Showing papers in "The FASEB Journal in 1996"

A decade of molecular biology of retinoic acid receptors.

Abstract: Retinoids play an important role in development, differentiation, and homeostasis. The discovery of retinoid receptors belonging to the superfamily of nuclear ligand-activated transcriptional regulators has revolutionized our molecular understanding as to how these structurally simple molecules exert their pleiotropic effects. Diversity in the control of gene expression by retinoid signals is generated through complexity at different levels of the signaling pathway. A major source of diversity originates from the existence of two families of retinoid acid (RA) receptors (R), the RAR isotypes (alpha, beta, and gamma) and the three RXR isotypes (alpha, beta, and gamma), and their numerous isoforms, which bind as RXR/RAR heterodimers to the polymorphic cis-acting response elements of RA target genes. The possibility of cross-modulation (cross-talk) with cell-surface receptors signaling pathways, as well as the finding that RARs and RXRs interact with multiple putative coactivators and/or corepressors, generates additional levels of complexity for the array of combinatorial effects that underlie the pleiotropic effects of retinoids. This review focuses on recent developments, particularly in the area of structure-function relationships.

Antioxidant and Redox Regulation of Gene Transcription

Abstract: Reactive oxygen species (ROS) are implicated in the pathogenesis of a wide variety of human diseases. Recent evidence suggests that at moderately high concentrations, certain forms of ROS such as H202 may act as signal transduction messengers. To develop a better understanding of the exact mechanisms that underlie ROS-dependent disorders in biological systems, recent studies have investigated the regulation of gene expression by oxidants, antioxidants, and other determinants of the intracellular reduction-oxidation (redox) state. At least two well-defined transcription factors, nuclear factor (NF) kappa B and activator protein (AP) -1 have been identified to be regulated by the intracellular redox state. The regulation of gene expression by oxidants, antioxidants, and the redox state has emerged as a novel subdiscipline in molecular biology that has promising therapeutic implications. Binding sites of the redox-regulated transcription factors NF-kappa B and AP-1 are located in the promoter region of a large variety of genes that are directly involved in the pathogenesis of diseases, e.g., AIDS, cancer, atherosclerosis and diabetic complications. Biochemical and clinical studies have indicated that antioxidant therapy may be useful in the treatment of disease. Critical steps in the signal transduction cascade are sensitive to oxidants and antioxidants. Many basic events of cell regulation such as protein phosphorylation and binding of transcription factors to consensus sites on DNA are driven by physiological oxidant-antioxidant homeostasis, especially by the thiol-disulfide balance. Endogenous glutathione and thioredoxin systems, and the exogenous lipoate-dihydrolipoate couple may therefore be considered to be effective regulators of redox-sensitive gene expression. The efficacy of different antioxidants to favorably influence the molecular mechanisms implicated in human disease should be a critical determinant of its selection for clinical studies.

S-Adenosylmethionine and methylation.

Abstract: S-Adenosylmethionine (AdoMet or SAM) plays a pivotal role as a methyl donor in a myriad of biological and biochemical events. Although it has been claimed that AdoMet itself has therapeutic benefits, it remains to be established whether it can be taken up intact by cells. S-Adenosylhomocysteine (AdoHcy), formed after donation of the methyl group of AdoMet to a methyl acceptor, is then hydrolyzed to adenosine and homocysteine by AdoHcy hydrolase. This enzyme has long been a target for inhibition as its blockade can affect methylation of phospholipids, proteins, DNA, RNA, and other small molecules. Protein carboxymethylation may be involved in repair functions of aging proteins, and heat shock proteins are methylated in response to stress. Bacterial chemotaxis involves carboxymethylation and demethylation in receptor-transducer proteins, although a similar role in mammalian cells is unclear. The precise role of phospholipid methylation remains open. DNA methylation is related to mammalian gene activities, s...

Beta-carotene, carotenoids, and disease prevention in humans.

