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1.
F J Hornicek G I Malinin H G Gratzner T I Malinin 《Chemico-biological interactions》1985,55(3):289-302
Nitroxyldisulfonate [Fremy's salt; (KSO3)2NO.] and bisulfite (NaHSO3) have abolished periodic acid (H5IO6)-induced blastogenesis of human peripheral blood lymphocytes (HPBL), but only inhibited the blastogenic response of H5IO6-oxidized rat and mouse lymphocytes, as determined by the rates of nucleic acids synthesis, BrdUrd incorporation and by cell numbers in S + G2 + M phases of the cell cycle. The viability of the intact human, rat and mouse lymphocytes remained essentially unimpaired by 30 min pulses of 1 mM Fremy's salt or bisulfite. The marked inhibition of periodic acid-induced blastogenesis, exerted by Fremy's salt and by bisulfite, was attributed to the effect of the corresponding carbonyl addition derivatives formed in situ of the oxidized cell membranes. Consequently, it is concluded that Fremy's salt like bisulfite possibly forms addition derivatives with membrane carbonyls of viable target cells. 相似文献
2.
Divergent activities of protein kinases in IL-6-induced differentiation of a human B cell line 总被引:3,自引:0,他引:3
H Goldstein D Koerholz L Chesky X D Fan J L Ambrus 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(3):952-961
Lymphokines including IL-2, IL-4, and IL-6 are involved in the induction of Ig production by activated B cells. We have investigated the role of protein kinases in IL-6-induced IgM secretion by SKW6.4 cells, an IL-6 responsive B cell line. IL-6-stimulated IgM production was inhibited by elevated intracellular cAMP induced either by the addition of dibutyryl cAMP or cholera toxin. The inhibitory effect of elevated intracellular cAMP was blocked by n-(2-(Methylamino)ethyl)-5-isoquinolinesulfonic dihydrochloride (H8), an inhibitor of protein kinase A. H8 did not affect IgM secretion induced by IL-6. In contrast, the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H7), an inhibitor of protein kinase C activity, markedly inhibited IL-6-stimulated IgM production by SKW6.4 cells. H7 and elevated intracellular cAMP inhibited IgM mRNA expression and subsequent IgM synthesis by SKW6.4 cells. SKW6.4 proliferation, as determined by [3H]thymidine incorporation, was not markedly affected by IL-6, dibutyryl cAMP, cholera toxin, H7 or H8. PMA, an activator of protein kinase C, directly stimulated significant IgM secretion by SKW6.4 cells. When added to PMA-stimulated SKW6.4 cells, IL-6 stimulated additional IgM production. This observation suggested that IL-6 could stimulate differentiation without activating protein kinase C. This was confirmed by demonstrating that IL-6 did not stimulate production of diacylglycerol, did not induce the translocation of protein kinase C from the cytosolic compartment to the plasma membrane and could induce SKW6.4 cells to produce IgM after depletion of their cellular protein kinase C by PMA. Taken together these results suggests that IL-6-stimulated IgM production requires utilization of an H7-inhibitable protein kinase that can be inhibited by a protein kinase A-dependent pathway. Despite the fact that PMA can stimulate IgM production in SKW6.4 cells, IL-6 appears to use a protein kinase pathway other than protein kinase C to induce IgM production. 相似文献
3.
Gruber R Nothegger G Ho GM Willheim M Peterlik M 《Biochemical and biophysical research communications》2000,270(3):1080-1085
Formation of osteoclast-like cells in mouse bone marrow cultures induced by either 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), parathyroid hormone (PTH) or prostaglandin E(2) (PGE(2)), respectively, shows partial dependence on interleukin-6 receptor (IL-6R) activation. This suggests that locally produced IL-6 could be relevant for osteoclast formation. Therefore, we evaluated the effects of 1,25-(OH)(2)D(3), PTH, and PGE(2) on IL-6 production in stromal/osteoblastic cell lines. It appeared that these bone resorptive factors differed widely in their ability to modulate IL-6 mRNA expression and, consequently, protein synthesis in each of the cell lines studied. While 1,25-(OH)(2)D(3) was marginally effective only in ST2 cells, and PTH caused a 2- to 20-fold increase in IL-6 levels MC3T3-E1 and UMR-106 cells, PGE(2) enhanced IL-6 production in the ST2 and MC3T3-E1 cell line by two to three orders of magnitude, respectively, and also induced IL-6 in fibroblastic L929 cells. PGE(2)-stimulated IL-6 release from mesenchymal cells seems to be important for autocrine/paracrine control of osteoclast formation in health and disease. 相似文献
4.
