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71.
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73.
Yaoya S Kanho H Mikami Y Itani T Umehara K Kuroyanagi M 《Bioscience, biotechnology, and biochemistry》2004,68(9):1837-1841
Hairy root cultures of Pharbitis nil treated with CuSO4 and methyl jasmonate (MeJA) produced umbelliferone (1) and scopoletin (2) in the culture medium, and skimmin (3), a beta-D-glucopyranoside of 1, was isolated from the hairy roots. While 1 in the medium increased and reached a maximal level 16 h after the treatment with CuSO4, the amount of 3 in the hairy roots decreased, reaching a minimal level after 8 h, before recovering to a level higher than the basal level after 24 h and then continuously increasing. These observations suggest that 1 was released by the hydrolysis of 3. Umbelliferone (1) inhibited hairy root growth, while skimmin (3) did not. This result suggests that, after the release of 1 as a phytoalexin, the hairy roots glycosylated 1 for the detoxification and re-use of 3 as a source of phytoalexin. 相似文献
74.
Wu ZL Zhang L Yabe T Kuberan B Beeler DL Love A Rosenberg RD 《The Journal of biological chemistry》2003,278(19):17121-17129
Fibroblast growth factor (FGF) signaling begins with the formation of a ternary complex of FGF, FGF receptor (FGFR), and heparan sulfate (HS). Multiple models have been proposed for the ternary complex. However, major discrepancies exist among those models, and none of these models have evaluated the functional importance of the interacting regions on the HS chains. To resolve the discrepancies, we measured the size and molar ratio of HS in the complex and showed that both FGF1 and FGFR1 simultaneously interact with HS; therefore, a model of 2:2:2 FGF1.HS.FGFR1 was shown to fit the data. Using genetic and biochemical methods, we generated HSs that were defective in FGF1 and/or FGFR1 binding but could form the signaling ternary complex. Both genetically and chemically modified HSs were subsequently assessed in a BaF3 cell mitogenic activity assay. The ability of HS to support the ternary complex formation was found to be required for FGF1-stimulated cell proliferation. Our data also proved that specific critical groups and sites on HS support complex formation. Furthermore, the molar ratio of HS, FGF1, and FGFR1 in the ternary complex was found to be independent of the size of HS, which indicates that the selected model can take place on the cell surface proteoglycans. Finally, a mechanism for the FGF.FGFR signaling complex formation on cell membrane was proposed, where FGF and FGFR have their own binding sites on HS and a distinct ternary complex formation site is directly responsible for mitogenic activity. 相似文献
75.
Hori H Kubota S Watanabe K Kim JM Ogasawara T Sawasaki T Endo Y 《The Journal of biological chemistry》2003,278(27):25081-25090
Transfer RNA (guanosine-2')-methyltransferase (Gm-methylase) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to 2'-OH of G18 in the D-loop of tRNA. Based on their mode of tRNA recognition, Gm-methylases can be divided into the following two types: type I having broad specificity toward the substrate tRNA, and type II that methylates only limited tRNA species. Protein synthesized by in vitro cell-free translation revealed that Gm-methylase encoded in the Aquifex aeolicus genome is a novel type II enzyme. Experiments with chimeric tRNAs and mini- and micro-helix RNAs showed that the recognition region of this enzyme is included within the D-arm structure of tRNALeu and that a bulge is essentially required. Variants of tRNALeu, tRNASer, and tRNAPhe revealed that a combination of certain base pairs in the D-stem is strongly recognized by the enzyme, that 4 bp in the D-stem enhance methyl acceptance activity, and that the Py16Py17G18G19 sequence is important for efficient methyl transfer. The methyl acceptance activities of all the A. aeolicus tRNA genes, which can be classified into 14 categories on the basis of their D-arm structure, were tested. The results clearly showed that the substrate recognition mechanism elucidated by the variant experiments was applicable to their native substrates. 相似文献
76.
Steen M Villoutreix BO Norstrøm EA Yamazaki T Dahlbäck B 《The Journal of biological chemistry》2002,277(51):50022-50029
Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme Factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIIIa. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at positions 467, 511, 652, or 1683 displayed attenuated FXa binding, whereas the prothrombin affinity was unaffected. The affinity toward FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicates that the enzyme:cofactor assembly of the prothrombinase and the tenase complexes are homologous and provide a useful platform for further investigation of specific structural elements involved in the FVa.FXa complex assembly. 相似文献
77.