Abstract: A growing body of literature exists regarding the effects of beta-carotene and other carotenoids on chronic diseases in humans. This article reviews and critically evaluates this literature and identifies areas for further research. This review is restricted to studies in humans, with a major emphasis on the most recent literature in the area of carotenoids and selected cancers. Effects of carotenoids on cardiovascular diseases, photosensitivity diseases, cataracts, and age-related macular degeneration are also discussed briefly. Numerous observational studies have found that people who ingest more carotenoids in their diets have a reduced risk of several chronic diseases. However, intervention trials of supplemental beta-carotene indicate that supplements are of little or no value in preventing cardiovascular disease and the major cancers occurring in well-nourished populations, and may actually increase, rather than reduce, lung cancer incidence in smokers. As a consequence of these findings, some of the ongoing trials of beta-carotene and disease prevention have been terminated or have dropped beta-carotene from their interventions. Researchers should now seek explanations for the apparently discordant findings of observational studies vs. intervention trials. The most pressing research issues include studies of interactions of carotenoids with themselves and with other phytochemicals and mechanistic studies of the actions of beta-carotene in lung carcinogenesis and cardiovascular disease. Paradoxically, the finding that lung carcinogenesis and cardiovascular disease can be enhanced by supplemental beta-carotene may ultimately lead to a clearer understanding of the role of diet in the etiology and prevention of these diseases. The conclusion that major public health benefits could be achieved by increasing consumption of carotenoid-rich fruits and vegetables still appears to stand; however, the pharmacological use of supplemental beta-carotene for the prevention of cardiovascular disease and lung cancer, particularly in smokers, can no longer be recommended.

Carotenoids 3: in vivo function of carotenoids in higher plants.

Abstract: The function of the long-chain, highly unsaturated carotenoids of higher plants in photoprotection is becoming increasingly well understood, while at the same time their function in other processes, such as light collection, needs to be reexamined. Recent progress in this area has been fueled by more accurate determinations of the photophysical properties of these molecules, as well as extensive characterization of the physiology and ecology of a particular group of carotenoids, those of the xanthophyll cycle, that play a key role in the photoprotection of photosynthesis under environmental stress. The deepoxidized xanthophylls zeaxanthin and antheraxanthin, together with a low pH within the photosynthetic membrane, facilitate the harmless dissipation of excess excitation energy directly within the light-collecting chlorophyll antennae. Evidence for this function as well as current contrasting hypotheses concerning its molecular mechanism are reviewed. In addition, the acclimation of the xanthophyll cycle content and composition of leaves to contrasting environments with different demands for photoprotection is summarized.

Sphingolipid metabolism and cell growth regulation.

Abstract: Sphingolipids have been implicated in the regulation of cell growth, differentiation, and programmed cell death. The current paradigm for their action is that complex sphingolipids such as gangliosides interact with growth factor receptors, the extracellular matrix, and neighboring cells, whereas the backbones--sphingosine and other long-chain or "sphingoid" bases, ceramides, and sphingosine 1-phosphate--activate or inhibit protein kinases and phosphatases, ion transporters, and other regulatory machinery. Tumor necrosis factor-alpha, interleukin 1beta, and nerve growth factor, for example, induce sphingomyelin hydrolysis to ceramide. Other agonists, such as platelet-derived growth factor, trigger further hydrolysis of ceramide to sphingosine and activate sphingosine kinase to form sphingosine 1-phosphate. These metabolites either stimulate or inhibit growth and may be cytotoxic (in some cases via induction of apoptosis), depending on which products are formed (or added exogenously), the cellular levels (...

Absorption, metabolism, and transport of carotenoids.

Abstract: Carotenoids are currently under intense scrutiny regarding their potential to modulate chronic disease risk and prevent vitamin A deficiency, and renewed emphasis has been placed on achieving a better understanding of the metabolic fate of these compounds in humans. The development of new animal models, and use of human metabolic studies and stable tracer methods have greatly improved our knowledge of how carotenoids are absorbed, metabolized, and transported to tissues; however, many important issues remain unresolved. For example, intestinal uptake of carotenoids occurs by passive diffusion, but the lumenal or intracellular factors limiting this process are obscure. The intestinal mucosa plays a key role in the metabolism of provitamin A carotenoids such as beta-carotene, thus greatly influencing their bioavailability. Most recent evidence supports a central oxidation mechanism of cleavage of beta-carotene to retinal in the intestinal mucosa, but the extent and site(s) of postabsorptive metabolism in th...