Giambartolomei GH Zwerdling A Cassataro J Bruno L Fossati CA Philipp MT 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(7):4635-4642
Inflammation is a hallmark of brucellosis. Although Brucella abortus, one of the disease's etiologic agents, possesses cytokine-stimulatory properties, the mechanism by which this bacterium triggers a proinflammatory response is not known. We examined the mechanism whereby heat-killed B. abortus (HKBA), as well as its LPS, induces production of inflammatory cytokines in monocytes/macrophages. Polymyxin B, a specific inhibitor of LPS activity, did not inhibit the production of TNF-alpha- and IL-6-induced HKBA in the human monocytic cell line THP-1. HKBA induced the production of these cytokines in peritoneal macrophages of both C3H/HeJ and C3H/HeN mice, whereas B. abortus LPS only stimulated cells from C3H/HeN mice. Anti-TLR2 Ab, but not anti-TLR4 Ab, blocked HKBA-mediated TNF-alpha and IL-6 production in THP-1 cells. Because bacterial lipoproteins, a TLR2 ligand, have potent inherent stimulatory properties, we investigated the capacity of two B. abortus lipoproteins, outer membrane protein 19 (Omp19) and Omp16, to elicit a proinflammatory response. Lipidated (L)-Omp16 and L-Omp19, but not their unlipidated forms, induced the secretion of TNF-alpha, IL-6, IL-10, and IL-12 in a time- and dose-dependent fashion. Preincubation of THP-1 cells with anti-TLR2 Ab blocked L-Omp19-mediated TNF-alpha and IL-6 production. Together, these results entail a mechanism whereby B. abortus can stimulate cells from the innate immune system and induce cytokine-mediated inflammation in brucellosis. We submit that LPS is not the cause of inflammation in brucellosis; rather, lipoproteins of this organism trigger the production of proinflammatory cytokines, and TLR2 is involved in this process. 相似文献
5.
Reactivities of hydroxylamine and sodium bisulphite with carbonyl-containing haems with the prosthetic groups of the erythrocyte green haemoproteins
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The reactivities of alkaline NH(2)OH and neutral NaHSO(3) with carbonyl and olefinic groups conjugated with the tetrapyrrole nucleus of haems were studied. The reactions were carried out with 2-3nmol of haem a, spirographis haem, isospirographis haem, 2,4-diacetyldeuterohaem and protohaem. Vinyl side chains were found to be insensitive to the chemical action of both alkaline NH(2)OH and neutral NaHSO(3). The formyl-containing haems reacted rapidly with both reagents at room temperature, as evidenced by sizable hypsochromic shifts of the reduced pyridine haemochrome spectrum. In less alkaline solution, the reactions of these formyl-containing haems with NH(2)OH were much slower. 2,4-Diacetyldeuterohaem reacted with alkaline NH(2)OH, but not with neutral NaHSO(3). These rapid, simple and straightforward tests are readily usable in differentiating among formyl, acetyl and other electron-withdrawing side chains conjugated with the tetrapyrrole ring of haems. We applied these observations to an investigation of the two unique prosthetic groups of the bovine erythrocyte green haemoproteins. The prosthetic groups of these two proteins were isolated and spectrally characterized. Under the conditions used, the haems did not react with either NH(2)OH or NaHSO(3), but were altered by dithionite, suggesting that the previous interpretation that a formyl group was present [Hultquist, Dean & Reed (1976) J. Biol. Chem.251, 3927-3932] may have been premature. These studies also provide evidence that the alpha-hydroxyfarnesylethyl side chain of haem a affects the alpha-band maximum, but not the beta- or Soret bands of the reduced pyridine haemochrome spectrum of haem a. 相似文献
6.
