senile dementias. Under- standing the function of amino acid receptors in the aging brain is an important area for future research. The availability of molecular probes for the individual subunits of these receptors should facilitate this re- search. Iron Metabolism and Regulation Iron deficiency remains the most common nutritional deficiency in the world, despite the fact that it is preventable with iron supplementa- tion. Much is known about the metabolism of iron, but we still need more research on physiological control mechanisms, regulation of the involved genes, and relative benefits of different forms of iron provided in the diet or as dietary supplements. These issues provide important opportunities for future research. Iron is an essential dietary constituent; from primitive to aclvanced

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 59 forms of life, it is required for aerobic metabolism. Nevertheless, iron is also deleterious because it can catalyze formation of reactive oxygen radi- cals. This property has been exploited in iron-containing drugs to treat .sn~?cific malignancies. Fllrth`?rmor'? high iron intake m:~v inn tier? , It, ~ , ~ , ~ . 1 ~ . · · · ~ . 1 · . 1 1- it_ . 1 growth ot certain microorganisms associated with cLlsease. Consequently, the uptake and metabolism of cellular iron must be carefully regulated. Iron absorption, metabolism, and storage in mammalian systems in- volve a poorly understood interplay among transferrin, the transferrin re- ceptor, ferritin, iron complexes, and transport and storage forms of this ligand. Over 50 years ago, nutritionists showed that absorption of iron is proportional to body needs. The major circulating forms of iron are in erythrocytes as hemoglobin and in plasma as transferrin. Within cells, iron is distributed to the proteins that make up the respiratory chain, enzymes of the cytochrome P-450 system, lipoxygenases, reductases, and ferritin. Transferrin has a high affinity for iron and is the main component of iron transport. Ferritin has a high capacity for iron and is the major compo- nent of iron storage. Transferrin and the Transferrin Receptor Transferrin is a glycoprotein with two iron-binding domains. Expres- sion of the human transferrin gene is stimulated by glucocorticoid hor- mone and cytokines. Regulation of the transferrin receptor is the key to understanding iron transport in plasma. In the transferrin cycle, transfer- rin binds to the transferrin receptor and undergoes endocytosis. In tissues that require a large amount of iron (e.g., erythroid cells and hepatocytes) and during cell proliferation, expression of the transferrin receptor is high. Furthermore, expression is increased when cellular iron concentrations are low, allowing iron-poor cells to acquire more transferrin-bound iron. The transferrin receptor mRNAs have specific sequences in the untranslated region called iron-responsive elements (IRE). A protein called the iron-responsive-element-binding protein (IRE-BP) binds to these IREs. The IRE-BP is also the cytosolic enzyme aconitase that may help regulate Tow molecular weight iron pools via citrate. Affinity of the IRE-BP for the IREs of transferrin receptor mRNA is increased when cellular iron is low (Figure 3.4~. When the IRE-BP binds to the IREs, the mRNA for trans- ferrin receptor becomes more stable, thereby increasing the mRNA con- centration and the rate of receptor synthesis. Increasing the number of receptors increases the uptake of iron bound to transferrin. When iron supplies are sufficient for cellular needs, IRE-BP binding to transferrin receptor mRNA is decreased, so the mRNA degrades more rapidly (i.e., translation decreases). Consequently, synthesis of the receptor is decreased.

60 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES Extracellular Tf-bound Fe | Receptor-mediated Cytoplasmic Fe Pool a_~` Utilized Fe Regulatory Fe Sequestered Fe ~/~ Stabilizes In Ferr~t~n IRE-BP off _ No RNA Binding Active Aconitase Ferritin mRNA IRE-BP (D~ AAAAM 40S AUG IRE occupied by IRE-BP inhibiting translation initiation TfR mRNA 1 1 Inhibits |5~ 1 Ferritin mRNA,Il. | Translation ( _ ~ I IRE-BP on RNA Binding Inactive Aconitase TfR mRNA ? Other IRE-containing mRNAs? IRF-RP One or more IREs occupied by IRE-BP protecting mRNA from rate-determining step in mRNA degradation AAMAA FIGURE 3.4 Cellular iron homeostasis is regulated posttranscriptionally through iron-dependent changes in the IRE-BP. Iron enters dividing cells of higher eu- karyotes via the transferrin receptor. Once inside the cell, the iron may be consid- ered to be in 1 or 3 pools, although overlap and interchange between these pools  may occur. Iron is utilized in a variety of metabolic processes, or iron may be sequestered in ferritin. Intracellular iron also serves to regulate the IRE-BP such that when iron is abundant (e.g., in hemin-treated cells) the IRE-BP is in its f4Fe- 4S] state, which has high aconitase enzymatic activity but low affinity for IREs (IRE-BPoff). When iron is scarce (e.g., in desferrioxamine-treated cells), the IRE- BP is in its high affinity state for RNA binding (IRE-BPon) with negligible aconi- tase activity. The IRE-BPon state results in decreased translation of ferritin mRNA and increased stability of the transferrin receptor mRNA by binding to the IREs contained in these transcripts. The IRE-BP system may also regulate other IRE-containing mRNAs (e.g., mRNAs encoding eALAS and mitocondrial aconi- tase). From Klausner et al. (1993) ~vith permission. Copyright 1993 by Cell Press.

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 61 This is an example of a nutrient that controls the concentration of a pro- tein by regulating the degradation rate of its mRNA. Regulation of Iron Storage as Ferritin The second way in which cells regulate iron metabolism is to control the amount of free iron within the cell. This is accomplished by regulating synthesis of ferritin, an iron-binding protein. Nearly two decades ago, dietary iron was found to stimulate the efficiency with which ferritin mRNA is translated. Within the last few years, investigators have shown that the factor that inhibits translation of ferritin mRNA is the same IRE-BP that binds to transferrin mRNA. In the case of ferritin mRNA, binding of the IRE-BP to the IRE of ferritin mRNA inhibits synthesis of ferritin by inhibiting the efficiency of translation, as opposed to altering the aegraaa- tion rate of the mRNA. When cellular iron concentrations are high, IRE- BP dissociates from the mRNA and translation of ferritin mRNA increases. Understanding the regulation of expression of transferrin and ferritin a _ --Of rid ~ . r . . . ~. ~1 . TO has increased our understanding ot iron nutrition and metabolism. ~ow- ever, both physiological and molecular questions about iron metabolism remain to be answered. For example, how do IRE-BP and IREs influence iron absorption in the intestine? Do they control the availability of iron involved in the production of H'O', a process that leads to formation of deleterious hyaroxy1 radicals? These and other aspects of the transferrin- rerr~n system aL~ok;~Yef~7h~pc)~gn~~~'~rp~Rv~5;~;~1011~' =~^i~i=~ {11_ ture research opportunities. TECHNOLOGIES CREATING NEW OPPORTUNITIES FOR BASIC RESEARCH IN NUTRITION SCIENCE Genetics Manipulation of the Man~mc~lian Genome In prokaryotes, genetics has been a powerful tool for establishing definitive cause-and-effect relationships between molecular mechanisms that regulate enzyme activity and flux through metabolic pathways. Mo- lecular nutrition as a field brings the tools of genetics and genetic engi- neerin~ to the analysis of metabolism in animals. Using the techniques of o __~ ) ~ - - - 1 ·, · 1 gene transfer with both cells In culture and whole animals, it Is now pos- sible to introduce functional promoter-regulatory DNA attached to re- porter genes to (1) analyze mechanisms involved in the regulation of gene expression, (2) express a gene's protein product in a specific subcellular compartment or organ, (3) express measured concentrations of gene prod

