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991.
Kadono S Sakamoto A Kikuchi Y Oh-eda M Yabuta N Yoshihashi K Kitazawa T Suzuki T Koga T Hattori K Shiraishi T Haramura M Kodama H Ono Y Esaki T Sato H Watanabe Y Itoh S Ohta M Kozono T 《Biochemical and biophysical research communications》2005,327(2):589-596
Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. Structure-based designs of the P3 moiety in the peptide mimetic factor VIIa inhibitor successfully lead to novel inhibitors with selectivity for FVIIa/TF and extrinsic coagulation the same as or even higher than those of previously reported peptide mimetic factor VIIa inhibitors. X-ray crystal structure analysis reveals that one of the novel inhibitors shows improved selectivity by forming interactions between the inhibitor and FVIIa as expected. Another of the novel inhibitors achieves improved selectivity through an unexpected hydrogen bond with Gln217, with a unique bent conformation in FVIIa/TF accompanied by conformational changes of the inhibitor and the protein. 相似文献
992.
Sasakura Y Kanda K Yoshimura-Suzuki T Matsui T Fukuzono S Shimizu T 《Biochemistry》2005,44(28):9598-9605
Ec DOS, a heme-regulated phosphodiesterase from Escherichia coli, is composed of an N-terminal heme-bound PAS domain and a C-terminal phosphodiesterase domain. The heme redox state in the PAS domain regulates Ec DOS phosphodiesterase activity. Interestingly, the isolated heme-bound PAS fragment enhances phosphodiesterase activity of full-length Ec DOS. The enhancement is also regulated by the heme redox state of the isolated PAS domain. In the present study, we used a newly developed protein microarray system to examine the relationship between catalytic activity and the interaction of full-length Ec DOS and the isolated PAS fragment. Adenosine 3',5'-cyclic monophosphate (cAMP), a substrate of the Ec DOS phosphodiesterase, was found to be indispensable for the interaction between Ec DOS and the PAS fragment, and two phosphodiesterase inhibitors, 3-isobutyl-methyl-xanthine and etazolate hydrochloride, hindered the interaction. In addition, an enzyme with a mutation in the putative cAMP-binding sites (H590 and H594) was unable to interact with Ec DOS and lacked enzymatic activity. These results strongly suggest a close relationship between Ec DOS phosphodiesterase activity and interaction with the isolated PAS fragment. Therefore, this study provides insights into the mechanism of how the isolated PAS domain activates Ec DOS, which has important implications for the general role of the isolated PAS domain in cells. Moreover, we found that multiple microscale analyses using the protein microarray system had several advantages over conventional affinity column methods, including the quantity of protein needed, the sensitivity, the variability of immobilized protein, and the time required for the experiment. 相似文献
993.
Hemmi H Kumazaki T Yoshizawa-Kumagaye K Nishiuchi Y Yoshida T Ohkubo T Kobayashi Y 《Biochemistry》2005,44(28):9626-9636
Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor from Anemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized alpha-helical (CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solution structure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by a central alpha-helix and a three-stranded antiparallel beta-sheet of a typical Kazal-type inhibitor such as silver pheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forming a disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3. Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB). Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motif could produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI (AEI analogue) was chemically synthesized when a Cys(4)-Cys(34) bond was changed to a Cys(6)-Cys(31) bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibited almost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structural difference was detected between the two, but the N-terminal loop from Pro(5) to Ile(7) near the putative reactive site (Met(10)-Gln(11)) in the analogue moved by 3.7 A toward the central helix to form the introduced Cys(6)-Cys(31) bond. Such a conformational change in the restricted region correlates with the specificity change of the inhibitor. 相似文献
994.
Umehara T Fukuda K Nishikawa F Kohara M Hasegawa T Nishikawa S 《Journal of biochemistry》2005,137(3):339-347
The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multifunctional enzyme with protease and helicase activities. It is essential for HCV proliferation and is therefore a target for anti-HCV drugs. Previously, we obtained RNA aptamers that inhibit either the protease or helicase activity of NS3. During the present study, these aptamers were used to create advanced dual-functional (ADD) aptamers that were potentially more effective inhibitors of NS3 activity. The structural domain of the helicase aptamer, #5Delta, was conjugated via an oligo(U) tract to the 3'-end of the dual functional aptamer NEO-III-14U or the protease aptamer G9-II. The spacer length was optimized to obtain two ADD aptamers, NEO-35-s41 and G925-s50; both were more effective inhibitors of NS3 protease/helicase activity in vitro, especially the helicase, with a four- to five-fold increase in inhibition compared with #5 and NEO-III-14U. Furthermore, G925-s50 effectively inhibited NS3 protease activity in living cells and HCV replication in vitro. Overall, we have demonstrated rational RNA aptamer design based on features of both aptamer and target molecules, as well as successfully combining aptamer function and increasing NS3 inhibition. 相似文献
995.
