Crystal structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase complexed with 17 beta-estradiol.
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Binding Sites↗
Biomedical subjects
A Azzi
Publications and source records attributed to A Azzi.
Correlation between human vascular smooth muscle cell proliferation and protein kinase C alpha-expression: effect of d-alpha-tocopherol.
Population doublings of four different human aortic vascular smooth muscle cell strains correlate with the amount of protein kinase C alpha present in these cells. d-alpha-Tocopherol inhibits, at different extents, protein kinase C activity in all cells studied. Furthermore, the extent of inhibition positively correlates with amount of protein kinase C alpha expression and not with that of the other isoforms. It is suggested that, in human aortic smooth muscle cells, protein kinase C alpha modulates cell proliferation and serves as a target for d-alpha-tocopherol inhibition.
Cell Division↗
Vitamin E inhibits proliferation of human Tenon's capsule fibroblasts in vitro.
Failure of glaucoma surgery is mostly due to fibrocellular scar formation, derived from Tenon's capsule fibroblasts. In high-risk cases, postoperative Tenon's capsule fibroblast proliferation is inhibited by mitomycin C or 5-fluorouracil. Toxicity to other ocular cell types and the risk of ocular hypotony limits the use of these agents. We have found that d-alpha-tocopherol (vitamin E) was able to inhibit proliferation of in vitro human Tenon's capsule fibroblasts obtained from seven different donors. At 48 h, inhibition of cell proliferation was 30-78% (mean 60%) for 50 microM d-alpha-tocopherol and 46-97% (mean 77%) for 100 microM d-alpha-tocopherol. This inhibition was statistically significant. No cytotoxic effects were observed.
Adolescent↗
Human polyomaviruses DNA detection in peripheral blood leukocytes from immunocompetent and immunocompromised individuals.
Peripheral blood leukocytes from immunocompetent and immunocompromised individuals were analyzed for human polyomarivus BK and JC DNA presence. A nested polymerase chain reaction which amplify the transcriptional control region of the genome of both viruses was employed. The immunocompromised patients included bone marrow transplantation recipients and AIDS patients. BKV sequences were detectable in 52.8-62.5% of the individuals included in this study, whereas the percentage of individuals with JCV sequences in peripheral blood lymphocytes varied from 38.8% to 50%. The frequency of reactivations of BKV and JCV were also determined by detection of shedding in urine of viral DNA. The highest frequency of reactivations of either BKV or JCV was demonstrable in the group of bone marrow transplantation recipients, but reactivations occurred also in immunocompetent individuals. JCV sequences amplified from urine samples showed a restriction pattern similar to the archetype one, whereas sequences obtained from lymphocytes showed rearranged pattern as well as archetype pattern. Finally all JCV sequences from cerebrospinal fluid seemed to be rearranged. These observations suggest that peripheral blood lymphocytes have a fundamental role in the persistence of polyomaviruses infection and in the dissemination at least of JCV within the organism allowing that rearranged variants, better adapted to grow in brain tissue, emerge.
Acquired Immunodeficiency Syndrome↗
The role of hydrogen peroxide and RRR-alpha-tocopherol in smooth muscle cell proliferation.
Oxidants can be considered early growth signals, since they have been shown to activate a number of pathways that are also stimulated by growth factors. In particular, H(2)O(2) activates the protein kinase C signal transduction pathway in smooth muscle cells. These events certainly play a role in the activation of the DNA synthesis machinery although it is still unclear whether they can also regulate the lethal response. Evidence exists of an oxidant-mediated increase in tyrosine protein phosphorylation as an early event in the signal transduction cascade of growth factor receptors, leading to augmentation of cell proliferation. Oxidants can also induce transcription of enzymes, such as ornithine decarboxylase and the phosphatase CL-100. CL-100 is the first example of a new class of protein phosphatases responsible for modulating the activation of MAP kinase following exposure of quiescent cells to growth factors and further implicates MAP kinase activation/deactivation in the cellular response to hydrogen peroxide. Moreover H(2)O(2) activates the MAP kinase cascade by stimulating the tyrosine kinase and protein kinase C pathways. JNK1, a relative of the MAP kinase group, is activated by dual phosphorylation at Thr and Tyr during the UV response. RRR-alpha-tocopherol and RRR-beta-tocopherol have different and competing effects on smooth muscle cell proliferation, indicating that they do not act as antioxidants. The earliest event brought by RRR-alpha-tocopherol in the signal transduction cascade contolling receptor mediated cell growth is the inhibition of the transcription factor AP-1, activated by phorbol esters. RRR-beta-tocopherol alone is without effect but in combination with RRR-alpha-tocopherol prevents the AP-1-inhibiting effect of the latter. Protein kinase C is inhibited by RRR-alpha-tocopherol and not by RRR-beta-tocopherol, which also in this case prevented the effect of RRR-alpha-tocopherol. The inhibition of RRR-alpha-tocopherol of protein kinase C is not the consequence of a direct interaction but is due to a diminution, produced by RRR-alpha-tocopherol of the kinase phosphorylation. A tocopherol binding protein appears to be at the basis of the RRR-alpha-tocopherol, that discriminates between RRR-alpha-tocopherol and RRR-beta-tocopherol and initiates a cascade of events at the level of cell signal transduction leading to cell proliferation inhibition.