62 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES ucts by using regulatable heterologous promoter-regulator DNAs joined to structural genes, and (4) delete specific proteins and replace them with ~1 1 . 1 ~ 1 ~ aiterea genes. These are a few examples of the experiments that modern molecular genetics has made possible in animals and that are applicable to important questions in nutrition approached at the molecular level. The most significant impact of these powerful tools on nutrition re- search has been in the analysis of the regulation of gene expression by hormones and diet. Other areas, such as regulation of theifunctional effi- ciency of proteins, have received less attention. In this section, we will describe methods for introducing normal and mutant DNA into cells and animals. In a later section, we show how these methods might be applied to nutritionally significant research problems. Nutrient absorption and utilization and disease-relatecI nutritional problems involve either altered gene expression or altered functional efficiency of key proteins. The new genetic approaches are critical to understanding in these new fields. Introduction of DNA into cells in culture There are several ways to in- troduce DNA into cells in culture. Agents that precipitate DNA or bind it to liposomes are effective, as is electroporation, a procedure that makes transient holes in the cell membrane. Once inside the cells, foreign DNA probably uses the same cellular machinery that guides infective viral DNA to the nucleus. Initially, the DNA is present as an episomal element, separate from genomic DNA. If the test DNA contains a structural gene with appropriate eukaryotic regulatory elements, it can be expressed tran- siently (i.e., for a few days). Thereafter, most of the DNA is lost by cell division and degradation. DNA taken up by cells in culture is inserted into the genome, usually at random sites, at a frequency of about 1 in 1,000. The low frequency of this event necessitates a selection process to identify and purify cells with integrated DNA. Usually this involves adding a poi- son that is detoxifiec! by a second gene on the test plasmid or by co-uptake of the plasmid containing the test DNA from a seconc! plasmic! containing the detoxification gene. Transiently and permanently expressed transgenes are used for different purposes. Introduction of genes using viral vectors Infective viruses contain nucleic acid encoding the viral genome coated with viral proteins. Depending on the type of virus, the nucleic acid may be either DNA or RNA. Viruses gain entrance to cells by mimicking the ligand for a natural cell-surface receptor. Once inside a cell, the genome of RNA viruses is reverse tran- scribed to produce a DNA copy. Viral DNA makes its way to the nucleus, where the viral genes can be transcribed. For some viruses, the DNA version of the viral genome can become integrated into the host cell's DNA. For some commonly used retroviral vectors, integration of viral

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 63 DNA appears to require ongoing replication of cellular DNA. This limits the usefulness of retroviral vectors to dividing cells. In gene therapy for cancer, this may be an advantage, because in many tissues only the tumor cells are undergoing rapid division. Viral DNAs often contain coding sequences for proteins that are not essential for replication or infection. Once a viral DNA has been cloned. the unnecessary DNA can be replaced with sequences that will generate a product of interest. Once integrated, the viral sequences become part of the cell's genome and are passed to daughter cells at each division. Infec- tion of cells with viral vectors is more efficient than transfection of naked DNA and results in better control of the number of DNA copies intro- duced into the cell. Replication-competent viral vectors, such as those described above, are infective and have the disadvantage that infected cells are continu- ously synthesizing and shedding virus particles. Viral vectors that undergo only a single round of infection are also available anti will be particularly useful for gene therapy of domestic animals and humans. Introduction of genes into animals Cells in culture are good models for testing hypotheses that involve levels of organization simpler than those of intact organs; they clearly are not adequate models for intact animals. Tests of the more complex interactions that take place in whole animals have been made possible by the development of procedures for making transgenic animals. There are two methods for introducing genes into the germline of animals. In one method, DNA is injected directly into the male pronucleus of the fertilized egg. The second method involves inser- tion of the DNA into pluripotent embryonic stem cells in culture, fol- Towed by injection of the transgenic cells into (leveloping embryos. The latter procedure is less efficient, but it can be used to inactivate specific host-cell genes by homologous recombination (gene knockout) and will be explained in a later section. Direct injection of fertilized eggs has been used in mammals, fish, and amphibians. Fertilized eggs of these organisms are large enough to permit injection of DNA directly into a pronucleus. Integration of the injected DNA into the host's genome occurs cluring subsequent cell divi- sion. Numerous lines of transgenic animals have been developed using this technology, and these animals are used for a variety of experimental and commercial purposes. Overexpression of growth factors or hormones can lead to changes in body size and composition. Overexpression of spe- cific apolipoproteins has profound effects on lipid metabolism, suggesting possible therapeutic strategies for human hereditary hyperlipiclemias. Other examples of disease-related advances will be discussed in a later section.

64 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES Why transgenes are useful Transgenes have two key uses. Defining thefurlction of upstream regulatory regions In most genes, DNA upstream (in the 5' direction with respect to the coding strand) from the transcription start site contains a promoter. This element binds RNA polymerase and initiation factors, determines the nucleotide at which tran- scription will start, and bestows a basal rate of transcription on the gene. Actct~t~ona~ upstream regions contain nuc~eot~ue sequences that act in cis (i.e., are present in the same molecule) to regulate transcription. These vary as a function of cell type, nutritional state, hormonal status, and pharmacological conditions. Cis-acting regulatory elements are, in turn, regulated by trans-acting proteins (diffusible regulatory molecules) that bind to the DNA sequences or to proteins bound to those sequences. The concentrations of most proteins are regulated by controlling the initiation of transcription of the corresponding genes. An important step in molecular analysis of the signaling pathways by which hormones or nutritional states regulate protein concentration, therefore, is identifica- tion and characterization of cis-acting DNA elements and trans-acting proteins that regulate transcription. Knowledge of the structure and func ~ 1 1-. - 1 . . . . 1 .. ~ _ - 1 tion of regulatory elements is also essential for constructing vectors that will drive expression of structural genes in specific cell types under spe- cific physiological or pharmacological conditions both in cells in culture and in intact animals. The cis-acting elements can be defined functionally by introducing them into an appropriate cell type linked to a reporter gene. The reporter gene usually encodes an easily assayed enzyme or protein that is not nor- mally expressed in vertebrate cells. The bacterial gene for chloramphenicol acetyTtransferase and the insect gene for luciferase are examples. By sys- tematically testing mutated versions of the regulatory DNA, we can define the nucleotide sequence required for a given response. A steroid response element confers sensitivity to cholesterol on the gene for 3-hydroxy-3- methylglutaryl coenzyme A (lIMG-CoA) reductase and has been defined in this manner. Testing the function of specific proteins 1 ~- (~ _ Different forms of struc- tural genes can be tested by introducing them into cells in culture using transient or permanent transfection techniques or by constructing transgenic animals. The expression of such genes can be regulated by an inducible promoter or can be maintained at a high, essentially unregulated level with viral promoters. The function of gene products can be studied by introducing the gene into a cell type that normally does not express that product. For example, a trans-acting factor postulated to activate tran