In vitro folding of inclusion body proteins.

Abstract: Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.

Forces contributing to the conformational stability of proteins

Abstract: For 35 years, the prevailing view has been that the hydrophobic effect is the dominant force in protein folding. The importance of hydrogen bonding was always clear, but whether it made a net favorable contribution to protein stability was not. Studies of mutant proteins have improved our understanding of the forces stabilizing proteins. They suggest that hydrogen bonding and the hydrophobic effect make large but comparable contributions to the stability of globular proteins.

Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function.

Abstract: This review focuses on the extracellular proteoglycans. Special emphasis is placed on the structural features of their protein cores, their gene organization, and their transcriptional control. A simplified nomenclature comprising two broad groups of extracellular proteoglycans is offered: the small leucine-rich proteoglycans or SLRPs, pronounced "slurps, " and the modular proteoglycans. The first group encompasses at least five distinct members of a gene family characterized by a central domain composed of leucine-rich repeats flanked by two cysteine-rich regions. The second group consists of those proteoglycans whose unifying feature is the assembly of various protein modules in a relatively elongated and often highly glycosylated structure. This group is quite heterogeneous and includes a distinct family of proteoglycans, the "hyalectans," that bind hyaluronan and contain a C-type lectin motif that is likely to bind carbohydrates, and a less distinct group that contains structural homologies but lacks ...

The vascular protective effects of estrogen.

Abstract: There is now strong epidemiological evidence that estrogen replacement therapy has a protective effect in postmenopausal women. The cardiovascular protective action of estrogen is reported to be mediated indirectly by an effect on lipoprotein metabolism and by a direct effect on the vessel wall itself. Estrogen is active both in vascular smooth muscle and endothelium. Functionally competent estrogen receptors have been identified in vascular smooth muscle cells, and specific binding sites have been demonstrated in endothelium. Estrogen administration promotes vasodilation both in human and experimental animals, in part by stimulating] prostacyclin and nitric oxide synthesis. Both the prostaglandin synthase and the constitutive nitric oxide synthase were recently reported to be induced by estrogen treatment. In vitro, estrogen exerts a direct inhibitory effect on the smooth muscle by inhibiting calcium influx. In addition, estrogen inhibits vascular smooth muscle cell proliferation. In vivo, estradiol-17 beta prevents neointimal thickening after balloon injury and in rabbit cardiac transplant allografts. These data are consistent with in vitro studies wherein estrogen inhibits [3H]thymidine uptake by arterial segments from porcine coronary artery as well as proliferation of rabbit aortic vascular smooth muscle cells induced by hyperlipedemic serum. Recent studies have also reported an effect of estrogen on directed vascular smooth muscle cell migration. Furthermore, like other steroids, the effect of estrogen on the vessel wall has a rapid nongenomic component involving membrane phenomena, such as alteration of membrane ionic permeability and activation of membrane-bound enzymes, as well as the classical genomic effect involving estrogen receptor activation and gene expression. The nature of these estrogen response genes in the vessel wall and their relation to vasodilation and antiproliferation remain to be determined.

The role of proteases during apoptosis.

Abstract: The importance of proteases during apoptosis is becoming increasingly apparent. Because apoptosis contributes to a diverse variety of disease processes, understanding the roles played by proteases and their inhibitors might provide insight into the pathogenesis of these conditions and suggest novel therapeutic strategies. In this review, we discuss the involvement and role of specific proteases, substrates, and protease inhibitors that appear to participate in the apoptotic process.

Liver-enriched transcription factors and hepatocyte differentiation.