Journal Article↗
Studies on the three-dimensional structure of estrogenic 17 beta-hydroxysteroid dehydrogenase.
The structure-function relationship of the estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), a pivotal enzyme in the synthesis of active sex hormones, has been studied via protein chemistry and crystallography. A highly active and homogeneous 17 beta-HSD1 was prepared with a rapid purification from human placenta. We then characterized the native and expressed enzyme, and concluded, for the first time, that 17 beta-HSD1 is formed by two identical subunits. The enzyme was also overproduced in insect cells with a baculovirus expression system. The highly active 17 beta-HSD1 preparation was successfully crystallized in the presence of NADP-, polyethylene glycol, beta-octylglucoside and glycerol, resulting in the first diffraction quality crystals of any steroid-converting enzyme from a human source. The three-dimensional structure of 17 beta-HSD1 was determined at 2.2 A resolution, showing that the overall structure of the enzyme is similar to the other enzymes in the short-chain dehydrogenase family, with a conserved Tyr-X-X-X-Lys sequence and a serine residue in the active site. It is distinguished from the other known structures reported for short-chain dehydrogenases by the insertion of two helix-turn-helix motifs that appear to govern membrane association and substrate specificity [corrected]. More recently, the complex of 17 beta-HSD1 with estradiol has been successfully crystallized and its structure determined. The latter demonstrates detailed information of the interactions between the substrate and residues Ser142, Tyr155, His221 and Glu282 of the enzyme. These interactions and the complementarity of the substrate with the hydrophobic binding pocket make critical contributions to the enzyme specificity. The above results provide a strong basis for the design of potent inhibitors of this pivotal steroid dehydrogenase.
Estradiol Dehydrogenases↗
B19 parvovirus withstands "super heating" in antihemophilic concentrates.
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Blood↗
d-alpha-tocopherol inhibition of vascular smooth muscle cell proliferation occurs at physiological concentrations, correlates with protein kinase C inhibition, and is independent of its antioxidant properties.
d-alpha-Tocopherol, but not d-beta-tocopherol, negatively regulates proliferation of vascular smooth muscle cells at physiological concentrations. d-alpha-Tocopherol inhibits protein kinase C (PKC) activity, whereas d-beta-tocopherol is ineffective. Furthermore d-beta-tocopherol prevents the inhibition of cell growth and of PKC activity caused by d-alpha-tocopherol. The negative regulation by d-alpha-tocopherol of PKC activity appears to be the cause and not the effect of smooth muscle cell growth inhibition. d-alpha-Tocopherol does not act by binding to PKC directly but presumably by preventing PKC activation. It is concluded that, in vascular smooth muscle cells, d-alpha-tocopherol acts specifically through a nonantioxidant mechanism and exerts a negative control on a signal transduction pathway regulating cell proliferation.
Amino Acid Sequence↗
Stimulation of protein kinase C activity by compactin in vascular smooth muscle cells.
The effect of compactin (a lovastatin analogue) on vascular smooth muscle cells was studied at the level of cell proliferation and protein kinase C. It was observed: a) an inhibition of cell proliferation by compactin at a micromolar range, which was prevented by simultaneous addition of mevalonate; b) a stimulation of DNA synthesis with a shift in the cell cycle kinetics, either in the presence or absence of fetal calf serum and c) an increase in protein kinase C activity in compactin-treated cells in the G1 phase of the cycle. This increase was similar to the one elicited by calyculin A, an inhibitor of protein phosphatases of type PP-1 and PP-2A. It is suggested that compactin behaves as a PP-1/PP-2A protein phosphatase inhibitor, inhibiting proliferation of smooth muscle cells by a block of the cell cycle after the S-phase.
Animals↗
Hydrogen peroxide-and fetal bovine serum-induced DNA synthesis in vascular smooth muscle cells: positive and negative regulation by protein kinase C isoforms.