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 65 scription in a particular tissue might be introduced into a different cell type along with an appropriate regulatory sequence linked to a reporter gene. Expression of the reporter gene would be evidence that the factor was required for transcription. By introducing mutant versions of the gene for the trar~s-acting factor, structural requirements for the function of the trans-acting factor could also be tested. A similar protocol could be de- vised to analyze structure, function, and regulation of any gene product for which a suitable assay system could be devised. This technology was used to identify retinoic acid receptors (see page 511. Identifying the mechanisms by which nutrient intake regulates the function of trans- acting factors will be critical to understanding how nutrients regulate gene expression. by, A, ~_ (, Deletion of specific gene products Just as important as the ability to express or overexpress a particular protein is the ability to suppress ex- pression. Three general methods have been developed: dominant negative mutations, antisense RNA or DNA, and homologous recombination. The ability to delete the protein products of specific genes provides the nutri- tion research community with opportunities to examine the roles of these proteins in nutrient function. Dominant negative mutations This strategy requires that the prod- uct of the targeted gene be a multimeric protein. A mutant version encod- ing a stable but inactive subunit is overexpressed by one of the means described above. If the ratio of mutant to wild-type subunits is high enough, virtually every multimer will contain one or more mutant subunits and the resulting multimers will be inactive. Antisense RNA or DNA To suppress gene expression using antisense RNA, researchers insert into the cell a transgene that expresses an RNA complementary to the target messenger RNA. At sufficiently high ratios of antisense RNA to mRNA, the mRNA will be partially double-stranded and translation will be inhibited. Generally, this technique is successful only for target mRNAs expressed at low levels. Antisense oligodeoxynucleotides work on a similar principle but are added to the outside of cells. Once inside the cell, the oligomers hybridize to the target RNA and inhibit translation or stimulate degradation of the mRNA. Stability and efficiency of uptake are potential problems, but new methods are being developed to resolve these difficulties without altering the specificity or affinity of the antisense DNA for its complementary RNA. Antisense oligodeoxynucleotides are important experimental agents at the present time, and they are widely expected to become important therapeutic agents in the future.

66 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES Ilomologous recombination This procedure permits researchers to target specific mutations to specific genes. DNA introduced into cells readily recombines with the host cell's genomic DNA. In most cases, the recombination does not have sequence specificity; the foreign DNA is inserted randomly. In rare instances, however, recombination is sequence- specific and the introduced DNA is inserted at exactly the same location as its endogenous homolog. Strategies have been developed to purify the cells with homologous recombination events from those containing the more frequent random, nonhomologous events. Additional developments were necessary before scientists could use homologous recombination to produce intact animals (mice) with targeted mutations. Primary cell lines were established from the pluripotent stem cells of the mouse embryo. These cell lines maintain an undifferentiated pluripotent state even after substantial manipulation. Embryo-derived stem (ES) cells containing a disrupted target gene are isolated by one of several procedures and injected into the blastocoele of a mouse embryo. The manipulated embryo is implanted in the uterus of a pseudopregnant fos- ter mother, and development proceeds to term. If successful, the result- ing mouse will be a chimera composed of cells derived from the mutated ES cell line and the recipient embryo. Typically, the ES cells are clerived from a mouse with one coat color and the recipient embryo from a mouse of another coat color so that the chimeric offspring will have patches of both colors and be easily detectable. If the chimerism extends to the germ cells, interbreeding will yield animals homozygous for the desired muta- tion. Gene knockout provides the opportunity to study the consequences of designed mutations of selected genes in an intact animal. This enables investigators to use genetic methods to dissect such complex integrative processes as development, nutrition, and disease pathophysiology. Mice with targeted disruptions, or knockouts, of several genes have been pro- duced. Genes presumed to have important roles in development and genes known to be defective in certain hereditary human diseases have been the favorite targets so far. Structure-function studies in the natural environ- ment of a gene product will be of great value in nutrition-related studies. Once a knockout has been achieved, mutant versions of the gene product can be introduced by standard transgenic methods. Tissue specificity can be ensured by using the appropriate promoter-enhancer region to drive transcription of the mutant transgene. Gene knockout work is still in an early stage, and we anticipate increased activity in the years to come with improvements in homologous recombination and ES cell technologies anc! the application of these approaches to adclitional animal models.

GENETIC, MOLEC ULAR, CELL ULAR, AND PHYSIOLOGICAL PROCESSES 67 Approaches to Analysis of Multifactorial Traits Genetic disorders are typically classified into three groups. The first includes the monogenic disorders, caused by defects in a single gene and inherited in classical Mendelian fashion as autosomal dominant, autoso- mal recessive, and X-linked traits. Over 5,000 monogenic traits have been recognized. Nearly all are rare, with frequencies in the general population of less than 1 per 10,000. Phenylketonuria, cystic fibrosis, and Tay-Sachs disease are well-known examples. The second group of genetic disorders involves abnormalities of chro- mosomal structure or number or both, in which many genes are present in abnormal amounts. Although as many as 10 percent of all conceptions yield a fetus with a chromosomal aberration, the vast majority of chromo- somally abnormal conceptuses are aborted spontaneously. Trisomy 21, or Down's syndrome, is an example that frequently survives this in utero selection. Multifactorial disorders account for the third and most challenging group of genetic diseases. For the most part, these are common diseases of adult life that tend to run in families but follow no simple inheritance pattern. Examples are obesity, hypertension, diabetes, and hyperlipidemia. Multifactorial diseases result from the interplay of multiple genes and environmental factors, many of which are nutritional. There are multiple susceptibility genes for each disorder, and the genetic determinants among indivicluals may differ. This combination of great complexity and high frequency makes the multifactorial disorders a daunting, challenge for medical and nutritional investigators. Recent advances have enhanced our ability to identify the genes that contribute to multifactorial phenotypes. First, the human gene map has grown more dense and informative. The recognition and characterization of a large number of highly polymorphic microsatellite markers at sites spread evenly about the map is particularly important. These markers can be analyzed rapidly and are highly informative, so genetic linkage studies can examine nearly every region of the genome with high resolution. The second major advance has been the development of statistical methods to study complex non-Mendelian traits. Earlier methods required large, well-characterized multigenerational families, a particularly difficult problem for disorders that appear in middle-aged people in a highly mo- bile society. The development of multipoint linkage analysis, using an affected-sib-pair method, makes it possible to analyze small nuclear fami- lies with two or more affected siblines and unaffected parents. The method is based on the notion that affected relatives share alleles for a suscepti- bility gene at a higher frequency than that predicted by their genetic relationship. The method requires a large number of nuclear pedigrees,

68 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES but provides a robust and workable method for detecting disease suscepti- bilitv Genes. ~ O Advances in the study of animal models, particularly mice, have also , ~ , been of great value for the identification of susceptibility genes in hu- mans. Elucidating the extensive similarity between human and murine genomes makes it possible to predict with reasonable confidence where a gene homolog will map in the one species, given its location in the other. Thus, identification of a susceptibility gene in mice quickly points to a chromosomal location in humans. Using this method, researchers are making progress in identifying genes involved in obesity, hypertension, and non- insulin-dependent diabetes mellitus. Clarifying the genetic factors that contribute to the development of multifactorial disorders will provide us with a better understanding of the environmental factors. The latter may be more amenable to change, and we can anticipate a time when individuals will be informed of their own particular genetic susceptibilities and advised as to how to adjust their diets and other behaviors to compensate for these susceptibilities. Identification, Isolation, and Tracking of Specific Cell Types Identifying specific cell types during development, pathology, or in culture systems where their normal morphology or relations with other cells may be disturbed is a particularly pressing problem for investigators. Frequently, cells must be removed from their normal environment for experiments, or they must be grown in culture to obtain adequate amounts of tissue for analysis or experimentation. Several techniques are used to Cent phenotype In such situations. Most of these depend on a particu- lar molecular species being diagnostic for a particular cell type. . 1 ..r 1 Monoclonal Antibodies 1 . . ~, Monoclonal antibodies are homogeneous immunogIobulins secreted by an immortal line of hybridoma cells formed by fusing a normal B- lymphocyte and one of severalwell-definecl myeloma (tumor) cell lines. Several attributes of monocTonal antibody technology make it useful for identifying specific cell types. For example, in order to generate a mono- clonal antibody, as opposed to a polyclonal antiserum, it is not necessary to purify the antigen. Purified antigen or crude homogenates containing the antigen of interest can be injected into mice or rats. Cells from the animal s spleen or lymph nodes or both are fused with a myeloma line that supplies enzymes critical to the use of an alternative pathway for synthesis of thymine. Successful somatic cell hybrids are selected by growth on a medium that selects cells capable of using the alternative pathway. Cell