Abstract: Liver-specific gene expression in adult hepatocytes relies on four families of evolutionary conserved transcription factors that are liver-enriched but not restricted to this tissue. These factors function in unique combinations, often synergistically, to stimulate cell-specific transcription. Each family is composed of several members displaying similar, if not identical, DNA recognition properties and sharing structural homology in their DNA binding domains. The homo- and heterodimerization between members of a particular transcription factor family adds an additional level of complexity in gene regulation. The consequences of inactivating different family members in the mouse by homologous recombination, together with recent studies of their regulation, suggest a model for liver differentiation involving a regulatory network rather than a completely hierarchical genetic circuitry. These studies also indicate that individual regulators appear to serve multiple developmental functions. Their possible role in the progression through different stages of hepatic cell commitment and differentiation is discussed.

Neurohormone melatonin prevents cell damage: effect on gene expression for antioxidant enzymes.

Abstract: It is well known that porphyrins cause a toxic light-mediated effect due to their capability to generate free radicals. Several reports have proved that melatonin is a potent free radical scavenger. The aim of this work has been to study the ability of melatonin to prevent the cell damage caused by porphyrins in the Harderian gland of female Syrian hamsters. Cell injury was evaluated estimating the percentage of damaged cells found in the gland and analyzing the degree of this damage at ultrastructural level. To explain the mechanism by which this hormone could prevent the cell damage caused by porphyrins, its capability to both decrease porphyrin synthesis and increase the mRNA levels for antioxidant enzymes was evaluated. Our results demonstrate that melatonin administration decreases the percentage of damaged cells, porphyrin synthesis, and aminolevulinate synthase (ALA-S) mRNA levels and increases the mRNA levels for manganese superoxide-dismutase and copper-zinc superoxide dismutase. When observed un...

Retinoids in cancer chemoprevention.

Abstract: Naturally occurring and synthetic vitamin A metabolites and analogs (retinoids) inhibit tumor development in a variety of cellular, animal, and patient studies. They suppress transformation of cells in vitro and inhibit carcinogenesis in various organs in animal models. In a mouse skin carcinogenesis model, topical retinoids exhibit suppressive effects on tumor promotion, but have no effect on tumor initiation. In other models, retinoids administered in the diet suppress tumor development even in an adjuvant setting after excision of the first tumor. Retinoids suppress carcinogenesis in individuals with premalignant lesions and a high risk to develop cancer of the aerodigestive tract. Likewise, retinoids prevent the development of second primary cancers in head/neck and lung cancer patients who had been treated for the first primary. The mechanisms underlying the anticarcinogenic activity of retinoids appear to be associated with the ability of retinoids to modulate the growth, differentiation, and apopto...

Spatial and temporal aspects of cellular calcium signaling

Abstract: Cytosolic Ca2+ signals are often organized in complex temporal and spatial patterns, even under conditions of sustained stimulation. In this review we discuss the mechanisms and physiological significance of this behavior in nonexcitable cells, in which the primary mechanism of Ca2+ mobilization is through (1,4,5)IP3-dependent Ca2+ release from intracellular stores. Oscillations of cytosolic free Ca2+ ([Ca2+]i) are a common form of temporal organization; in the spatial domain, these [Ca2+]i oscillations may take the form of [Ca2+]i waves that propagate throughout the cell or they may be restricted to specific subcellular regions. These patterns of Ca2+ signaling result from the limited range of cytoplasmic Ca2+ diffusion and the feedback regulation of the pathways responsible for Ca2+ mobilization. In addition, the spatial organization of [Ca2+]i changes appears to depend on the strategic distribution of Ca2+ stores within the cell. One type of [Ca2+]i oscillation is baseline spiking, in which discrete [Ca2+]i spikes occur with a frequency, but not amplitude, that is determined by agonist dose. Most current evidence favors a model in which baseline [Ca2+]i spiking results from the complex interplay between [Ca2+]i and (1,4,5)IP3 in regulating the gating of (1,4,5)IP3-sensitive intracellular Ca2+ channels. Sinusoidal [Ca2+]i oscillations represent a mechanistically distinct type of temporal organization, in which agonist dose regulates the amplitude but has no effect on oscillation frequency. Sinusoidal [Ca2+]i oscillations can be explained by a negative feedback effect of protein kinase C on the generation of (1,4,5)IP3 at the level of phospholipase C or its activating G-protein. The physiological significance of [Ca2+]i oscillations and waves is becoming more established with the observation of this behavior in intact tissues and by the recognition of Ca2+-dependent processes that are adapted to respond to frequency-modulated oscillatory [Ca2+]i signals. In some cells, these [Ca2+]i signals are targeted to control processes in limited cytoplasmic domains, and in other systems [Ca2+]i waves can be propagated through gap junctions to coordinate the function of multicellular systems.

