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Lipocortin-85 (L-85, calpactin-I/lipocortin-II heterotetramer) binds to F-actin in the presence of calcium with high affinity and in a cooperative manner. Quantitative analysis of binding curves indicate an apparent Kd (L-85) of 0.226 microM +/- 0.153 (2 S.D., n = 3), a stoichiometry of L-85/actin of 1:1.9 and a Hill coefficient of 1.37 +/- 0.14 (2 S.D., n = 3). Large anisotropic bundles were visualized by electron microscopy under these conditions, and quantitation of bundling by both low speed sedimentation and light scattering yielded apparent Kd values between 0.12 and 0.27 microM L-85. Filament bundling was dependent upon calcium, and the calcium sensitivity was increased by raising the molar ratio of lipocortin-85/F-actin. At saturating levels of L-85, apparent K0.5 values of 0.1-2 microM Ca2+f were obtained. The monomeric heavy chain, lipocortin-II, bundled F-actin to a much lesser extent and at much higher concentrations than for lipocortin-85. Bundling of F-actin by lipocortin-I was not detected at molar ratios of lipocortin-I to actin as high as 2.5 mol/mol (lipocortin-I/actin). At 5-10 microM Ca2+f and saturating levels of L-85, F-actin bundling progressed very rapidly with a t0.5 of 6 s. The process was quickly reversed by the addition of excess EGTA, and bundles could be reformed by the addition of a second burst of 5-10 microM Ca2+f. Thus, our data suggest that lipocortin-85 can rapidly regulate F-actin bundling in a calcium-dependent manner at physiologically relevant calcium levels. 相似文献
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Liposome-encapsulated superoxide dismutase suppresses liposome-mediated augmentation of TNF-alpha production from peripheral blood leucocytes 总被引:1,自引:0,他引:1
Shibuya-Fujiwara N Hirayama F Ogata Y Matsuda A Sekiguchi S Ikeda H Ikebuchi K 《Life sciences》2001,69(17):2007-2015
Liposome-encapsulated hemoglobin (LEH), a candidate for a red cell substitute, has been reported to be cleared from circulation primarily by the phagocytic system and modulate the production of inflammatory cytokines, such as TNF-alpha and IL-6, both in vivo and in vitro. In this study, we investigated the effects of liposome vesicles on the LPS-induced TNF-alpha production using a whole blood culture system. We also studied the effects of superoxide dismutase (SOD) encapsulated in liposome on the cytokine production. The pre-treatment of whole blood with liposome vesicles potentiated the LPS-induced TNF-alpha production. The encapsulation of SOD in the liposome vesicles suppressed the liposome-mediated augmentation of TNF-alpha production in a dose-dependent manner. These results suggest that encapsulation of SOD in LEH decreases the production of inflammatory cytokines from the phagocytic system which may be caused or augmented by LEH infusion in vivo. 相似文献
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Huang Y Takeoka S Sakai H Abe H Hirayama J Ikebuchi K Ikeda H Tsuchida E 《Biotechnology progress》2002,18(1):101-107
Hemoglobin (Hb) has been widely studied as a raw material for various types of oxygen carriers. In the purification of Hb from red blood cells including virus inactivation and denaturation of other proteins and the long-term storage of Hb vesicles (HbV), a deoxygenation process is one of the important processes because of the high stability of deoxygenated Hb to heating and metHb formation. Though an oxygenated Hb solution can be deoxygenated with an artificial lung, it is difficult to reduce the oxygen partial pressure of the Hb solution to less than 10 Torr. We developed an electrochemical system for complete deoxygenation of the Hb solution at the cathode compartment using hydrogen containing nitrogen gas at the anode compartment. Oxygen in the Hb solution was reduced to OH(-) at the cathode compartment within several minutes at a potential value of -1.67 V and was finally converted to water by neutralization with H(+) from the anode in the whole system. The resulting completely deoxygenated Hb could tolerate heat treatment at 62 degrees C for 10 h with no denaturation of deoxygenated Hb. The metHb formation rate of reoxygenated Hb at 37 degrees C was not changed after heat treatment. Furthermore, vesicular stomatitis virus (VSV) could be inactivated at an inactivation degree of more than 5.96 log by heat treatment. 相似文献
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Nakayama A Matsuo H Takada T Ichida K Nakamura T Ikebuchi Y Ito K Hosoya T Kanai Y Suzuki H Shinomiya N 《Nucleosides, nucleotides & nucleic acids》2011,30(12):1091-1097
The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3'-azido-3'-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10(-5)). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion. 相似文献
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Ryoyo Ikebuchi Satoru Konnai Yuji Sunden Misao Onuma Kazuhiko Ohashi 《Microbiology and immunology》2010,54(5):291-298
Recent work has shown that PD‐1, an immune inhibitory receptor, is involved in mechanisms for down‐regulating immune responses during tumor progression or chronic viral infection. However, in the case of bovine diseases, there have been no reports on this molecule due to lack of information about bovine PD‐1. In this study, we performed identification and preliminary characterization of the bovine PD‐1 gene in two breeds of cattle. We cloned full cDNA sequences encoding for PD‐1 from both Holstein‐Friesian and Japanese Black breeds, and found that both of the genes encoded a 282‐amino acid protein, which had a signal sequence, transmembrane domain and an immunoreceptor tyrosine‐based inhibitory motif. This bovine PD‐1 showed 72.9% and 65.6% homology to human and mouse PD‐1, respectively, both of which have been well characterized and documented. Quantitative real‐time PCR analysis showed that bovine PD‐1 is expressed predominantly in T‐cells (such as CD4+ and CD8+ cells) and among PBMCs, and is strongly upregulated on T‐cell stimulation via ConA. A limited number of cattle were tested yet, as expected, the degree of PD‐1 mRNA expression in CD4+ and CD8+ T‐cells was greater in cattle with bovine leukemia virus‐induced lymphoma than in uninfected cattle. Further studies to characterize the functions of bovine PD‐1 are therefore warranted, in order to elucidate the mechanism of the immunosuppression associated with progression of several diseases and therapy in cattle. 相似文献