D A Bazylinski R A Arkowitz T C Hollocher 《Archives of biochemistry and biophysics》1987,259(2):520-526
The reaction between hydroxylamine (NH2OH) and human hemoglobin (Hb) at pH 6-8 and the reaction between NH2OH and methemoglobin (Hb+) chiefly at pH 7 were studied under anaerobic conditions at 25 degrees C. In presence of cyanide, which was used to trap Hb+, Hb was oxidized by NH2OH to methemoglobin cyanide with production of about 0.5 mol NH+4/mol of heme oxidized at pH 7. The conversion of Hb to Hb+ was first order in [Hb] (or nearly so) but the pseudo-first-order rate constant was not strictly proportional to [NH2OH]. Thus, the apparent second-order rate constant at pH 7 decreased from about 30 M-1 X s-1 to a limiting value of 11.3 M-1 X s-1 with increasing [NH2OH]. The rate of Hb oxidation was not much affected by cyanide, whereas there was no reaction between NH2OH and carbonmonoxyhemoglobin (HbCO). The pseudo-first-order rate constant for Hb oxidation at 500 microM NH2OH increased from about 0.008 s-1 at pH 6 to 0.02 s-1 at pH 8. The oxidation of Hb by NH2OH terminated prematurely at 75-90% completion at pH 7 and at 30-35% completion at pH 8. Data on the premature termination of reaction fit the titration curve for a group with pK = 7.5-7.7. NH2OH was decomposed by Hb+ to N2, NH+4, and a small amount of N2O in what appears to be a dismutation reaction. Nitrite and hydrazine were not detected, and N2 and NH+4 were produced in nearly equimolar amounts. The dismutation reaction was first order in [Hb+] and [NH2OH] only at low concentrations of reactants and was cleanly inhibited by cyanide. The spectrum of Hb+ remained unchanged during the reaction, except for the gradual formation of some choleglobin-like (green) pigment, whereas in the presence of CO, HbCO was formed. Kinetics are consistent with the view advanced previously by J. S. Colter and J. H. Quastel [1950) Arch. Biochem. 27, 368-389) that the decomposition of NH2OH proceeds by a mechanism involving a Hb/Hb+ cycle (reactions [1] and [2]) in which Hb is oxidized to Hb+ by NH2OH. 相似文献
7.
The combined action of ammonia monooxygenase, AMO, (NH(3)+2e(-)+O(2)-->NH(2)OH) and hydroxylamine oxidoreductase, HAO, (NH(2)OH+H(2)O-->HNO(2)+4e(-)+4H(+)) accounts for ammonia oxidation in Nitrosomonas europaea. Pathways for electrons from HAO to O(2), nitrite, NO, H(2)O(2) or AMO are reviewed and some recent advances described. The membrane cytochrome c(M)552 is proposed to participate in the path between HAO and ubiquinone. A bc(1) complex is shown to mediate between ubiquinol and the terminal oxidase and is shown to be downstream of HAO. A novel, red, low-potential, periplasmic copper protein, nitrosocyanin, is introduced. Possible mechanisms for the inhibition of ammonia oxidation in cells by protonophores are summarized. Genes for nitrite- and NO-reductase but not N(2)O or nitrate reductase are present in the genome of Nitrosomonas. Nitrite reductase is not repressed by growth on O(2); the flux of nitrite reduction is controlled at the substrate level. 相似文献
8.