Partial night sleep deprivation reduces natural killer and cellular immune responses in humans.

Abstract: Prolonged and severe sleep deprivation is associated with alterations of natural and cellular immune function. To determine whether alterations of immune function also occur after even a modest loss of sleep, the effects of early-night partial sleep deprivation on circulating numbers of white blood cells, natural killer (NK) cell number and cytotoxicity, lymphokine-activated killer (LAK) cell number and activity, and stimulated interleukin-2 (IL-2) production were studied in 42 medically and psychiatrically healthy male volunteers. After a night of sleep deprivation between 10 P.M. and 3 A.M., a reduction of natural immune responses as measured by NK cell activity, NK activity per number of NK cells, LAK activity, and LAK activity per number of LAK precursors (CD16,56, CD25) was found. In addition, concanavalin A-stimulated IL-2 production was suppressed after sleep deprivation due to changes in both adherent and nonadherent cell populations. After a night of recovery sleep, NK activity returned to baseline levels and IL-2 production remained suppressed. These data implicate sleep in the modulation of immunity and demonstrate that even a modest disturbance of sleep produces a reduction of natural immune responses and T cell cytokine production.

Supervising the fold: functional principles of molecular chaperones.

Abstract: Molecular chaperones are a set of conserved protein families that share the remarkable ability to recognize and selectively bind nonnative proteins under physiological and stress conditions. Thus, they prevent irreversible aggregation reactions and keep proteins on the productive folding pathway. Evidence suggests that the cell has developed several functionally distinct chaperone families to support protein folding. The importance of molecular chaperones under stress conditions is highlighted by the finding that all the major heat shock protein families (Hsp104, Hsp90, Hsp70, Hsp60/GroEL, and small Hsps) suppress irreversible unfolding reactions. Under heat shock, only the increased expression of a repertoire of different chaperones seems to convey thermotolerance and guarantee survival. The molecular mechanism by which chaperones in general influence protein folding processes is still far from clear. However, significant progress has been achieved in understanding some of the partial reactions of the chaperone folding cycles and in functionally differentiating between the different chaperone families.

The molten globule state of alpha-lactalbumin.

Abstract: The molten globule state of alpha-lactalbumin is the best-characterized folding intermediate of globular proteins and has been studied intensively by various spectroscopic and physiochemical techniques, including stopped-flow CD and fluorescence spectroscopies, a hydrogen-exchange technique, 1H-NMR spectroscopy, disulfide-exchange chemistry, site-directed mutagenesis, and calorimetric techniques. This review summarizes recent studies. Major findings about the structure of the molten globule state are: 1) It is highly heterogeneous, having a highly structured alpha-helical domain with the beta-sheet domain being significantly unfolded; and 2) it is not a nonspecific, collapsed polypeptide but already has a native-like tertiary fold. These structural characteristics are essential to fully understand the thermodynamic properties of the molten globule state which are described in connection with a recently proposed computational approach to predict the structure of the molten globule state of a protein. Mutant proteins in which the stability of the molten globule state was changed were constructed. Studies of the equilibrium unfolding and kinetic refolding of the mutant proteins will provide further insight into the molten globule state as a folding intermediate. In spite of an initial expectation that the structure recognized by an Escherichia coli chaperone, GroEL, is the molten globule, the interaction of GroEL with alpha-lactalbumin in the molten globule state is much weaker than the interaction with more unfolded states of alpha-lactalbumin, a disulfide-reduced form, and disulfide rearranged species.