Hydrogen peroxide and fetal bovine serum stimulate DNA synthesis in growth-arrested smooth muscle cells with remarkably similar kinetics and cell density dependence. However, while stimulation with fetal bovine serum results in cell proliferation, that by H2O2 is followed by cell death. Depletion of conventional and novel protein kinase C isoforms, resulting from a long treatment with phorbol-12-myristate-13-acetate, further increases H2O2-induced DNA synthesis. On the other hand, the specific protein kinase C inhibitor calphostin C abolished the increased DNA synthesis promoted by fetal bovine serum or H2O2. H2O2 increases protein kinase C activity in smooth muscle cells. This effect is markedly reduced, but not abolished, by down-regulation of the alpha, delta and epsilon protein kinase C isoforms. Thus, the zeta isoform of protein kinase C, which is not down-regulated, may be responsible for the residual H2O2 stimulation of protein kinase C. In conclusion, the results obtained show that H2O2 stimulates protein kinase C activity and DNA synthesis in growth-arrested smooth muscle cells: these events are not followed by cell proliferation but rather by cell death. This H2O2 stimulated DNA synthesis appears to be negatively controlled by alpha, delta and epsilon isoforms and positively controlled by the zeta isoform of protein kinase C.
Cell Count↗
Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-alpha-mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain.
Cellular signalling by the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) has been suggested to involve generation of low levels of reactive oxygen species (ROS). Certain antioxidants and metal chelators can inhibit cytotoxicity and gene expression in response to TNF alpha in numerous cell types. However, neither the source nor function of TNF alpha-induced oxidant generation is known. Using specific inhibitors, we ruled out involvement of several oxidant-generating enzymes [cyclo-oxygenase (indomethacin), cytochrome P-450 (metyrapone), nitric oxide synthase (NG-methyl-L-arginine), NADPH oxidase (iodonium diphenyl), xanthine oxidase (allopurinol), ribonucleotide reductase (hydroxyurea)] in TNF alpha-mediated apoptosis of the murine fibrosarcoma line, L929. We also demonstrated no role for mitochondrial-derived radicals/respiratory chain in the lytic pathway using specific inhibitors/uncouplers (rotenone, KCN, carboxin, fluoroacetate, antimycin, malonate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and chloramphenicol-derived respiration-deficient cells. Significant ROS (H2O2, O2-.) generation was not observed in response to TNF alpha in L929 cells using four separate assays. Also, prevention of intracellular H2O2 removal by inhibition of catalase did not potentiate TNF alpha-mediated cell death. These data suggest that neither H2O2 nor O2-. plays a direct role in TNF alpha cytotoxicity. Finally, we suggest a central role for lipoxygenase in TNF alpha-mediated lysis. Three inhibitors of this radical-generating signalling pathway, including an arachidonate analogue (5,8,11,14-eicosatetraynoic acid), could protect cells against TNF alpha. The inhibitor nordihydroguaiaretic acid is also a radical scavenger, but it could not protect cells from ROS toxicity at concentrations that effectively prevented TNF alpha killing. Therefore protection by nordihydroguaiaretic acid cannot be due to scavenging of cytotoxic H2O or O2-.. The lipoxygenase product, (12S)-hydroxyeicosatetraenoic acid, was also significantly protective. As this analogue can act as a substrate for certain lipoxygenases, this effect may be due to prevention of generation of physiological products.
Animals↗
Tocopherols and 6-hydroxy-chroman-2-carbonitrile derivatives inhibit vascular smooth muscle cell proliferation by a nonantioxidant mechanism.
The effects of two groups of similar compounds, a series of tocopherols and one of 6-hydroxy-chroman-2-carbonitrile, have been studied in vascular smooth muscle cells. A poor correlation has been found between antiproliferative and antioxidant properties of these molecules. D-alpha-Tocopherol inhibits cell proliferation, while D-alpha-tocopherylquinone has been found neither to inhibit nor to activate. D-beta-Tocopherol, a poor inhibitor of smooth muscle cell proliferation, has been shown to be capable of preventing and reversing the inhibition by D-alpha-tocopherol. It is concluded that the tocopherols and carbonitrile derivatives tested here appear to inhibit smooth muscle cell proliferation by a nonantioxidant mechanism. The competition between D-alpha-tocopherol and D-beta-tocopherol suggests the existence of a common binding site for the two molecules.
Animals↗
Role of oxidants in TNF-alpha-mediated cytotoxicity.
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5,8,11,14-Eicosatetraynoic Acid↗
Vitamin E: a sensor and an information transducer of the cell oxidation state.