Interleukin (IL)-1alpha and IL-1beta share low amino acid homology, but exhibit a very similar array of biological activities. The authors previously showed negative regulation of IL-1alpha-induced prostaglandin (PG) production by corticotropin releasing factor (CRF). In this study, the authors compared the effect of CRF on IL-1alpha- and IL-1beta-induced PG synthesis. IL-1alpha (100 U/ml) increased prostacyclin (PGI2) (measured as 6-keto PGF1alpha[6K]) synthesis in endothelial cells and the production of PGE2in fibroblasts. The PG response to IL-1alpha was suppressed by simultaneous exposure to CRF (2.5x10(-11)-2.5x10(-8) M) in both cell types. IL-1alpha enhanced both phospholipase A2(PLA2) and prostaglandin H synthase (PGHS) activities, and the two effects were completely abrogated by CRF. IL- 1beta (100 U/ml) was as active as IL-1alpha in triggering release of PGI2 from endothelial cells and PGE2 from fibroblasts. However, CRF (2.5x10(-11)-2.5x10(-8) M) failed to alter the IL-1beta-induced PG synthesis in both cell types. Following IL-1beta PGHS activity, and to a lesser extent PLA2 activity, were enhanced, however CRF only inhibited PGHS and not PLA2 activity. It is concluded that although IL-1alpha and IL-1beta usually produce similar biological effects, here they seem to act via different mechanisms. The different regulation of IL-1alpha and IL-1beta pro-inflammatory activities by CRF may attribute special precision and specificity to the neuroendocrine-immune control of inflammatory processes. 相似文献
9.
Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus. 总被引:2,自引:3,他引:2
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Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated. Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source. Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h. The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity. This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein. H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase. H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited. Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely. In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels. 相似文献
10.
A number of ruthenium complexes were tested for their ability to induce filamentation in Escherichia coli. These included monomeric and dimeric complexes with ruthenium in the II or III oxidation states, as well as mixed-valence complexes with ruthenium in the (II,III) oxidation states. In general, dimeric mixed-valence Ru(II,III) complexes were the most active class of compound, although some complexes of this type were relatively inactive. These were pyrazine- or bipyridyl-bridged complexes which are known to involve strong metal-ligand interaction, which stabilizes the Ru(II) oxidation state. Some Ru(III) complexes were also significantly active in induction of filamentous growth in E. coli. One of these was [Ru(NH3)5Cl]Cl2, which did not inhibit electron transport, Mg2+-ATPase activity or DNA synthesis in E. coli, but like [Ru2(NH3)6Br3]Br2 X H2O was a potent inhibitor of respiration-driven calcium transport in the organism. Filament-inducing activity of the complex was reduced in the presence of NaCl, but not in the presence of added Ca2+, ethanol, calcium pantothenate, or E. coli 'division promoting extract'. This behaviour is also similar to that of [Ru2(NH3)6Br3]Br2 X H2O. It is suggested that both complexes may induce filamentation in E. coli by a common mechanism, which may involve interference with calcium metabolism, or a wall or membrane target, rather than interaction with DNA. 相似文献
11.
Studies on electron transfer from methanol dehydrogenase to cytochrome cL, both purified from Hyphomicrobium X. 总被引:1,自引:0,他引:1
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Ferricytochrome cL isolated from Hyphomicrobium X is an electron acceptor in assays for homologous methanol dehydrogenase (MDH), albeit a poor one compared with artificial dyes. The intermediates of MDH seen during the reaction are identical with those observed with Wurster's Blue as electron acceptor, indicating that the reaction cycles are similar. The assay showed a pH optimum of approx. 7.0 and scarcely any stimulation by NH4Cl, this being in contrast with assays with artificial dyes, where strong activation by NH4Cl and much higher pH optima have been reported. From the results obtained with stopped-flow as well as steady-state kinetics, combined with the isotope effects found for C2H3OH, it appeared that the dissimilarities between the electron acceptors can be explained from different rate-limiting steps in the reaction cycles. Ferricytochrome cL is an excellent oxidant of the reduced MDH forms at pH 7.0, but the substrate oxidation step is very slow and the activation by NH4Cl is very poor at this pH. At pH 9.0 the reverse situation exists: ferricytochrome cL is a poor oxidant of the reduced forms of MDH at this pH. No C2H3OH isotope effect was observed under these conditions, indicating that substrate oxidation is not rate-limiting, so that activation by NH4Cl cannot be found. Since just the opposite holds for assays with artificial dyes, the poor electron-acceptor capability and the different pH optimum of ferricytochrome cL as well as the insignificant activating effect of NH4Cl (all compared with artificial assays) can be explained. Although different views have been reported on the rate-limiting steps in the systems from Methylophilus methylotrophus and Methylobacterium sp. strain AM1, these are most probably incorrect, as rate-limiting electron transfer between ferrocytochrome cL and horse heart ferricytochrome c can occur. Therefore the conclusions derived for the Hyphomicrobium X system might also apply to the systems from other methylotrophic bacteria. Comparison of the assays performed in vitro (at pH 7.0) having ferricytochrome cL and Wurster's Blue as electron acceptor with methanol oxidation by whole cells shows that the former has similarity whereas the latter has not, this being although ferricytochrome cL is a poor electron acceptor in the assay performed in vitro. The reason for this is the absence of a (natural) activator able to activate the (rate-limiting) substrate oxidation step at physiological pH values. 相似文献
12.