Heparan sulfate: a piece of information.

Abstract: The sulfated glycosaminoglycans, heparan sulfate and heparin, are increasingly implicated in cell-biological processes such as cytokine action, cell adhesion, and regulation of enzymic catalysis. These activities generally depend on interactions of the polysaccharides with proteins, mediated by distinct saccharide sequences, and expressed at various levels of specificity, selectivity, and molecular organization. The formation of heparin/ heparan sulfate in the cell requires an elaborate biosynthetic machinery, that is conceived in terms of a novel model of glycosaminoglycan assembly and processive modification. Recent advances in the identification and molecular analysis of the enzymes and other proteins involved in the biosynthesis provide novel tools to study the regulation of the process, presently poorly understood, at the subcellular and cellular levels. The potential medical importance of heparin-related compounds is likely to promote the biotechnological exploitation of components of the biosynthetic machinery.

The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose

Abstract: The biological interconversion of galactose and glucose takes place only by way of the Leloir pathway and requires the three enzymes galactokinase, galactose-1-P uridylyltransferase, and UDP-galactose 4-epimerase. The only biological importance of these enzymes appears to be to provide for the interconversion of galactosyl and glucosyl groups. Galactose mutarotase also participates by producing the galactokinase substrate alpha-D-galactose from its beta-anomer. The galacto/gluco configurational change takes place at the level of the nucleotide sugar by an oxidation/reduction mechanism in the active site of the epimerase NAD+ complex. The nucleotide portion of UDP-galactose and UDP-glucose participates in the epimerization process in two ways: 1) by serving as a binding anchor that allows epimerization to take place at glycosyl-C-4 through weak binding of the sugar, and 2) by inducing a conformational change in the epimerase that destabilizes NAD+ and increases its reactivity toward substrates. Reversible hydride transfer is thereby facilitated between NAD+ and carbon-4 of the weakly bound sugars. The structure of the enzyme reveals many details of the binding of NAD+ and inhibitors at the active site. The essential roles of the kinase and transferase are to attach the UDP group to galactose, allowing for its participation in catalysis by the epimerase. The transferase is a Zn/Fe metalloprotein, in which the metal ions stabilize the structure rather than participating in catalysis. The structure is interesting in that it consists of single beta-sheet with 13 antiparallel strands and 1 parallel strand connected by 6 helices. The mechanism of UMP attachment at the active site of the transferase is a double displacement, with the participation of a covalent UMP-His 166-enzyme intermediate in the Escherichia coli enzyme. The evolution of this mechanism appears to have been guided by the principle of economy in the evolution of binding sites.

Basic concepts in RNA virus evolution.

Abstract: A hallmark of RNA genomes is the error-prone nature of their replication and retrotranscription. The major biochemical basis of the limited replication fidelity is the absence of proofreading/repair and postreplicative error correction mechanisms that normally operate during replication of cellular DNA. In spite of this unique feature of RNA replicons, the dynamics of viral populations seems to follow the same basic principles that classical population genetics has established for higher organisms. Here we review recent evidence of the profound effects that genetic bottlenecks have in enhancing the deleterious effects of Muller's ratchet during RNA virus evolution. The validity of the Red Queen hypothesis and of the competitive exclusion principle for RNA viruses are viewed as the expected result of the highly variable and adaptable nature of viral quasispecies. Viral fitness, or ability to replicate infectious progeny, can vary a million-fold within short time intervals. Paradoxically, functional and str...

Lipid mediator networks in cell signaling: update and impact of cytokines.

