Opportunities in the Nutrition and Food Sciences Research Challenges and the Next Generation of Investigators (1994) / Chapter Skim
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3 UNDERSTANDING GENETIC, MOLECULAR, CELLULAR, AND PHSYSIOLOGICAL PROCESSES
3 UNDERSTANDING GENETIC, MOLECULAR, CELLULAR, AND PHSYSIOLOGICAL PROCESSES
Pages 47-97
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From page 47... ...
Experiments with i5N-labeled amino acids provided early insights into the dynamics of protein synthesis. The knowledge of nutrient metabolism gained during the last half century would not have been possible without use of radioisotopes.
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From page 48... ...
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 function at the cellular level include: (1)
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From page 49... ...
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.
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From page 50... ...
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 essential roles in reproduction and in maintaining normally differentiated epithelial cells in many organs throughout the body.
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From page 51... ...
In cultured cells expressing the new receptor, addition of retinoic acid activated specific genes (see box)
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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.)
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From page 53... ...
Several natural and synthetic molecules were added as putative ligands for the chimeric receptor. Only retinoic acid was a strong activator.
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54 OPPORTUNITIES IN THE NUTRITION AND FOOD SCIENCES HOW 7-Dehydrocholesterol | DIET | SKIN / HO'v a' Vitamin D3 LIVER | | KIDNEY| '~` I' OH a' HOW OH 1,25-Dihydroxyvitamin D3 HOW ~ I OH HOW 25-Hydroxyvitamin D3 /\ \ HOW> Vitamin D2 | KIDNEY | >. I other tissues | OH ~ 1>H° 24,25-Dihydroxyvitamin D3 FIGURE 3.2 Vitamin D metabolism in the skin, liver, and kidney.
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From page 56... ...
the significance of the trans-acting vitamin D receptor system in differentiating tissues, or (3) how this receptor system interacts with cytokines and growth factors during development or nutritional deprivation or in diseases such as osteoporosis.
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From page 57... ...
Ionpassing 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 enzymes which, in turn, activate signaling pathways that lead to specific end effects.
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From page 58... ...
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.
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binds to these IREs. The IRE-BP is also the cytosolic enzyme aconitase that may help regulate Tow molecular weight iron pools via citrate.
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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 ?
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~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.
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. Thereafter, most of the DNA is lost by cell division and degradation.
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From page 63... ...
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.
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An important step in molecular analysis of the signaling pathways by which hormones or nutritional states regulate protein concentration, therefore, is identification and characterization of cis-acting DNA elements and trans-acting proteins that regulate transcription. Knowledge of the structure and func ~ 1 1-.
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From page 65... ...
This technology was used to identify retinoic acid receptors (see page 511. Identifying the mechanisms by which nutrient intake regulates the function of transacting factors will be critical to understanding how nutrients regulate gene expression.
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From page 66... ...
with targeted mutations. Primary cell lines were established from the pluripotent stem cells of the mouse embryo.
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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.
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cell lines. Several attributes of monocTonal antibody technology make it useful for identifying specific cell types.
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From page 69... ...
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)
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Much of the activity with ion imaging has concentrated on subcellular analysis of cell motility and transmission of neural signals. However, there is an enormous potential for new information at the interface of nutrition science and cell biology.
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Nontoxic, fluorescently tagged dextran is injected 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.
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somatic cell hybridization of normal, terminally differentiated cells with a definecI tumor cell line, followed by selection and subcloning in a selective medium, and (5) immortalization of a terminally clifferentiated cell type by introduction of an oncogene, followed by subcloning and selection of a line on the basis of some phenotypic characteristic.
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Alternatively, as described earlier in this chapter, specific target genes can be clisruptecl or modified by homologous recombination (gene knockout) in embryonic stem cells.
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~4 .~ Cat an Or E~ C)
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The recent explosion of interest in strategies for gene therapy is closely tied to availability of animal models, as discussed elsewhere in this chapter.
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Approaches to Determining the Structure of Macromolecules Understanding the molecular basis of the function of enzymes, receptors, 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,
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From page 77... ...
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 provided 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.
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From page 78... ...
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 causeand-effect relationships between organ-specific metabolic processes, extra- and intracellular effecters, molecular changes in key enzymes, and altered flow of metabolites through metabolic pathways.
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From page 79... ...
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 identification of those amino acids in the overall sequence that are essential
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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 identitied in a manner analogous to that described above for identifying pacesetting enzymes in metabolic pathways.
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From page 81... ...
Nevertheless, the majority of genetic diseases 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 introduction of a functional gene to replace or supplement the activity of a defective gene.
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From page 82... ...
By contrast, somatic gene therapy experiments for human genetic disease are under way. The first transfer of cells with intentionally altered genes took place in 1989.
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Given the complexity of the systems involved, many genetic diseases may continue to resist our therapeutic efforts. Nevertheless, somatic gene therapy will likely prove to be effective for some disorders and may truly cure others.
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Trans-acting proteins bind to cis-acting sequence elements and connect intracellular signaling pathways to regulation of transcription. Two types of binding assays help to define the exact nucleotide sequence of cis
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From page 85... ...
Identification of the cis-acting sequence elements and the trans-acting proteins to which they bind does not solve the problem of how intracellular signaling mechanisms control transcription initiation. Two problems are relevant.
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From page 86... ...
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 mutant forms of the involved proteins in a variety of cell types provides a powerful approach to understanding the molecular basis of regulatory phenomena. There are numerous examples of nutrient-related posttranslational regulation.
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Identification of these genes must be a priority for nutritional research. 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.
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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.
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The asymmetric cell division of stem cells probably 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!
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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.
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From page 91... ...
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. Morphologically, these reticuloendothelial cells show tight junctions completely blocking the intracellular spaces, and they are not interrupted by gap junctions.
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The specialized properties of brain endothelial cells apparently derive 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.
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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.
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, 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)
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From page 95... ...
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.
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From page 96... ...
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 pathways through which bioactive retinoids are formed from their nutrient precursors. These enzymic transformations take place in the cell's cytopiasm, where certain retinoid-binding proteins are also known to exist.
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From page 97... ...
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 .
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