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111.
J Jesty A K Spencer Y Nakashima Y Nemerson W Konigsberg 《The Journal of biological chemistry》1975,250(12):4497-4504
Bovine Factor X can be activated by two alternative pathways. The first, favored at high concentrations of the complex of tissue factor and Factor VII, is initiated by the action of Factor VII on Factor X to cleave an activation peptide from the NH2 terminus of the heavy chain, to produce alpha-Xa. This is then converted autocatalytically to another form of Factor Xa, beta-Xa, by the loss of a 17-residue glycopeptide from the COOH terminus of the heavy chain, in a lipid-dependent reaction. The alternative pathway, favored at lower activator concentrations, is initiated by the action of Factor Xa on Factor X, in the presence of lipid, to release the same COOH-terminal peptide as is produced in the conversion of alpha-Xa to beta-Xa. The intermediate produced by the loss of this peptide from Factor X,I1, can be activated directly to beta-Xa by the tissue factor-Factor VII complex, with the loss of the same NH2-terminal peptide as is produced in the conversion of Factor X to alpha-Xa. The autocatalytic activation of Factor X by Factor Xa described previously occurs to a marked extent only at very low activator concentrations, and has been shown to proceed largely by the loss of the normal NH2-terminal peptide from the heavy chain of I1-Initial experiments show that neither peptide affects the rate of coagulation by either the extrinsic or intrinsic pathways. The amino acid sequences have been determined on both sides of the peptide cleavages, and it has been shown that the cleavage sites are the same, regardless of the pathway of activation. The amino acid sequence and carbohydrate composition of the COOH-terminal peptide have been determined. The carbohydrate moiety is attached via an O-glycosidic linkage at a threonine residue, and contains galactosamine but no glucosamine. 相似文献
112.
113.
K Nakashima T Tsukada H Hibasami S Maekawa 《Biochemical and biophysical research communications》1986,141(2):718-722
N-Chlorosulfonyl dicyclohexylamine (CSD) was synthesized as a potent inhibitor of spermidine synthase and analyzed for antiproliferative effects on leukemic cells. The compound specifically inhibited spermidine synthase in a competitive mode with the substrate putrescine (Ki, 1.8 X 10(-7) M). When human leukemia Molt4B cells were cultured in the presence of the inhibitor, the intracellular level of spermidine and the rate of cell proliferation were markedly depressed. In these polyamine depleted and growth retarded cells the synthesis of protein, but not of DNA or RNA, was found to be significantly diminished. 相似文献
114.
Yoneda T Kumagai T Nagatomo I Furukawa M Yamane H Hoshino S Mori M Takeda Y Horai T Nishida S Watanabe D Kijima T Yoshida M Osaki T Tachibana I Greene MI Kawase I 《DNA and cell biology》2006,25(9):530-540
EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance. 相似文献
115.
Y Banno S Nakashima T Tohmatsu Y Nozawa E G Lapetina 《Biochemical and biophysical research communications》1986,140(2):728-734
The hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by cytosolic phospholipase C from human platelets was determined. Cytosolic fractions were prepared from platelets that had or had not been preactivated with thrombin. Thrombin pretreatment did not affect cytosolic phospholipase C activity. In both cytosolic fractions, phospholipase C was activated by GTP and GTP gamma S. This action is observed in the presence of 2 mM EGTA. GDP was as effective as GTP in stimulating cytosolic phospholipase C in the presence of Ca2+ or EGTA. Partially purified phospholipase C obtained from platelet cytosol is activated by GTP, but not by GTP gamma S, in the presence of 2 mM EGTA. However, in the presence of 6 microM Ca2+, both GTP and GTP gamma S stimulated the partially purified phospholipase C. Our present information indicates that GTP and GDP have a direct effect on the cytosolic phospholipase C. 相似文献
116.
Suzuki T Hara I Nakano M Zhao G Lennarz WJ Schindelin H Taniguchi N Totani K Matsuo I Ito Y 《The Journal of biological chemistry》2006,281(31):22152-22160
Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner. 相似文献
117.
