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1.
Convenient extraction and radioimmunoassay methods for measurement of leukotrienes C4 and D4 (LTC4 and LTD4) in biological fluids are described. LTC4 or LTD4 in plasma was extracted with acetonitrile, and the extract was washed with dichloromethane then adjusted to pH 3.5 or 6.0, respectively. Each leukotriene was partially purified by using a C18-bonded silica cartridge and quantitated by radioimmunoassay. Amounts of LTC4 and LTD4 in the range of 0.025-1.6 ng could be assayed in plasma. This procedure was employed to examine the increase in plasma LTC4 (0.249 +/- 0.036 ng/ml) and LTD4 (1.399 +/- 0.235 ng/ml) of guinea pigs during intravenous challenge-induced anaphylactic bronchoconstriction, and the suppression of the increase of bronchoconstriction and leukotrienes by the administration of 5-lipoxygenase inhibitors such as E6080 (6-hydroxy-2-(4-sulfamoylbenzyl-amino)- 4,5,7-trimethylbenzothiazole hydrochloride), AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone ) and phenidone. On the other hand, LTC4 and LTD4 were not detected in plasma after an inhaled challenge, though significant bronchoconstriction was provoked. It was concluded that the present study validates a new technique for quantitating plasma leukotrienes on the basis of pH and a suitable method for evaluating the pharmacological efficacy of 5-lipoxygenase inhibitors. 相似文献
2.
Regulatory relationship and gain control between cytosolic free Ca2+ concentration (Cai) and cytosolic pH (pHi) were evaluated by two different cell types, gastric parietal cells, and blood platelets. Studies were carried out in both single cells and populations of cells, using Ca2+-indicative probe fura-2 (1-(2-(5′-carboxyoxazol-2′-yl)-6-aminobenzofuran-5-oxy)-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid) and pH-indicative probe BCECF (2′,7′-bis(carboxyethyl) carboxyfluorescein). Stimulation of single and populational parietal cells and platelets with gastrin and thrombin, respectively, resulted in an increase in Cai. In both populational cell types, an initial change in pHi during agonist stimulation occurred almost simultaneously with the mobilization of Ca2+; an initial transient decrease in pHi was followed by a slower increase in pHi above the prestimulation level. When populational platelets were preloaded with the Ca2+ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′tetraacetic acid), the thrombin-induced initial large increase in Cai was apparently inhibited, whereas the pHi decrease induced by thrombin was not altered. This suggests that the initial Cai change is not a prerequisite for the pHi change. The effect of pHi on Cai was examined next. In both single and populational cell types, application of the K+-H+ ionophore nigericin, which induced a transient decrease in pHi, led to the release of Ca2+ from intracellular stores. In single parietal cells double-labeled with fura-2 and BCECF, a temporal decrease in pHi preceded the rise in Cai after stimulation with nigericin. A decrease in pHi, and an increase in Cai occurred at 1.5 and 4 s, respectively. In single parietal cells, replacement of medium Na+ with N-methyl-
-glucamine (NMG+), which also induced a decrease in pHi, resulted in repetitive Ca2+ spike oscillations. The source of Ca2+ utilized for the Ca2+ oscillation that was induced by NMG+ originated from the agonist-sensitive pool. Thus, several maneuvers, which were capable of decreasing pHi, led to an increase in Cai. Cytosolic acidification may be a part of the trigger for Ca2+ mobilization from intracellular stores in both parietal cells and platelets. 相似文献
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Masako Nomaguchi Masaru Yokoyama Ken Kono Emi E. Nakayama Tatsuo Shioda Naoya Doi Sachi Fujiwara Akatsuki Saito Hirofumi Akari Kei Miyakawa Akihide Ryo Hirotaka Ode Yasumasa Iwatani Tomoyuki Miura Tatsuhiko Igarashi Hironori Sato Akio Adachi 《Journal of virology》2013,87(21):11447-11461
Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells. 相似文献
5.
Kôichi Takinami Hiroshi Okada Toshinao Tsunoda 《Bioscience, biotechnology, and biochemistry》2013,77(12):858-863
It has been found that although Brevibacterium lactofermentum No. 2256 is incapable of accumulating l-glutamic acid in a biotin sufficient medium, it produces a large quantity of the acid in the presence of sucrose fatty acid ester. In a biotin deficient medium, however, the ester brought the unfavorable diminution of l-glutamic acid accumulation caused by the decrease of glucose consumption in an incubation period. The undesirable effects were practically lost when the ester was added to the culture medium after more than eight hours in the course of incubation. This fact suggests that the ester is concerned with the growth of microorganism. It is very interesting to elucidate the interrelation between sucrose fatty acid ester and biotin. For the maximum accumulation of l-glutamic acid corresponding increase in amount of the ester to the increasing concentration of biotin was necessary. The proportional relation did not extend to excedingly high levels of the two implicating factors. The further observations concerning the effects of the individual fatty acid esters such as sucrose stearate remain unsatisfactory. 相似文献
6.