The soy isoflavones, genistein (5,7,4'-trihydroxyisoflavone) and daidzein (7,4'-dihydroxyisoflavone), are representative phytoestrogens that function as chemopreventive agents against cancers, cardiovascular disease, and osteoporosis. However, recent studies indicated that genistein and/or daidzein induced cancers of reproductive organs in rodents, such as the uterus and vulva. To clarify the molecular mechanisms underlying the induction of carcinogenesis by soy isoflavones, we examined the ability of genistein, daidzein, and their metabolites, 5,7,3',4'-tetrahydroxyisoflavone (orobol), 7,3',4'-trihydroxyisoflavone (7,3',4'-OH-IF), and 6,7,4'-trihydroxyisoflavone (6,7,4'-OH-IF), to cause DNA damage and cell proliferation. An E-screen assay revealed that genistein and daidzein enhanced proliferation of estrogen-sensitive breast cancer MCF-7 cells, while their metabolites had little or no effect. A surface plasmon resonance sensor showed that binding of isoflavone-liganded estrogen receptors (ER) to estrogen response elements (ERE) was largely consistent with cell proliferative activity of isoflavones. Orobol and 7,3',4'-OH-IF significantly increased 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in human mammary epithelial MCF-10A cells, while genistein, daidzein, and 6,7,4'-OH-IF did not. Experiments using isolated DNA revealed a metal-dependent mechanism of oxidative DNA damage induced by orobol and 7,3',4'-OH-IF. DNA damage was enhanced by the addition of endogenous reductant NADH, formed via the redox cycle. These findings suggest that oxidative DNA damage by isoflavone metabolites plays a role in tumor initiation and that cell proliferation by isoflavones via ER-ERE binding induces tumor promotion and/or progression, resulting in cancer of estrogen-sensitive organs. 相似文献
996.
Manabe S Nishimura N Yamamoto Y Kitamura H Morimoto S Imai M Nagahiro S Seino S Sasaki T 《Biochemical and biophysical research communications》2004,316(1):218-225
The Rab3 family small G proteins (Rab3A-D) are involved in the regulated secretory pathway of brain and secretory tissues. Among Rab3-interacting proteins, Rabphilin-3, Rim, and Noc2, all of which contain a conserved Rab3-binding domain (RBD3), are generally recognized Rab3 effector proteins in neurons and secretory cells. Although Rab3B was also detected in epithelial cells, its function remained unknown. We isolated cDNA sequences from human epithelial Caco2-cell mRNA by degenerate RT-PCR based on the conserved amino acid sequence of RBD3. Multiple cDNA clones were identified as encoding Noc2. Northern blot analysis revealed that Noc2 mRNA was expressed not only in secretory tissues but also in epithelial tissues and cell lines. A pull-down assay demonstrated that Noc2 bound to Rab3B in a GTP-dependent manner. When Noc2 was co-expressed with the GTP-bound form of Rab3B, it was recruited from the cytosol to perinuclear membranes. Furthermore, overexpression of Noc2 inhibited the cell-surface transport of basolateral vesicular stomatitis virus glycoprotein. These results suggest that Noc2 functions as a potential Rab3B effector protein in epithelial cells. 相似文献
997.
998.
Fas-mediated apoptosome formation is dependent on reactive oxygen species derived from mitochondrial permeability transition in Jurkat cells 总被引:5,自引:0,他引:5
Sato T Machida T Takahashi S Iyama S Sato Y Kuribayashi K Takada K Oku T Kawano Y Okamoto T Takimoto R Matsunaga T Takayama T Takahashi M Kato J Niitsu Y 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(1):285-296
Generation of reactive oxygen species (ROS) and activation of caspase cascade are both indispensable in Fas-mediated apoptotic signaling. Although ROS was presumed to affect the activity of the caspase cascade on the basis of findings that antioxidants inhibited the activation of caspases and that the stimulation of ROS by itself activated caspases, the mechanism by which these cellular events are integrated in Fas signaling is presently unclear. In this study, using human T cell leukemia Jurkat cells as well as an in vitro reconstitution system, we demonstrate that ROS are required for the formation of apoptosome. We first showed that ROS derived from mitochondrial permeability transition positively regulated the apoptotic events downstream of mitochondrial permeability transition. Then, we revealed that apoptosome formation in Fas-stimulated Jurkat cells was clearly inhibited by N-acetyl-L-cysteine and manganese superoxide dismutase by using both the immunoprecipitation and size-exclusion chromatography methods. To confirm these in vivo findings, we next used an in vitro reconstitution system in which in vitro-translated apoptotic protease-activating factor 1 (Apaf-1), procaspase-9, and cytochrome c purified from human placenta were activated by dATP to form apoptosome; the formation of apoptosome was markedly inhibited by reducing reagents such as DTT or reduced glutathione (GSH), whereas hydrogen peroxide prevented this inhibition. We also found that apoptosome formation was substantially impaired by GSH-pretreated Apaf-1, but not GSH-pretreated procaspase-9 or GSH-pretreated cytochrome c. Collectively, these results suggest that ROS plays an essential role in apoptosome formation by oxidizing Apaf-1 and the subsequent activation of caspase-9 and -3. 相似文献
999.