Keiko Tanaka Izumi Shibuya Yasuhito Uezono Yoichi Ueta Yumiko Toyohira Nobuyuki Yanagihara Futoshi Izumi †Tomio Kanno Hiroshi Yamashita 《Journal of neurochemistry》1998,70(4):1652-1661
Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca2+ release and Ca2+ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca2+ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP3), cyclic AMP, and the intracellular Ca2+ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP3 and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP3, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP3 production, the Ca2+ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP3 channels. The potencies of the peptides to cause Ca2+ release in the presence of cinnarizine were similar. The Ca2+ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca2+ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca2+ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP3 production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca2+ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca2+ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP3 and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca2+ in adrenal chromaffin cells. 相似文献
78.
Tetsuhiko Yoshimura Sohsuke Shidara Tomio Ozaki Hitoshi Kamada 《Archives of microbiology》1993,160(6):498-500
The denitrifying bacterium, Achromobacter xylosoxidans NCIB 11015, was cultivated in meat extract-peptone medium and in Mn-free synthetic medium under denitrifying or non-denitrifying conditions. Electron paramagnetic resonance (EPR) spectra for the whole cells of the bacteria thus obtained were measured at 77K. The characteristic three-line signal was observed in the whole cells of the bacteria under denitrifying conditions, but not under non-denitrifying conditions. The three-line signal was more distinctly observed in the cells cultured in Mn-free medium. This signal could be assigned to nitrosylcytochrome c containing a five-coordinated nitrosylheme. The elemental composition in these cells is also discussed. 相似文献
79.
Ontogenetic development of specific [3H]spiperone binding to crude synaptic membranes and its regulation by Na+ and GTP was investigated in the rat striatum. (d)-Butaclamol more effectively inhibited [3H]spiperone binding than (l)-butaclamol. The ratio of inhibitory activity of (d)- and (l)-butaclamol for [3H]spiperone binding was not different between 1-, 7-, and 70-day-old animals but eight- to ninefold lower at 18 days of gestation than during the postnatal period. A Scatchard plot of specific binding indicated the presence of two types of binding: low-affinity (KD = 1.51 nM) and high-affinity (KD = 0.09 nM) binding on day 70. Only one component (KD = 0.075 nM) was observed on days 1 and 7 and both types of binding were found on day 15. Bmax gradually increased with age and reached a peak on day 30, followed by a decline on days 70 and 360. Na+, 100 mM, significantly increased specific binding on days 1, 7, 15, and 70. GTP, 50 microM, completely reversed the Na+-induced decrease in IC50 of apomorphine on both days 15 and 70, but not on day 7. It is suggested that receptors could recognize ligand stereospecificity on day 1. The density in dopamine receptors in the striatum reaches a peak on day 30, followed by a decrease on days 70 and 360. In addition, regulation by Na+ and GTP in agonist binding to dopamine receptors seems to become functional between 1 and 2 weeks after birth. 相似文献
80.
Masataka Ikeda Hideo Ariyoshi Jun-ichi Kambayashi Kazumasa Fujitani Nobutoshi Shinoki Masato Sakon Tomio Kawasaki Morito Monden 《Journal of cellular biochemistry》1996,63(1):23-36
Ca2+ concentration inside human umbilical vein endothelial cells was studied separately in cytosol and nucleus by a confocal laser scanning microscopy using fluo-3. The in vivo calibration curve for cytosol and nucleus showed good linearity between fluorescence intensity and Ca2+ concentration in cytosol ([Ca2+]i) and nuclei ([Ca2+]n). After calibration, [Ca2+]n was constantly higher than [Ca2+]i before and after the chelation of extracellular Ca2+ suggesting an active Ca2+ accumulation system on nuclear membrane. [Ca2+]n was also constantly higher than [Ca2+]i after the stimulation of thrombin (0.05 U/ml), FCS (10%), and thapsigargin (Tsg, 1μM). The temporal change of [Ca2+]n and [Ca2+]i was identical, and [Ca2+]i gradient towards the nucleus and peripheral or central [Ca2+]n rise was observed after these stimulations. From these results, [Ca2+]n is not only regulated by the active Ca2+ accumulation system on nuclear membrane at rest but also the generation of Inositol-triphosphate. FCS caused heterogeneous [Ca2+]n or [Ca2+]i rise from cell to cell; single spike or oscillatory change of [Ca2+]n and [Ca2+]i was observed in about 56% of cells, which were completely abolished by the chelation of extracellular Ca2+, suggesting that FCS stimulated [Ca2+]n and [Ca2+]i rise solely depending on Ca2+ influx from extracellular medium. The higher concentration of [Ca2+]n and heterogeneous [Ca2+]n rise may have important roles in nuclear-specific cellular responses. © 1996 Wiley-Liss, Inc. 相似文献