Abstract: Biomembranes serve barrier functions and serve as a store for precursors of rapidly generated, structurally diverse intracellular and extracellular lipid-derived mediators (LM). Cell activation is accompanied by remodeling of membrane components that appear to be essential in signal transduction. Phospholipases (PLA2, PLC, PLD, sphingomyelinase) are pivotal in the generation of these LM including eicosanoids, platelet activating factor (PAF), diacylglycerides, ceramide, and other newly discovered bioactive autacoids. Cytokines exert a dramatic multilevel impact both in regulating enzymes in individual LM pathways and in generating LM central to their action. Here, we provide an overview and update of recent progress in this area with emphasis on the effect of cytokines on LM networks. The generation of eicosanoids (prostaglandins, leukotrienes, and lipoxins), oxygenated lipids, and PAF remain the focus of rational drug design targets given their established roles in cell-cell communication and as mediator...

The denatured state (the other half of the folding equation) and its role in protein stability.

Abstract: Experimental studies of the physical interactions that stabilize protein structure are complicated by the fact that proteins do not unfold to a simple reference state. When their folded structure breaks down, protein chains do not become random coils. Instead, they enter a poorly understood ensemble of partially folded states known collectively as the denatured state. Although it has long been held that agents that promote protein unfolding act specifically on the denatured state, the idea that mutations can exert their destabilizing (or in some cases, stabilizing) effects directly on this state is not widely accepted. A large body of thermodynamic data on mutant proteins plus a limited amount of structural information describing mutational effects on denatured states indicate that 1) the denatured state plays a central role in all aspects of protein stability, including mutant effects, and 2) a quantitative understanding of how amino acid sequence encodes protein structure will probably depend on a more ...

Molecular mechanisms of retinoid actions in skin.

Abstract: For more than 40 years, it has been appreciated that vitamin A is a critical regulator of growth and differentiation of developing and adult mammalian and avian skin. Vitamin A deficiency and hypervitaminosis A cause disruption of normal cellular homeostatic mechanisms, resulting in impairment of skin barrier function. More recent studies demonstrating all-trans retinoic acid as the major biologically active form of vitamin A, and nuclear retinoid receptors as the major mediators of all-trans retinoic acid actions, have provided exciting new insights into the molecular basis of vitamin A actions. These recent insights have been the driving force for important advances in the many areas of retinoid research made during the past 6 years. Nowhere has this new knowledge been more extensively applied than toward understanding the molecular basis of retinoid physiology and pharmacology in skin. This article will review these recent findings and attempt to synthesize their meaning to provide a view into the mech...

Regulation of gene expression by alternative promoters.

Abstract: Promoters have been defined as modulatory DNA structures containing a complex array of cis-acting regulatory elements required for accurate and efficient initiation of transcription and for controlling expression of a gene. It is becoming increasingly evident that they also constitute prime target elements through which diversity and flexibility in the complex patterns of gene expression in multicellular organisms are created. The use of multiple promoters and transcription start sites is apparently a frequently used mechanism, whereas at the same time there is considerable variation and complexity in the patterns of alternative promoter usage. This review discusses the use of alternative promoters as a versatile mechanism to create diversity and flexibility in the regulation of gene expression. Alternative promoter usage can influence gene expression in very diverse ways. The level of transcription initiation can vary between alternative promoters, the turnover or translation efficiency of mRNA isoforms with different leader exons can differ, alternative promoters can have different tissue specificity and react differently to some signals, and finally, alternative promoter usage can lead to the generation of protein isoforms differing at the amino terminus.

Retinoic acid biosynthesis and metabolism.