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Ohte S Shin M Sasanuma H Yoneyama K Akita M Ikebuchi K Jimi E Maruki Y Matsuoka M Namba A Tomoda H Okazaki Y Ohtake A Oda H Owan I Yoda T Furuya H Kamizono J Kitoh H Nakashima Y Susami T Haga N Komori T Katagiri T 《Biochemical and biophysical research communications》2011,(1):213-218
Naphthoquinone derivatives have been reported to possess various pharmacological activities, such as antiplatelet, anticancer, antifungal, and antiviral properties. In this study, we investigated the effects of a newly-synthesized naphthoquinone derivative, 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone (2-decylamino-DMNQ), on VSMC proliferation and examined the molecular basis of the underlying mechanism. In a dose-dependent manner, 2-decylamino-DMNQ inhibited PDGF-stimulated VSMC proliferation with no apparent cytotoxic effect. While 2-decylamino-DMNQ did not affect PDGF-Rβ or Akt, it did inhibit the phosphorylation of Erk1/2 and PLCγ1 induced by PDGF. Moreover, 2-decylamino-DMNQ suppressed DNA synthesis through the arrest of cell cycle progression at the G0/G1 phase, including the suppression of pRb phosphorylation and a decrease in PCNA expression, which was related to the downregulation of cell cycle regulatory factors, such as cyclin D1/E and CDK 2/4. It was demonstrated that both U0126, an Erk1/2 inhibitor, and U73122, a PLCγ inhibitor, increased the proportion of cells in the G0/G1 phase of the cell cycle. Thus, these results suggest that 2-decylamino DMNQ has an inhibitory effect on PDGF-induced VSMC proliferation and the mechanism of this action is through cell cycle arrest at the G0/G1 phase. This may be a useful tool for studying interventions for vascular restenosis in coronary revascularization procedures and stent implantation. 相似文献
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Asami Nishimori Satoru Konnai Ryoyo Ikebuchi Tomohiro Okagawa Chie Nakajima Yasuhiko Suzuki Claro N. Mingala Shiro Murata Kazuhiko Ohashi 《Microbiology and immunology》2014,58(7):388-397
Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)‐1 receptor and its ligand, PD‐L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD‐L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD‐L2 was analyzed in vitro. Recombinant PD‐L2 (PD‐L2‐Ig), which comprises an extracellular domain of bovine PD‐L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD‐L2. Bovine PD‐L2‐Ig bound to bovine PD‐1‐expressing cells and addition of soluble bovine PD‐1‐Ig clearly inhibited the binding of PD‐L2‐Ig to membrane PD‐1 in a dose‐dependent manner. Cell proliferation and IFN‐γ production were significantly enhanced in the presence of PD‐L2‐Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD‐L2‐Ig significantly enhanced IFN‐γ production from virus envelope peptides‐stimulated PBMCs derived from bovine leukemia virus‐infected cattle. Interestingly, PD‐L2‐Ig‐induced IFN‐γ production was further enhanced by treatment with anti‐bovine PD‐1 antibody. These data suggest potential applications of bovine PD‐L2‐Ig as a therapy for bovine diseases. 相似文献
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A French population was investigated for genetic polymorphism of alpha 2HS-glycoprotein (A2HS; nomenclature according to Human Gene Mapping 7, Los Angeles, 1983) using isoelectric focusing and immunoblotting. Three variants were observed together with two common alleles A2HS 1 and A2HS 2, whose frequencies were significantly different from the data in Canadians and Egyptians. An anodal variant to A2HS 1 was identical to a variant with two different nomenclatures reported by three different groups, indicating that there is a confusion in the A2HS nomenclature. The others were new variants with cathodal isoelectric points to A2HS 2 in the native state. 相似文献
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Simultaneous induction of Pb-metallothionein-like protein and Zn-thionein in the liver of rats given lead acetate. 总被引:1,自引:0,他引:1 下载免费PDF全文
Administration of a sublethal dose of lead acetate to rats induced the simultaneous synthesis of a Pb-metallothionein (Pb-MT)-like protein (Pb-BP) and Zn-thionein (Zn-BP) in the liver. The Pb-BP had an apparent molecule mass of 6900 Da and seemed to bind preferentially to lead in the liver cytosol. The Zn-BP was identified by comparison of the Mr, elution profiles from Sephadex G-75 and DEAE-Sephadex A-25 columns, and polyacrylamide-gel-electrophoretic mobility, with those of rat liver Zn-MT-II. The Pb-BP accumulated in the liver to a maximum 6 h after the intraperitoneal injection of lead acetate and accounted for about 60% of the lead in the liver cytosol at this stage. However, after that, it gradually decreased in the liver, until it was close to the basal amount 24 h after the induction. In contrast, the amount of Zn-MT increased gradually, reached a maximum 12 h after the administration of lead acetate and maintained a constant value until at least 24 h after the induction. Amino acid analysis of the Pb-BP indicated that it contained about 28% half-cysteine. These results strongly suggest that lead acetate induces the synthesis of Pb-MT as well as Zn-MT in rat liver. 相似文献