Sakamoto K Hayashi A Sakamoto A Kiga D Nakayama H Soma A Kobayashi T Kitabatake M Takio K Saito K Shirouzu M Hirao I Yokoyama S 《Nucleic acids research》2002,30(21):4692-4699
A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter. 相似文献
118.
Takahiro Shibata Fumie Nakashima Kazuya Honda Yu-Jhang Lu Tatsuhiko Kondo Yusuke Ushida Koichi Aizawa Hiroyuki Suganuma Sho Oe Hiroshi Tanaka Takashi Takahashi Koji Uchida 《The Journal of biological chemistry》2014,289(47):32757-32772
Toll-like receptors (TLRs) play a key role in linking pathogen recognition with the induction of innate immunity. They have been implicated in the pathogenesis of chronic inflammatory diseases, representing potential targets for prevention/treatment. Vegetable-rich diets are associated with the reduced risk of several inflammatory disorders. In the present study, based on an extensive screening of vegetable extracts for TLR-inhibiting activity in HEK293 cells co-expressing TLR with the NF-κB reporter gene, we found cabbage and onion extracts to be the richest sources of a TLR signaling inhibitor. To identify the active substances, we performed activity-guiding separation of the principal inhibitors and identified 3-methylsulfinylpropyl isothiocyanate (iberin) from the cabbage and quercetin and quercetin 4′-O-β-glucoside from the onion, among which iberin showed the most potent inhibitory effect. It was revealed that iberin specifically acted on the dimerization step of TLRs in the TLR signaling pathway. To gain insight into the inhibitory mechanism of TLR dimerization, we developed a novel probe combining an isothiocyanate-reactive group and an alkyne functionality for click chemistry and detected the probe bound to the TLRs in living cells, suggesting that iberin disrupts dimerization of the TLRs via covalent binding. Furthermore, we designed a variety of iberin analogues and found that the inhibition potency was influenced by the oxidation state of the sulfur. Modeling studies of the iberin analogues showed that the oxidation state of sulfur might influence the global shape of the isothiocyanates. These findings establish the TLR dimerization step as a target of food-derived anti-inflammatory compounds. 相似文献
119.
Kenichiro Nakashima Youko Hidaka Tomomi Yoshida Naotaka Kuroda Shuzo Akiyama 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1994,661(2)
High-performance liquid chromatographic determination of four short-chain aliphatic aldehydes using fluorescence detection was carried out with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). DBD-H derivatives with three aliphatic aldehydes — formaldehyde, acetaldehyde and propionaldehyde — were synthesized and their fluorescence properties were examined. Relative fluorescence intensities of these compounds in acetonitrile were ca. ten-fold larger than those in aqueous acetonitrile. DBD-hydrazones could be separated by reversed-phase chromatography using aqueous acetonitrile as eluent and detection at 560 nm with excitation at 445 nm. Submicromolar levels of formaldehyde, acetaldehyde, propionaldehyde and butylaldehyde could be determined. The HPLC procedure using propionaldehyde as internal standard was applied to the measurement of acetaldehyde levels in normal human plasma before and 30 min after ingestion of ethanol. 相似文献
120.
Osamu Nunobiki Daisuke Sano Kyoko Akashi Taro Higashida Toshitada Ogasawara Hikari Akise Shinji Izuma Kiyo Torii Yoshiaki Okamoto Ichiro Tanaka Masatsugu Ueda 《Human cell》2016,29(2):91-95
To investigate the clinical significance of ALDH2 genetic polymorphisms in cervical carcinogenesis. ALDH2 polymorphisms together with human papillomavirus (HPV) types were examined in a total of 195 cervical smear in exfoliated cervical cell samples using Real-Time polymerase chain reaction (PCR) System. The frequency for the AG+AA genotype was seven in the normal group (70.0 %), 16 in the LSIL group (57.1 %), and 27 in the HSIL group (90.0 %). A significant difference was found between the LSIL and HSIL groups (P = 0.0064). Patients with HSIL lesions frequently had high-risk HPV infections and concurrently belonged to the AG+AA group. ALDH2 genotype in cervical cell samples may be associated with more severe precancerous lesions of the cervix in a Japanese population. 相似文献