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 69 extracts are assayed by enzyme-linked immunosorbent assays (ELISA) or immunohistochemistry to determine which of the surviving colonies pro- duce specific antibodies. Monoclonal antibodies thus provide a method for identifying cell types that express rare or difficult-to-purify antigens. Monoclonal antibodies are also used to screen libraries of recombinant bacteria containing eukar,votic cDNAs or genomic DNAs. They can iden- tify recombinant bacterial clones that express specific proteins. In addition to identifying specific cell types, monoclonal antibodies can detect specific antigens in serum and tissue extracts and facilitate assessments of nutrient status. As we learn more about plasma antigens as tools for assessment of nutrient status, banks of monoclonal antibodies will become extremely valuable tools to the nutrition scientist. Visuati~ation Histochemistry, immuno1~istochemistry, and radioimmunoassay If a cell type produces a particular enzyme or hormone, its phenotype can be identified by histochemistry, antibodies against the specific molecule, or radioimmu- noassay. For example, histochemistry for alkaline phosphatase and radio- immunoassay for gonadotrophic hormone have been used to identify the phenotypes of placental cell lines. Antibodies against different keratins have been used to identify various epithelial cell lines. In situ hybridization In situ hybridization uses antisense RNA as a probe to localize cells expressing mRNA for a particular gene of interest. In this technique, radioactively or chemically labeled nucleotide sequences (probes) hybridize specifically to their complementary nucTeotide strands. These probes can be used to examine a complex tissue or organ to determine which cells are expressing a particular gene. In situ hybridization is used, for example, to show where in the brain transcripts for various neurotransmitter subunits are expressed and how they change with time. The new limits of microscopy Over the past two decades, the explosion in computer and laser technology has produced a revolution in micros- copy that affects all fields of biological science. The new technology makes possible resolution and analyses with light microscopes that are well be- yond the capabilities of standard optical microscopes. Most of these tech- niques use the light microscope to obtain an image, then digitize the image using a video microscope, and finally process the digitized image to improve spatial resolution and increase signal cletection. The initial stages of signal gathering (a microscope and good objec- tives) are similar for all varieties of image analysis. Coherence of the light representing the image, filtering of diffracted and scattered light, spatial -

70 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES resolution, spectral sensitivity, and temporal response of the system are variables that differ dramatically in the new forms of digitized microscopy. Silicon-intensifier video cameras are used for signals with low light inten- sity, such as those obtained in many kinds of fluorescence microscopy. High-resolution and charge-coupled crevice cameras are used for high spa- tial resolution. Depending on the computer hardware and software avail- able, the signal can be amplified relative to "noise" at a chosen level, and various aspects of the image, its movement, fluorescence, or density can be analyzed. The processed image can be saved either on an optical moni- toring disk recorder or on a videotape recorder. This technology makes possible dynamic imaging of cell migration. Scientists can follow and experimentally manipulate organelle movement in the cytoplasm of cells and can watch the polymerization of actin and microtubules. Silicone-intensifier cameras permit biologists to analyze liv- ing cells at exceptionally low light levels, thus avoiding the heat ant! light damage usually encountered with standard microscopy. These same cam- eras, in association with fluorophores specifically designed to fluoresce in the presence of certain ions, also permit cletailec3 visual analyses of fluxes through membrane channels or from intracellular stores. Much of the activity with ion imaging has concentrated on subcellular analysis of cell motility and transmission of neural signals. However, there is an enor- mous potential for new information at the interface of nutrition science and cell biology. In particular, these techniques will apply to specific transport systems and to receptor-mediated endocytosis in polarized epi- thelial cells. The laser-scanning confocal microscope reduces the collection of scattered light by sandwiching the specimen between two lenses that focus an en- trance and an exit pinhole on the same (confocal) point in the specimen. Resolution is further enhanced by the coherence of laser illumination. These microscopes produce thin optical sections of fluorescence images that eliminate out-of-focus fluorescence. In larger fields, we can examine fine detail in thick sections and living tissue. Moreover, series of optical sections can be stored in the memory of the image processor so that, in a matter of seconds, alternative views, or stereopairs, of an image can be presented on a monitor screen. Confocal microscopy can also produce digital movies of subcellular events such as mitosis and changes in cellular Ca++. These new techniques, taken together, reveal the organization of cells and tissues with resolution and specificity that were unimaginable even a decade ago. It is now possible to see the transmembrane proteins of the brush border cells of the intestine with submicron resolution and to follow the intracellular migrations of vesicles and vacuoles as cells take up mate- rials from the outside. These and other applications will provide exciting

GENETIC, MOLEC ULAR, CELL ULAR, AND PHYSIOLOGICAL PROCESSES 71 new opportunities to analyze the interactions of nutrients, nutrient trans- port, and cell function. Tczgging acid following cells in living tissue Tracking the movements of or morphological changes in specific cell types in complex tissue is a difficult problem. Ideally, the markers should be suitable for use with living tissue, give high resolution, and allow continuous monitoring. One approach, if cell surface antibodies are available, is to label these antibod- ies with a fluorescent tracer. In thin embryonic tissues or in organs or tissue slices in culture, these labeled cells can then be followed with a confocal microscope without phototoxic damage. A more direct approach to following cells is being used to trace the development and migration of cells in the nervous system. Nontoxic, fluorescently tagged dextran is in- jected into individual cells in organs or slices in culture. It also may be possible to label cells using retroviral vectors to deliver specific reporter genes to specific cell types. Fluorescence-ActirczLed Cell Sorting Fluorescence-activated cell sorting (FACS) is a technique that sepa- rates a population of heterogeneous cells into separate groups on the basis of their relative fluorescence. If particular cells or cell type-specific mol- ecules can be labeled fluorescently with any of the techniques mentioned above, and if sufficient quantities of cells are available, an investigator can obtain a reasonably pure population of the labeled cells. The FACS tech- nique, underutilized at present, could be tremendously powerful in con- junction with techniques for immortalizing cells, because it would permit the small number of specifically tagged cells recovered from FACS of a complex tissue to be expanded for biochemical, genetic, molecular, or cell biological analyses. Cell and Tissue Culture Systems Nutrition requires an eclectic approach to studies at the cellular and molecular levels. Cell lines and culture systems make possible experimen- tal control of multiple variables and the gathering of large amounts of homogeneous tissue. Useful culture systems are those that closely mimic the state of the cells of interest in the whole animal. Nevertheless, find- ings must be verified by comparing them with observations in the intact organism. Three kinds of culture systems are used. Mass maintenance cultures are used for differentiated cells that do not grow in culture. They are usually limited to a few days in culture and may be obtained by colla- genase perfusion of intact organs. Primary cells grow in culture and sur