Yasuto Watanabe Tatsuhiko Ohe Makoto Morita 《Bioscience, biotechnology, and biochemistry》2013,77(12):2735-2741
The effect of glucose on the formation of uricase by a strain of Streptomyces sp. incubated under conditions of nitrogen limitation was investigated. Glucose stimulated uricase formation in the presence of potassium ion and inhibited it in the absence of the ion. Glucose metabolism by the organism was altered in the absence of the ion, and this appeared to cause the inhibition of the enzyme formation. The stimulatory effect of glucose in the presence of potassium ion was to shorten the lag period. Comparisons of the enzyme formation with and without urate in the presence and absence of glucose revealed that glucose promoted the utilization of exogenous urate as the inducer. The effect of glucose appeared to require protein synthesis, since it was prevented by chloramphenicol. Cyclic adenosine 3′,5′-mono-phosphate showed apparently no effect on uricase formation of this organism. 相似文献
7.
Chie Hashimoto Tetsuo Narumi Hiroyuki Otsuki Yuki Hirota Hiroshi Arai Kazuhisa Yoshimura Shigeyoshi Harada Nami Ohashi Wataru Nomura Tomoyuki Miura Tatsuhiko Igarashi Shuzo Matsushita Hirokazu Tamamura 《Bioorganic & medicinal chemistry》2013,21(24):7884-7889
To date, several small molecules of CD4 mimics, which can suppress competitively the interaction between an HIV-1 envelope glycoprotein gp120 and a cellular surface protein CD4, have been reported as viral entry inhibitors. A lead compound 2 (YYA-021) with relatively high potency and low cytotoxicity has been identified previously by SAR studies. In the present study, the pharmacokinetics of the intravenous administration of compound 2 in rats and rhesus macaques is reported. The half-lives of compound 2 in blood in rats and rhesus macaques suggest that compound 2 shows wide tissue distribution and relatively high distribution volumes. A few hours after the injection, both plasma concentrations of compound 2 maintained micromolar levels, indicating it might have promise for intravenous administration when used combinatorially with anti-gp120 monoclonal antibodies. 相似文献
8.
9.
Daiki Miki Hidenori Ochi Atsushi Takahashi C. Nelson Hayes Yuji Urabe Hiromi Abe Tomokazu Kawaoka Masataka Tsuge Nobuhiko Hiraga Michio Imamura Yoshiiku Kawakami Hiroshi Aikata Shoichi Takahashi Norio Akuta Fumitaka Suzuki Kenji Ikeda Hiromitsu Kumada Yoshiyasu Karino Joji Toyota Tatsuhiko Tsunoda Michiaki Kubo Naoyuki Kamatani Yusuke Nakamura Kazuaki Chayama 《PloS one》2013,8(12)
Hepatitis C virus (HCV) establishes a chronic infection in 70-80% of infected individuals. Many researchers have examined the effect of human leukocyte antigen (HLA) on viral persistence because of its critical role in the immune response against exposure to HCV, but almost all studies have proven to be inconclusive. To identify genetic risk factors for chronic HCV infection, we analyzed 458,207 single nucleotide polymorphisms (SNPs) in 481 chronic HCV patients and 2,963 controls in a Japanese cohort. Next, we performed a replication study with an independent panel of 4,358 cases and 1,114 controls. We further confirmed the association in 1,379 cases and 25,817 controls. In the GWAS phase, we found 17 SNPs that showed suggestive association (P < 1 × 10-5). After the first replication study, we found one intronic SNP in the HLA-DQ locus associated with chronic HCV infection, and when we combined the two studies, the association reached the level of genome-wide significance. In the second replication study, we again confirmed the association (P
combined = 3.59 × 10−16, odds ratio [OR] = 0.79). Subsequent analysis revealed another SNP, rs1130380, with a stronger association (OR=0.72). This nucleotide substitution causes an amino acid substitution (R55P) in the HLA-DQB1 protein specific to the DQB1*03 allele, which is common worldwide. In addition, we confirmed an association with the previously reported IFNL3-IFNL4 locus and propose that the effect of DQB1*03 on HCV persistence might be affected by the IFNL4 polymorphism. Our findings suggest that a common amino acid substitution in HLA-DQB1 affects susceptibility to chronic infection with HCV in the Japanese population and may not be independent of the IFNL4 genotype. 相似文献
10.
Takefumi Yamashita Eiichi Mizohata Satoru Nagatoishi Takahiro Watanabe Makoto Nakakido Hiroko Iwanari Yasuhiro Mochizuki Taisuke Nakayama Yuji Kado Yuki Yokota Hiroyoshi Matsumura Takeshi Kawamura Tatsuhiko Kodama Takao Hamakubo Tsuyoshi Inoue Hideaki Fujitani Kouhei Tsumoto 《Structure (London, England : 1993)》2019,27(3):519-527.e5