Abstract: Metabolic activation of retinol into the hormone retinoic acid and metabolism of retinoic acid entail essential aspects of retinoid biology that seem interdependent with functions of retinoid binding-proteins. Cellular retinol binding protein and cellular retinoic acid binding protein enjoy widespread expression and, where expressed, their liganded forms represent the major physiological forms of retinol and retinoic acid, respectively. These retinoid binding proteins may protect cells from the amphipathic properties of retinoids and protect the structurally sensitive retinoids from the cellular milieu. Starting from the perspective that the enzymes most likely to metabolize retinoids in vivo might recognize the major forms of retinoids that occur in vivo, several laboratories have produced results that support a model of retinoid metabolism with prominent roles for the cellular retinoid binding proteins. In this model, liganded cellular retinoid binding proteins serve as substrates for the metabolism of some retinoids (retinol, retinoic acid), restricting access to those enzymes that recognize both the binding protein and the retinoid. Other retinoids (3,4-didehydroretinol, 4-oxo-retinoic acid) liganded to binding-proteins have their metabolism arrested. In its unliganded form, at least one retinoid binding protein (cellular retinol binding protein) serves as a retinoid concentration-sensitive modulator of enzymes that catalyze retinol metabolism. This review will describe the model and the intrinsic relationships among retinoid-specific enzymes and retinoid binding proteins.

Desmosomes and hemidesmosomes: structure and function of molecular components.

Abstract: Desmosomes and hemidesmosomes are the major cell surface attachment sites for intermediate filaments at cell-cell and cell-substrate contacts, respectively. The transmembrane molecules of the desmosome belong to the cadherin family of calcium-dependent adhesion molecules, whereas those in the hemidesmosome include the integrin class of cell matrix receptors. In each junction, the cytoplasmic domains of certain transmembrane junction components contain unusually long carboxy-terminal tails not found in those family members involved in linkage of actin to the cell surface. These domains are thought to be important for the regulation of junction assembly and specific attachment of intermediate filaments via associated adapter proteins. Recent developments have suggested the exciting possibility that these junctions, in addition to playing an important structural function in tissue integrity, are both acceptors and affectors of cell signaling pathways. Many desmosomal and hemidesmosomal constituents are phosphoproteins and in certain cases the function of specific phosphorylation sites in regulating protein-protein interactions is being uncovered. In addition, a more active role in transmitting signals that control morphogenesis during development and possibly even regulate cell growth and differentiation are being defined for cytoplasmic and membrane components of these junctions.

Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration.

Abstract: Extracellular matrix (ECM) geometry is an important modulator of cell polarity and function. For example, 3-dimensional matrices often more effectively induce differentiated cell function than traditional 2-dimensional substrates. The effect of ECM topology can be investigated in a controlled fashion using a technique whereby cells cultured on a single surface are overlaid with a second layer of ECM, thereby creating a "sandwich" configuration. Confluent monolayers of epithelial or endothelial cells overlaid in this fashion often reorganize into structures that are reminiscent of their native tissue. In the case of hepatocytes, the overlay causes a dramatic reorganization of the cytoskeleton, adoption of in vivo-like morphology and polarity, and expression of a wide array of liver-specific functions. In this short review, we use the sandwiched hepatocyte culture system to illustrate the effect of ECM geometry on cellular function. Pertinent studies are summarized in the context of defining the parallels, strengths, and limitations of this culture system as an in vitro model to study the physiology and morphogenesis of liver tissue. We also explore some of its potential uses as a model to study liver pharmacology and toxicology, and for the development of liver preservation techniques and liver-assist devices.

Immobilization stress causes oxidative damage to lipid, protein, and DNA in the brain of rats.

Abstract: Immobilization stress of male Sprague-Dawley rats induces oxidative damage to lipid, protein, and DNA in the brain. Significant increases in lipid peroxidation were found in the cerebral cortex, cerebellum, hippocampus, and midbrain compared to the unstressed controls. Significant increases in levels of protein oxidation were also found in the cortex, hypothalamus, striatum, and medulla oblongata. Oxidative nuclear DNA damage increased after stress in all brain regions, although only the cerebral cortex showed a significant increase. Depletion of glutathione showed some stimulation to oxidative damage in the unstressed control and stressed animals. Further studies of the mitochondrial and cytosol fractions of cerebral cortex demonstrated that mitochondria showed a significantly greater increase in lipid peroxidation and protein oxidation than cytosol. Data from plasma and liver showed oxidative damage similar to that of the brain. These findings provide evidence to support the idea that stress produces ox...