~2 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES vive numerous passages; ultimately, however, they senesce and die. Per- manent cell lines contain immortal cells; these cells do not senesce and can be maintained in culture indefinitely. Immortczli~.ation of Cells Five methods are used to obtain continuously propagating cell lines: (1) growth in culture of cells isolated from tumors, (2) isolation of immor- tal cell lines from primary cell cultures, (3) transformation of cells in culture with carcinogens, (4) somatic cell hybridization of normal, termi- nally differentiated cells with a definecI tumor cell line, followed by selec- tion and subcloning in a selective medium, and (5) immortalization of a terminally clifferentiated cell type by introduction of an oncogene, fol- lowed by subcloning and selection of a line on the basis of some pheno- typic characteristic. The technologies described in the previous section can be used to develop cell lines with methods 4 and 5 because they provide a means of selecting, from complex tissue, the type of terminally differentiated cell to be hybridized or transformed. They also provide means of selecting continuously propagating lines on the basis of the mol- ecules they express. In addition, the availability of cloned oncogenes and oncogenic viruses makes it possible to use method 5 to develop cell lines from terminally differentiated cells. Many cell lines have been derived from tissues of potential interest to the nutrition scientist. One established cell line, Caco-2, deserves special mention in the context of nutritional studies. This human intestinal cell line was established from a human adenocarcinoma and has many of the properties of differentiated intestinal absorptive cells, or enterocytes. Many other tumor- or intestine-derived cell lines fail to maintain a clifferenti- ated state. Caco-2 cells, however, spontaneously assemble a brush border when cultured on a filter and grown to confluence. They express normal intestinal brush border proteins such as hydrolases, alkaline phosphatase, and amino- and dipeptidases. They have secretory proteins, growth factor responses, intestinal polypeptide receptors, an(l transport systems that ap- pear similar to normal human fetal enterocytes. Additional well-differenti- ated cell lines are essential to progress in several areas of nutrition and food safety research. The American Type Culture Collection maintains a large collection of cell lines. Complex Culture Systems Normal endodermaT cells may differentiate into intestinal epithelial cells if the endoclermal cells are cultured with fibroblasts or undifferenti- ated mesenchymal cells. Unfortunately, actual contact between the two

GENETIC. MOLEC ULAR, CELLULAR, AND PHYSZOLOGZCAL PROCESSES 73 cell types is required, making it difficult to obtain pure cell types for biochemical studies. Undoubtedly, changes in the extracellular matrix caused by one cell type influence differentiation of the cocultured cell type. Another complex culture system has been used to study differentiat- ing neural epithelia. In this system, 200- to 400-micron slices of tissue are cultured on a plasma clot, usually at an air-water interface. Neurons and glia from various regions of the fetal or neonatal central nervous system and retina appear to undergo relatively normal differentiation in such cultures, providing the investigator access to the cells for visual monitor- ing or perturbation. Differentiation and transport properties of various epithelial tissues may be amenable to analysis in such systems. Animal Models The ultimate goal of biological research is to understand how indi- vidual genes and their products function in the intact organism, interact- ing with other genes and gene products in the complex networks required to program and achieve normal development and physiological homeosta- sis. Similarly, medical researchers want to understand how defects in one or a few genes disrupt normal development and physiological homeostasis to produce genetic disease and how to devise effective therapies. The integrative approaches required to reach these goals will depend heavily on animal models of genetic diseases. Great progress has been made in the methods for producing animal models and in the ways in which these models can be used to provide insight into normal and abnormal human biology. Much of this progress has involved the laboratory mouse, Mus musculus. Over the last century, mouse geneticists identified and characterized scores of moclels resulting from naturally occurring mutations or from selective breeding strategies aimed at producing animals with phenotypes such as obesity or hyperten- sion. Ambitious strategies to mutagenize mice with agents such as N- ethyl-N-nitrosourea and scrutinize their offspring for mutant phenotypes have paid off with identification of several important new models (Table 3.1~. More recently, the rapid development of technologies to manipulate mouse gametes and early embryos has opened the way to rational, preplanned development of specific models and greatly reduced dependence on the serendipity of other methods. Introduction of disease-producing genes into mouse zygotes has enabled researchers to develop animal models of atherosclerosis, anemia, and other common ailments. Alternatively, as de- scribed earlier in this chapter, specific target genes can be clisruptecl or modified by homologous recombination (gene knockout) in embryonic stem cells. Models of cystic fibrosis, Gaucher's disease, anct several other disorders have been proclucec! in this fashion. Although most of this work

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GENETIC, MOLEC ULAR, CELL ULAR, AND PHYSIOLOGICAL PROCESSES 75 has been done with mice, important models have been developed in other species. Animal models are extraordinarily valuable and are used in madly ways. First, as outlined earlier (see page 66), genes responsible for a variety of monogenic and multifactorial disorders can be identified. Animal models also are valuable in sorting out the genetic and environmental contribu- tions to complex multifactorial traits. For example, investigators are be- ginning to use genetic linkage methods to identify the genes responsible for hypertension and obesity in rat and mouse models. The contribution of these same genes to the corresponding human condition can then be examined. The final proof that genes identified in this fashion contribute to a particular multifactorial trait can be obtained from transgenic and gene knockout experiments in mice. Several genes thought to be involved in atherosclerosis have been tested in this fashion; overexpression of the human LDL receptor gene protected mice against massive cholesterol feeding, while knockout of the apolipoprotein E gene confirmed its role in cholesterol homeostasis and atherogenesis. A third use of animal models is to elucidate the pathophysiology of disease. For many human genetic disorders we know the clinical features and have identified the responsible gene and many causative mutations. Nevertheless, there is still much uncertainty regarding pathophysiological mechanisms. Genetic heterogeneity, variation in clinical severity, and dif- ficulty in obtaining samples of the affected tissues at all stages of the illness contribute to this lack of understanding. For example, the clinical features, biochemical abnormalities, and molecular defects in phenylketonuria (PKU) are well known, but the mechanisms) by which elevated blood phenylalanine produces mental retardation, the principal phenotypic fea- ture of PKU, remains unknown. The recently described Pah~lP~l~5 mouse appears to be an excellent model for PKU. lIomozygous animals have less than 3 percent of normal hepatic phenylalanine hydroxylase activity, and phenylalanine in the drinking water causes hyperphenylalaninemia and urinary excretion of phenylketones. Studies of neurotransmitter metabo- lism and myelinization in these animals may provide insight into the patho- physiology of the mental retardation in human PKU. A final important use of animal models is to develop and test disease treatments. This includes a variety of conventional therapies, including nutritional manipulations, pharmacological interventions, organ transplan- tation, and other forms of surgery. The results and experiences with trials in animal models guide the design of studies in humans with the same disorders. The recent explosion of interest in strategies for gene therapy is closely tied to availability of animal models, as discussed elsewhere in this chapter.

76 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES Imaging Technologies for Metabolic Studies Traditional approaches to elucidating the relationships between nutri- tional variables and intracellular metabolism have involved repeated sam- plin~ of accessible body fluids such as blood and urine or direct assays of tissue samples. These methods have limited value for studying metabolism in humans because most tissues cannot be easily and repeatedly sampled. Understanding the rapidly changing, region-specific metabolism of the central nervous system is a prime example of this challenge. Two new technologies offer investigators the opportunity to examine metabolism of the human brain and other tissues in real time. Positron emission tomography (PET) uses positron-emitting radionuclides CHIC, i50, i3N, and others) that are incorporated into metabolically important mol- ecules such as water or glucose. After administration of tracer amounts, these compounds are detected by paired, integrated crystal detectors po- sitioned outside the body. With the assistance of a computer, these signals are reconstructed into a three-climensional image of the organ. Quantita- tive estimates of the metabolism of labeled compounds can be determined from metabolic rate constants, which, in turn, can be calculated from consecutive scans. PET is sensitive and painless, but it does expose the subject to radiation and requires an on-site cyclotron to produce the short- lived positron-emitting isotopes. Nuclear magnetic resonance (NMR) is the second technique. Certain atomic nuclei become energized when placed in a magnetic field. When the field is relaxed, a detectable signal is created as the nuclei dissipate their energy by oscillating briefly at a characteristic radiofrequency. The resulting data can yield three-dimensional images (diagnostic NMR imag- ing) or quantifiable spectra. The latter can be used for regional assess- ment of metabolism (NMR spectroscopy>. For clinical NMR spectros- copy, 3iP and iU metabolites are observed in defined regions (e.g., the brain). NMR is less sensitive than PET, but it is harmless and can be repeated many times. PET and NOR continue to be refined to enhance sensitivity, expand applications, and, in the case of PET, reduce exposure to radiation. Even at their current stages of development, however, both technologies provide real-time analysis of regional metabolism in complex tissues and organs. Approaches to Determining the Structure of Macromolecules Understanding the molecular basis of the function of enzymes, recep- tors, structural proteins, and other important macromolecules requires that we understand their three-dimensional structures. The most powerful method for determining the three-dimensional structures of molecules,