Using microarrays to analyze differential gene expression as a function of p53 status and radiation quality, we observed downregulation of a large set of histone genes in p53 wild-type TK6 cells 24 h after exposure to equitoxic doses of high-LET (1.67 Gy 1 GeV/amu (56)Fe ions) or low-LET (2.5 Gy γ rays) radiation. Quantitative real-time PCR of specific subtypes of core (H2A, H2B, H3 and H4) and linker (H1) histones confirmed this result. DNA synthesis and histone gene expression are tightly coordinated during the S phase of the cell cycle, and both processes are regulated by cell cycle checkpoints in response to DNA damage caused by ionizing radiation. However, we observed similar repression of histone gene expression in both TK6 cells and their p53-null derivative NH32 after radiation exposure, although the histone gene expression was not decreased to the same extent in NH32 cells as it was in TK6 cells. We also found decreased histone gene expression that was dose- and time-dependent in the colon cancer cell line HCT116 and its p53-null derivative. These results show that both high- and low-LET radiation exposure negatively regulate histone gene expression in human lymphoblastoid and colon cancer cell lines independent of p53 status. 相似文献
13.
14.
The YadA protein of Yersinia pseudotuberculosis promotes tight adhesion and invasion into mammalian cells through beta(1)-integrins. In this work, we demonstrate that YadA also triggers the production of interleukin-8 (IL-8) in host cells and we identify intracellular signal transduction mechanisms involved in YadA-initiated cell invasion and/or IL-8 synthesis. Tyrosine protein kinases, including the focal adhesion kinase (FAK) and c-Src, as well as the small GTPase Ras, were shown to play a significant role in both YadA-promoted cell processes. YadA-mediated cell contact led to autophosphorylation of FAK at position Tyr397 and induced GTP-loading of Ras. Furthermore, IL-8 production and invasion induced by YadA were strongly reduced in FAK- and c-Src-deficient cells and in cells overexpressing dominant interfering forms of FAK, c-Src or Ras. We also demonstrate that YadA activates the Ras-dependent Raf-MEK1/2-ERK1/2 pathway and mitogen-activated protein kinases (MAPKs) p38 and JNK. Moreover, inhibition of ERK1/2 by pharmacological agents or overexpression of dominant negative FAK, c-Src or Ras abrogated IL-8 release, whereas invasion remained unaffected. In contrast, actin polymerization and phosphatidylinositol 3-kinase (PI3K) activity is essential for YadA-promoted cell entry, but not for cytokine secretion. We conclude that YadA triggers FAK-Src complex formation and subsequent Ras activation, which leads to the stimulation of MAPKs-dependent IL-8 production or to PI3K-dependent invasion. 相似文献
15.
16.