We studied the effects of RRR-alpha-tocopherol and RRR-beta-tocopherol in smooth muscle cells from rat (line A7r5) and human aortas. RRR-alpha-Tocopherol, but not RRR-beta-tocopherol, inhibited smooth muscle cell proliferation in a dose-dependent manner at concentrations in the range from 10 to 50 mumol/L. RRR-beta-Tocopherol added simultaneously with RRR-alpha-tocopherol prevented growth inhibition. The earliest event brought about by RRR-alpha-tocopherol in the signal transduction cascade controlling receptor-mediated cell growth was the activation of the transcription factor AP-1. RRR-beta-tocopherol alone was without effect but in combination with RRR-alpha-tocopherol prevented the AP-1 activating effect of the latter. Protein kinase C was inhibited by RRR-alpha-tocopherol and not by RRR-beta-tocopherol, which also in this case prevented the effect of RRR-alpha-tocopherol. Calyculin A, a protein phosphatase inhibitor, prevented the effect of RRR-alpha-tocopherol on protein kinase C. The data can be rationalized by a model in which a tocopherol-binding protein discriminates between RRR-alpha-tocopherol and RRR-beta-tocopherol and initiates a cascade of events at the level of cell signal transduction that leads to the inhibition of cell proliferation.
Animals↗
New roles of low density lipoproteins and vitamin E in the pathogenesis of atherosclerosis.
Accumulation of oxidized low density lipoproteins in macrophages and smooth muscle cells causes foam cell formation, an initial step in atherosclerosis. Active oxygen species are considered important in the pathogenesis of the disease. Antioxidants, such as tocopherols and tocotrienols have been considered to prevent the deleterious effects of active oxygen species. We found native low density lipoproteins can stimulate directly smooth muscle cell proliferation, it is associated with an increase of protein kinase C activity. d-alpha-Tocopherol, biologically most active form of vitamin E, inhibits both cell proliferation and protein kinase C activity. The effect of d-alpha-tocopherol is not related to its radical scavenging properties. Transforming growth factor-beta secreted by smooth muscle cells as growth inhibitor. Low density lipoproteins decrease the release of transforming growth factor-beta from smooth muscle cells thus activating growth. d-alpha-Tocopherol activates the cellular release of transforming growth factor-beta. These new aspects explain the important role of low density lipoproteins and vitamin E in increasing and decreasing the risk of atherosclerosis, respectively.
Amino Acid Sequence↗
Severe aplastic anaemia due to B19 parvovirus infection in renal transplant recipient.
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Anemia, Aplastic↗
Inhibition of protein kinase C activity and vascular smooth muscle cell growth by d-alpha-tocopherol.
The inhibition by d-alpha-tocopherol of protein kinase C activity has been studied in synchronised A7r5 rat smooth muscle cells during the cell cycle. Cell protein kinase C activity has been found to oscillate, with a minimum in the G0 phase, a maximum in the late G1 phase and a new minimum in the S phase. An inhibition of protein kinase C activity by d-alpha-tocopherol appears to be at the basis of cell growth inhibition. Nevertheless, the amount of the different protein kinase C isoenzymes present in smooth muscle cells, measured by their specific antibodies, does not change during the cell cycle in both untreated and d-alpha-tocopherol-treated cells. The possible mechanisms of protein kinase C modulation during the cell cycle and of its inhibition by d-alpha-tocopherol are discussed.
Animals↗
Modulation of activator protein-1 (AP-1) transcription factor and protein kinase C by hydrogen peroxide and D-alpha-tocopherol in vascular smooth muscle cells.
The effects of hydrogen peroxide D-alpha-tocopherol and of D-beta-tocopherol on proliferation, protein kinase C and activator protein-1 (AP-1) activation have been studied in vascular smooth muscle cells. Cell proliferation, when activated by foetal calf serum, was inhibited by D-alpha-tocopherol. Protein kinase C activity was stimulated by hydrogen peroxide in a manner similar to phorbol myristate acetate; in the latter case, but not in the former, D-alpha-tocopherol inhibited the reaction. Hydrogen peroxide prevented phorbol-myristate-acetate-stimulated AP-1 binding to DNA but stimulated it if protein kinase C was down-regulated or inhibited. D-alpha-Tocopherol promoted AP-1 activation in quiescent cells but prevented its activation by phorbol myristate acetate. None of the described effects of D-alpha-tocopherol were shared by D-beta-tocopherol, suggesting a non-antioxidant mechanism as the basis of its action. The data show that hydrogen peroxide and D-alpha-tocopherol affect more than one element in the cell signal-transduction cascade.
Animals↗