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 77 with or without bound effecters, is X-ray diffraction. This approach re- quires a homogeneous protein and formation of a protein crystal, and it allows us to characterize the structure at near-atomic resolution. In this technique, an X-ray beam is diffracted by the electron cloud surrounding the atomic nuclei in the crystal with an intensity proportional to the number of electrons around the nucleus. From the distribution of electrons within the molecule, we infer positions of the nuclei. The dif- fraction patterns are recorded on photographic film or electronic area detectors. The amplitude and phase angle measurements of the diffracted radiation are mathematically reconstructed by Fourier synthesis, which in turn generates a three-dimensional electron-density map of the diffracted object. When thousands of reflections are collected and analyzed, resolu- tion at 1.4 angstroms can be achieved. Using advanced computer graphic techniques, the known amino acid sequence of the protein is fitted to the electron-density pattern. The primary structure adds a large number of constraints in the form of allowable bond angles and known volumes occu- pied by side chains. Refinement of the three-dimensional structure is obtained by aligning the amino acid sequence to the electron-density map to obtain the best fit. Proteins do not function in viva in a crystalline environment. The physical structure of proteins in solution has been more difficult to ap- proach, but it is now being attacked by techniques such as nuclear mag- netic resonance (NMR) imaging. Like X-ray crystallography, high-resolu- tion structural analysis of proteins in solution requires knowledge of the protein's amino acict sequence. At present, NMR spectroscopy is limited to small proteins, but it is an advancing field and should be capable of greater and greater resolution with more and more complex proteins. NMR measurements suggest that the crystal structures are good approximations of protein structures in solution. Prior to the advent of recombinant DNA technology, amino acid se- quences were determined directly, a laborious procedure that required many months for even small proteins. Primarily for this reason, only a few three-dimensional structures had been discovered. Now, the amino acid sequence of a protein can be deduced from the nucleotide sequence of its cloned complementary DNA. The recombinant DNA revolution also pro- vided a way to produce large quantities of proteins, even those that are present at very low concentrations in tissues or are difficult to purify from natural sources. The cloned complementary DNAs are linked to promoter- regulatory DNAs of either bacterial, insect, yeast, or vertebrate origin and inserted into the appropriate host. Under appropriate conditions, the host will overproduce the encoded protein, facilitating purification and result- ing in high yields of the purified protein. These developments greatly accelerated the pace of structural determinations. Two other develop

IS OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES meets also stimulated the increase in structural analyses automated data collection and advances in computerized analysis of the collected clata. FUTURE OPPORTUNITIES We have chosen some examples of future opportunities for research In basic nutritional science. Earlier in this chapter, we described recent accomplishments in the basic biological sciences relevant to nutrition and some of the new technologies that are, in part, responsible for these re- cent breakthroughs. It is this new understanding of biochemistry, molecu- lar biology, and cell physiology and the development of powerful new technologies that make it possible to describe these opportunities. Many of these opportunities involve increasing our understanding of molecular, cellular, and physiologic processes that are influenced by nutritional state. In some cases, the opportunities involve the development and perfection of techniques that will lead to greater understanding of the basic pro- cesses as well as to effective therapeutic strategies to treat human disease. Many important health and disease problems linked to nutrition are dis- cussed in detail in Chapter 5. In this chapter, however, we have not linked each opportunity identified below to a specific human disease or health problem. Manipulation of the Mammalian Genome to Understand Gene Function and Normal Metabolism The ultimate goal in analyzing metabolic regulation is to determine the molecular basis of physiological events. This requires a physiological milieu for conducting the analysis and methods for establishing cause- and-effect relationships between organ-specific metabolic processes, ex- tra- and intracellular effecters, molecular changes in key enzymes, and altered flow of metabolites through metabolic pathways. New molecular genetic approaches can be used to accomplish those goals. Some examples particularly relevant to nutrition are described below. Rate-limiting En~.y~nes in Metabolic Pathways Conversion of a nutrient to a metabolically useful product often re- quires the concerted action of many enzymes. Each enzyme usually cata- lyzes a single chemical transformation, but the pathway leads ultimately to a product greatly different from the starting nutrient. Tightly controlled regulation of the flux of carbon and nitrogen through metabolic pathways makes it possible for the constant energy demands of a living organism to be met by its irregular eating patterns, which vary greatly with respect to

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 79 both quality and quantity. Understanding the molecular means bv which the catalytic activity of pace-setting enzymes is regulated is crucial to understanding the regulation of flux through an overall pathway and to designing ways to correct pathophysiological changes that cause disease. Correlations between the concentrations of intermediates in meta- bolic pathways and the flux of carbon through those pathways have pro- vided indirect evidence that some key enzymes are rate-limitin~ in their ~ ~ ~ ._1~ ~1: ~ ~ ~11~ . ~ ~1_ ~ ·1 · . . · . - . mera~o~c patnways.` 1ne anility to ~nachvate a single gene by homologous recombination should permit unequivocal identification of such enzymes and extensive characterization of the molecular mechanisms involved. The process would start with insertion of a mutation into the Acne fir ~ n~t~- ~-~ rem . - . 1 · · . · r~1 1 . ~ , , ~ , rive rare-~m~t~ng enzyme. the resulting transgenic animal should have a null phenotype with respect to that enzyme activity. The issue of rate limitation could be tested in one or both of two ways. First, the structural gene for a normal enzyme could be linked to a promoter-regulatory region regulated by a mechanism not usually used by the gene. This transgene would be inserted into the genome of the null mouse by the egg-injection method. Graded levels of expression of the protein would be obtained by treating the mouse with an agent specific to the promoter-regulatory re- gion linked to the structural gene. The other possibility would be to link the promoter-regulatory region of a constitutively expressed gene to a set of structural genes that specified mutant enzymes of different catalytic efficiencies. This would create strains of mice that express different levels of enzyme activity. In either model, changes in pathway function that paralleled changes in catalytic activity of the putative rate-limiting en- zyme, when those activities were in the same range as those in normal mice, would provide compelling evidence that the enzyme was a rate- limiting step in the pathway in Volvo. - . . Physiological Relevance of Regulatory Phenomena Characterized with Purified Proteins The catalytic activity of many purified enzymes is regulated by me- tabolites that bind to the enzyme noncovalently. Often, physiologically induced changes in the concentrations of the relevant metabolite in vivo correlate with changes in flux through a metabolic pathway, consistent with the allosteric regulation deduced from action of the metabolite on the purified enzyme in vitro. Such correlations, however, do not consti- tute proof that flow through a pathway is regulated by controlling the catalytic activity of the enzyme involved. The new analyses would begin with characterization of the physical and kinetic properties of purified proteins by site-specific mutagenesis. This process could also lead to iden- tification of those amino acids in the overall sequence that are essential

so OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES for specific, high-affinity binding of the metabolite to the enzyme. Ho- mologous recombination would then be used to knock out the natural gene in intact mice. A mutant form of the enzyme that cannot bind the effecter could be reinserted into the null mice by egg injection. If the effecter is physiologically important, the normal regulatory phenomenon should be absent in null mice expressing a site-specific mutant that cannot bind the effecter. The physiological relevance of phosphorylation-dephos- phorylation another common mode of regulation of rate-limiting enzymes in intermediary metabolism, in transcription regulation, and in other cel- lular processes could be analyzed using a similar strategy. Identification of the physiologically relevant regulatory mechanisms would be only the first step in such an analysis. Characterization of the structural require- ments for function of both the regulated protein and the regulatory pro- tein or ligand would then follow. r. 1 . Intracellular Signaling Pathways The influence of diet on intracellular metabolism is often mediated by changes in the concentrations of hormones in the blood. When a hormone binds to a specific receptor on the exterior surface of a cell's plasma membrane, it sets in motion a series of events that ultimately leads to a biological response. That response could be a change in catalytic effi- ciency of a specific enzyme, in rate of transcription of a specific gene, or in the rates of numerous other processes. A series of molecular events along a signaling pathway connects the extracellular event to the eventual biological response. Proteins involved in intracellular signaling can be identi- tied in a manner analogous to that described above for identifying pace- setting enzymes in metabolic pathways. The physiological significance of putative molecular mechanisms can be tested as described above for regu- lation of the catalytic efficiency or phosphorylation of pace-setting en- zymes. Such information is essential for designing drugs or nutritional therapies to restore normal signals in pathways disrupted by disease or genetic mutations. Importance of Tissue-specific Expression in Metabolism Many enzymes en cl transcription factors, and in some cases their isoforms, are expressed tissue-specifically. Different isoforms of the same enzyme or transcription factor may be selectively responsive to the metabolites characteristic of a given tissue. Eliminating the gene for a specific isoform of an enzyme by homologous recombination would provide mice with a null background for testing the validity of this hypothesis. Mixing and matching the structural genes for the different isoforms linked to tissue

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES S1 specific promoter-regulatory regions and expression of them in a null back- ground will provide the test itself. Similarly, expression of enzymes and transcription factors in inappropriate tissues can test hypotheses about their roles in interorgan metabolism. Gene Therapy Treatment of genetic diseases by conventional strategies has proven difficult. Many of the successes have involved nutritional approaches to inborn errors of metabolism. Nevertheless, the majority of genetic dis- eases have resisted our therapeutic efforts. The opportunities provided by the advances in molecular biology have focused attention on a more direct approach to treating genetic disease, namely, gene therapy the introduc- tion of a functional gene to replace or supplement the activity of a defec- tive gene. Two gene therapy strategies that differ in the nature of the recipient cells have been contemplated germline and somatic. In the germline approach, foreign DNA is introduced into the zygote or early embryo with the expectation that it will contribute to the germline of the recipient and be passed on to the next generation. In somatic gene therapy, genetic material is introduced only into somatic cells and will not be transmitted to the next generation. Somatic gene therapy could be implemented in the perinatal period or any time thereafter. A third approach to gene therapy involves activation of an endog- enous genefs) to augment or circumvent a defective gene. One experi- ment already in progress involves inhibition of the normal developmental silencing of the fetal globin gene to treat genetic defects of the adult beta- globin gene, particularly sickle cell anemia. Agents such as hydroxyurea and sodium butyrate increase production of fetal globin to levels in the range required to ameliorate the symptoms of sickle cell anemia. Many interesting questions remain to be answered. What is the mechanism for these effects and what are their specificities; In what other gene families are there fetal forms that could be activated to replace a defective adult form? What are the side effects of these perturbations in gene regulation? Considerable experience with germline gene therapy has been ac- quired with transgenic mice. Germline gene therapy has been used to treat several monogenic diseases of mice, including deficiency of growth hormone, myelin basic protein, and beta-globin. In general, the disease phenotype is markedly ameliorated. These experiments have provided enor- mous amounts of information on the regulation of gene expression and the pathogenesis of genetic disease, but application to humans is still problematic. Only 15 to 20 percent of injected eggs produce transgenic animals, and of these, only 20 to 30 percent express the introduced gene.

so OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES Furthermore, random insertion of foreign DNA poses the risk of damage to resident genes. In most instances, the certainty of having an unaffected child, as established by prenatal diagnosis, is preferable to the risks and uncertainty of the transgenic approach. For these reasons, germline gene therapy is unlikely to be applied to human genetic disease. Nevertheless, future research on transgenic experimental animals will continue to yield new understanding of the nutritional regulation of gene expression and its role in pathogenesis. By contrast, somatic gene therapy experiments for human genetic dis- ease are under way. The first transfer of cells with intentionally altered genes took place in 1989. The first attempt to correct a genetic disease began in 1990. In this latter experiment, the T cells of two young girls with adenosine deaminase (ADA) deficiency were isolated and transduced in culture (ex viva: with a retroviral vector containing a human ADA gene. The transformed T cells were then infused into the patients. Preliminary results indicate that gene therapy caused an improvement in the girls' immune function, although the long-term consequences of this approach have yet to be reported. Additional experiments in somatic gene therapy of familial hypercholesterolemia, alpha-1-antitrypsin deficiency, and cystic fibrosis have begun or will begin soon. Many of them will have important relevance to nutrition. In order to develop rational somatic gene therapy strategies, it is important to understand the biology and pathophysiology of the target disease. These research questions provide important opportunities. Is the disease local (within the cell manifesting deficient activity of the gene product) or systemic (with cell injury resulting from metabolic distur- bances caused by functional deficits in a remote cell type)? Neuronal death caused by local deficiency of hexoseaminidase A activity in Tay- Sachs disease is an example of the former. An example of the latter is phenylketonuria, where neuronal damage is secondary to high extracellu- lar phenylalanine levels caused by the deficiency of hepatic phenylalanine hydroxylase (PH) and dietary intake of phenylalanine. In the former, gene therapy requires expressing the introduced gene in the neurons them- selves. In the latter, expression of PH in any population of cells with access to the extracellular fluid should reduce phenylalanine levels and prevent neural damage. Considerations of this type influence the choice of recipient cell for the introduced DNA. Initially, attention focused on bone marrow stem cells, but these have proved difficult to isolate and transduce in adequate amounts. More recently, investigators have worked with hepatocytes, endothelial cells, skin cells, and myoblasts, depending on the requirements imposed by the particular disorder. Another important methodological variable concerns access to the tar- get cell. In the ex vivo strategy, cells are removed from the body, trans

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 83 cluced in culture, and then reintroduced into the patient. The ADA re- placement experiment described above is an example of this approach. Alternatively, the target cells are transduced in situ, without being re- moved from the body. This direct delivery strategy leaves the target tissue intact, but the new genes may not reach an adequate number of cells. What we know about gene therapy is dwarfed by what remains to be learned. We need more research on vector design, delivery systems, ma- nipulation of the recipient cells, biology of the affected cells and tissues, and pathophysiology of the disease processes. Given the complexity of the systems involved, many genetic diseases may continue to resist our thera- peutic efforts. Nevertheless, somatic gene therapy will likely prove to be effective for some disorders and may truly cure others. It is likely to be used to treat common diseases such as cancer and acquired immune defi- ciency syndrome (AIDS). Generally, somatic gene therapy approaches will introduce genes whose protein products are toxic to the diseased cells or make these cells more sensitive to pharmacologic agents. The Human Genome Project and Nutrition The Human Genome Project was launched in 1988. Its short-term goals are to produce several types of maps of the human genome and to enhance technologies required for the task. The long-term goal is formi- dable: to determine the nucleotide sequence of the human genome by the year 2005. The human genome is estimated to contain 50,000 to 100,000 genes encoded in 3 billion base pairs. Completion of the sequencing will require 3,000 to 10,000 person-years of effort. A considerable informatics capability will be necessary to collect and maintain the sequences in us- able forms. With industrial-scale use of current technologies, a focused group of 300 people may be able to obtain the entire sequence in 10 years. The short-term goals are also impressive. The first of these will be a genetic map of the human genome at a relatively high resolution. In addi- tion, two types of physical maps will be constructed. One will show the location of certain landmark sequences or sequence-tagged sites at inter- vals of about 100,000 base pairs. The other will be an assembly of overlap- ping sets of cloned DNAs covering large portions of the genome. These physical maps will allow manipulation of the genome and identification of genes of interest. Early versions of the map have assisted with identifica- tion and cloning of genes for adrenoleuLodystrophy, amyotrophic lateral sclerosis, Menkes' syndrome, choroideremia, Norrie's disease, and Huntington's disease. The third short-term goal is to obtain limited sequence information in humans and certain model organisms. The experience gained with the