Chronic hypertriglyceridemia in young watanabe heritable hyperlipidemic rabbits impairs endothelial and medial smooth muscle function 总被引:1,自引:0,他引:1
Shishido T Tasaki K Takeishi Y Takasaki S Miyamoto T Itoh M Takahashi H Kubota I Ito T Katano Y Wakabayashi I Tomoike H 《Life sciences》2004,74(12):1487-1501
Several studies have suggested that hypertriglyceridemia is a common risk factor for coronary heart disease. Although increasing serum levels of triglyceride correlate with hypercoagulability, little is known about the contribution of hypertriglyceridemia to vascular function. We successfully segregated two lines of rabbits with genetically-determined severely high (TGH; 2764 +/- 413 mg/dl) and moderately high (TGL; 191 +/- 12 mg/dl) levels of triglyceride, but with comparable levels of total cholesterol, from Watanabe heritable hyperlipidemic rabbits. To determine whether hypertriglyceridemia was involved in alterations of vascular function, we conducted isometric tension studies and analyzed protein expression on thoracic aortic rings isolated from young (3-4 month) TGH, TGL and Japanese White rabbit (JW). No difference in percentage of plaque area in the thoracic aorta was found between TGH and TGL. Relaxing responses, evoked by sodium nitroprusside were similar in JW, TGL and TGH, but endothelium-dependent relaxation to acetylcholine was impaired in TGH compared with JW or TGL (maximal relaxation in JW; 83.5 +/- 2.7%, TGL; 79.9 +/- 5.3%, TGH; 59.1 +/- 5.7%, p<0.05). Relaxation to A23187 was also attenuated in TGH compared with JW, but not significantly different between TGL and JW. Endothelium-independent relaxation elicited by isoproterenol in TGH was significantly decreased compared with JW or TGL (maximal relaxation in JW; 95.2 +/- 2.6% TGL; 91.0 +/- 4.9%, TGH; 75.1 +/- 5.2%, p<0.05). Protein expression of angiotensin II type-1 receptor was increased in TGH and that of nitric oxide synthases-3 was attenuated in TGH compared with TGL. This is the first study showing that endothelium-dependent and -independent vascular relaxation under the condition of combined hyperlipidemia was severely impaired as compared to that under only hypercholesterolemia. These results suggest that hypertriglyceridemia aggravates functional impairment induced by hypercholesterolemia in endothelial and smooth muscle cells. 相似文献
1000.
Propyl gallate (PG), widely used as an antioxidant in foods, is carcinogenic to mice and rats. PG increased the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60, but not in HP100, which is hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. Although PG induced no or little damage to 32P-5'-end-labeled DNA fragments obtained from genes that are relevant to human cancer, DNA damage was observed with treatment of esterase. HPLC analysis of the products generated from PG incubated with esterase revealed that PG converted into gallic acid (GA). GA induced DNA damage in a dose-dependent manner in the presence of Fe(III)EDTA or Cu(II). In the presence of Fe(III) complex such as Fe(III)EDTA or Fe(III)ADP, GA caused DNA damage at every nucleotide. Fe(III) complex-mediated DNA damage by GA was inhibited by free hydroxy radical (*OH) scavengers, catalase and an iron chelating agent. These results suggested that the Fe(III) complex-mediated DNA damage caused by GA is mainly due to *OH generated via the Fenton reaction. In the presence of Cu(II), DNA damage induced by GA occurred at thymine and cytosine. Although *OH scavengers did not prevent the DNA damage, methional inhibited the DNA damage. Cu(II)-mediated DNA damage was inhibited by catalase and a Cu(I) chelator. These results indicated that reactive oxygen species formed by the interaction of Cu(I) and H2O2 participates in the DNA damage. GA increased 8-oxodG content in calf thymus DNA in the presence of Cu(II), Fe(III)EDTA or Fe(III)ADP. This study suggested that metal-mediated DNA damage caused by GA plays an important role in the carcinogenicity of PG. 相似文献