Differentiation and maturation of monocytes are accompanied by the expression of specific surface glycoproteins, the secretion of cytokines, and the capacity to respond to ligands. These changes may be influenced by interactions with hormones, soluble lymphocytic products, or direct contact with lymphocytes. We have studied two distinct pathways in the differentiation of a human monocytic cell line, THP-1: one being induced by IFN-gamma and the other by 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In THP-1 cells, IFN-gamma induces cell surface expression of HLA-DR and CD54 and production of IL-1 beta, TNF-alpha, and IL-6. In contrast, 1,25(OH)2D3 increases cell surface expression of CD11b and CD14, but fails to stimulate cytokine production. Direct contact of THP-1 with stimulated fixed T cells markedly induces IL-1 beta, TNF-alpha, and IL-6 production by THP-1. Production is higher when THP-1 have been previously exposed to 1,25(OH)2D3 as compared to prior exposure to IFN-gamma. mAb raised against certain relevant cell surface glycoproteins on THP-1 were tested for their ability to block the response of THP-1 to T cells. Antibodies to CD11a, CD11b, and CD11c, alone or in combination, only partially blocked IL-1 beta production by THP-1, whereas antibodies to CD54 and CD14 did not. Thus other unknown structures on the THP-1 cells may be involved in the induction of THP-1 cytokine production by T cell contact. 相似文献
17.
Illumination of PSII core preparations can cause the production of H2O2 at rates which approach 60 mumol of H2O2 (mg of Chl.h)-1. The rate of peroxide production is maximal at pH 7.2 at low sucrose concentrations and at concentrations of Cl- (1.5-3.0 mM) that limit the rate of the oxidation of water to O2. The rate of H2O2 production increased with pH from pH 6.8 to 7.2 and was inversely proportional to the oxidation of water to O2 from pH 6.8 to 7.5. While EDTA does not inhibit H2O2 production, this reaction is abolished by 5 mM NH2OH and inhibited by the same concentrations of NH3 that affect water oxidation which indicates that the oxygen-evolving complex is responsible for the production of peroxide generated upon illumination of PSII core preparations. These results support a mechanism in which bound Cl- in the S2 state is displaced by OH- ions which are then oxidized by the OEC to form H2O2. Thus, the OEC requires Cl- to prevent access to the active site of the OEC until four oxidizing equivalents can be generated to allow the oxidation of water to O2. 相似文献
18.
Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli 总被引:4,自引:2,他引:2
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(35)S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H(2)O(2), NH(2)OH, I(2), HNO(2), KMnO(4) and NaIO(4), under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH(2)OH, I(2) and NaIO(4) reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 2-thiocytidine (s(2)C) and 2-methylthio-N(6)-isopentenyladenosine (ms(2)i(6)A), to various extents. CNBr, HNO(2) and NaHSO(3) reacted with s(4)U, mnm(5)s(2)U and s(2)C, but not with ms(2)i(6)A. KMnO(4) and H(2)O(2) were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of (35)S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli. 相似文献
19.
Side chain metabolism of vitamin D3 in osteosarcoma cell line UMR-106. Characterization of products 总被引:1,自引:0,他引:1
Previous work has shown that 25-hydroxyvitamin D3 (25-OH-D3) and 1 alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) may be metabolized in the mammalian kidney through a side chain oxidation pathway resulting in C23-C24 cleavage, yielding 24,25,26,27-tetranor-23-OH-D3. In the present study, we have used UMR-106 clonal osteoblast cells to demonstrate that products of the side chain oxidation pathway are produced by an osteoblast-like cell. Cells cultured on microcarrier beads and incubated in the presence of pharmacological levels of substrate (1.4 microM, either 25-OH-D3 or 1,25-(OH)2D3) produced sufficient quantities of metabolite to allow identification through mass spectrometry. In addition, putative metabolites were identified through comigration with authentic standards on three high pressure liquid chromatography systems, chemical modification by NaBH4 and periodate, and UV spectral characterization. The pathway was undetectable unless the cells had been exposed to 1,25-(OH)2D3 prior to incubation with substrate. We have shown that 1,25-(OH)2D3 induces the 24-hydroxylase and perhaps also the other enzymes of this pathway in the bone cell. Although we used pharmacological concentrations of substrate to demonstrate the existence of the side chain oxidation pathway in bone cells, physiological levels of 25-OH-D3 or 1,25-(OH)2D3 were also metabolized through the pathway, at least as far as the penultimate product. We speculate that the side chain oxidation pathway may be ubiquitous among vitamin D target tissues. 相似文献