~4 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES model organisms will be utilized to improve sequencing and informatics technology and provide information for evolutionary comparisons of gene sequences and genome organization. Adclitional short-term goals include developing informational, training, and technological resources necessary to support the effort as well as study of the ethical legal and social implications of the information to be acquired. - O 1 What opportunities might this rapic! expansion of our knowledge of the human genome provide to the nutritional sciences? First, new genes will be iclentifiect, cloned, and mapped at an ever-increasing pace. Many of them will be of importance to nutrition research, including genes en- coding proteins involved in epithelial transport of small molecules, en- zymes in intermediary metabolism, transcriptional regulators, cytokines, and cell cycle regulators. Some will be genes directly involved in nutri- tional diseases. Others will be genes whose products play important physi- ologicaT roles not yet associated with particular diseases. Many genes may be unanticipated by our current view of the genome. For example, even in such a thoroughly studied organism as yeast, Saccharomyces cerevisiae, approximately 60 percent of the genes identified by sequencing chromo- some 3 encode unknown proteins. With molecular reagents in hand, nu- trition investigators can ask how the encoded proteins function anc3 inter- act to produce normal growth and physiological homeostasis. Regulation of Gene Expression Transcriptional Mechanisms The phenotype of a cell is the sum of the developmentally regulated expression of a tissue-specific set of genes and the influence of environ- mental factors such as nutrients, hormones, and growth factors on expres- sion of those genes. Understanding the molecular mechanisms by which development and environment regulate the expression of specific genes is thus crucial to understanding how nutrition influences metabolism and other processes. For many genes, the quantitatively most important type of regulation is exerted when transcription is initiated. Earlier in this chapter we described the importance of cis-acting DNA sequence ele- ments flanking the 5' region of genes vis-a-vis regulation of transcription initiation. Specific sequence elements, usually fewer than 20 base pairs in length, (determine which genes will be expressed in which tissues after treatment with which hormones (or growth factor, drug, or, in some cases, micronutrient). How do intracellular signaling pathways interact with these cis-acting elements to regulate gene expression? Trans-acting proteins bind to cis-acting sequence elements and con- nect intracellular signaling pathways to regulation of transcription. Two types of binding assays help to define the exact nucleotide sequence of cis

GENETIC, MOLECULAR, CELLULAR, AND PHYSIOLOGICAL PROCESSES 85 elements and protein involved. In one assay, binding of the protein to a radiolabeled DNA fragment alters the mobility of the DNA cluring gel electrophoresis. In the second assay, binding of the protein to the DNA protects a region of the DNA from degradation by DNAse or chemical agents. Aclding unlabeled DNA fragments of the same sequence will cre- ate competition for the relevant protein and prevent the alteration in mobility or the protection from degradation. By systematically altering the base sequence of the unlabeled competitor fragments, one can define the length and nucleotide sequence of the DNA that constitutes the binding site for a trans-acting factor. If the protein binding in the in vitro assay is physiologically relevant, introduction of the same mutations in DNA tested in the functional assay should lead to proportional changes in the ability of that DNA to activate transcription of a linked reporter gene. Numerous trans-acting factors involves! in tissue-, hormone-, and micronutrient-spe- cific expression of genes have been identified and purified using these binding assays. The nutritional significance of many of the already puri- fied proteins is not known. Furthermore, many nutritionally significant DNA-binding proteins remain to be identified and characterized. The experimental work described in the previous section usually uses definecl cell lines in culture and transient transfection systems. Unfortu- nately, the chromatin structure of transiently expressed genes is not the same as that of natural genes. It is thus important to test cis-acting ele- ments in their natural environment by creating permanent cell lines or transgenic mice carrying the test genes. Such experiments are much more time-consuming than transient expression experiments, but they are es- sential to un(lerstancling the physiologically relevant events. Identification of the cis-acting sequence elements and the trans-act- ing proteins to which they bind does not solve the problem of how intra- cellular signaling mechanisms control transcription initiation. Two prob- lems are relevant. First, the molecular mechanisms whereby binding of a trans-acting factor regulates transcription initiation are obscure. Additional proteins that "connect" the DNA-binding protein with the general tran- scription apparatus appear to be involved. Second, little is known about how intracellular signaling pathways regulate activity of the trans-acting factors. In some cases, the amount of the DNA-binding protein is regu- lated, and in others the "catalytic" activity is regulated by covalent modifi- cation or allosteric mechanisms. Either the DNA-binding protein itself or the "connecting" proteinfs) could interact with the signaling pathway. In some cases, binding of the protein to DNA is altered by activation of the signaling pathway, whereas in others the ability of the bound protein to activate transcription appears to be the regulated event. There is much to be learned about regulation of transcription in general and dietary regula- tion of gene expression in particular.

S6 Posttranscriptionai Mechanisms OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES Initiation of transcription is only the first of several steps that lead to the production of a mature, biologically active protein. Regulation at any of the subsequent steps has the potential to regulate production of the mature protein. In the nucleus, transcription initiation is followed by elonga- tion and then termination of transcription; all three are potential steps at which gene expression may be regulated. Little is known about regulation of elongation or termination in higher animals. Similarly, little is known about regulation of the processing of primary transcripts that precedes transport from the nucleus to the cytoplasm. Transport of the mature transcript to the cytoplasm may also be a regulated step, but little is known about this process. In the cytoplasm, protein synthesis can be regulated by two general mechanisms. First, the rate of degradation of the mature mRNA will in- fluence the steady-state concentration of mRNA and thus the rate of syn- thesis of the corresponding protein. Some examples of this type of regula- tion involve hormones. Examples of regulation of this reaction by specific nutrients have also been reported (such as that for iron, discussed on page 58~; however, the molecular mechanisms involved remain a mystery. Sec- ond, selective changes in the rate of translation of a specific mRNA will influence production of the protein product. As noted in an earlier sec- tion, regulation of ferritin concentrations by dietary iron is controlled by a protein that binds to a specific sequence in the 5' end of the ferritin mRNA and regulates its rate of translation. Substantial regulation also occurs after the protein product has been synthesized. Among the post- translational processing events that cause altered activities of proteins are proteolytic cleavage, phosphorylation, glycosylation, methylation, acylation, isoprenylation, and carboxylation. In each case, the ability of recombinant DNA techniques to facilitate identification of the modified amino acid residues, to create site-specific mutations, and to express normal and mu- tant forms of the involved proteins in a variety of cell types provides a powerful approach to understanding the molecular basis of regulatory phe- nomena. There are numerous examples of nutrient-related posttransla- tional regulation. Gene-Environmental Interactions in Complex Disease Phenotypes Complex disease phenotypes may involve both genetic and environ- mental components. Dietary intake of a particular nutrient is an environ- mental component that we can potentially control. Identifying genes that are involved in these diseases and characterizing their functions should

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

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

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

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.

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.

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

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 -

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 ~/

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

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

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. - .