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141.
Yoshizawa Itsuo Nagata Kyoko Ohuchi Ryoko Itoh Shinji Kanaiwa Yoshio Amiya Takashi 《Steroids》1980,36(1):87-96
Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III). 相似文献
142.
High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge 总被引:2,自引:1,他引:2
The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 107 cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement. 相似文献
143.
144.
Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia 总被引:2,自引:0,他引:2
Ueyama T Lennartz MR Noda Y Kobayashi T Shirai Y Rikitake K Yamasaki T Hayashi S Sakai N Seguchi H Sawada M Sumimoto H Saito N 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(7):4582-4589
Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis. 相似文献
145.
Youki Ueda Midori Takeda Kyoko Mori Hiromichi Dansako Takaji Wakita Hye-Sook Kim Akira Sato Yusuke Wataya Masanori Ikeda Nobuyuki Kato 《PloS one》2013,8(8)
Background
Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed.Methodology/Principal Findings
Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect.Conclusions/Significance
We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin. 相似文献146.
Yoshie Maitani Sae Yano Yoshiyuki Hattori Masahiko Furuhata Kyoko Hayashi 《Journal of liposome research》2013,23(4):359-372
For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy. 相似文献
147.
Masakazu Atobe Natsumi Yamakawa Yasuhiro Wada Tokuhito Goto Masashi Kawanishi Takeshi Ito Makoto Saito Masahiro Takeda Takanori Tabata Hirokazu Arai 《Bioorganic & medicinal chemistry letters》2017,27(21):4828-4831
A series of indazole derivatives were identified as Sirt 1 activators though high-throughput screening. Optimization of each substituent on the indazole ring led to the identification of compound 13. Compound 13 appeared to give the best Sirt 1 activity of the compounds tested and also showed osteogenesis activity in a cell assay. Sirt 1 activators are therefore potential candidates for the treatment of osteoporosis. 相似文献
148.
Kunio Takeda Kyoko Ogawa Masami Ohara Satoshi Hamada Yoshiko Moriyama 《The protein journal》1995,14(8):679-684
Four disulfide bridges of bovineα-lactalbumin (α-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and OSSα-lact, respectively). The original helicity was almost maintained in 3SSα-lact missing only the Cys6-Cysl20 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SSα-lact, from 26% to 31% in 2SSα-lact, and from 18% to 37% in OSSα-lact, as against from 34% to 44% in intactα-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution. 相似文献
149.
Ohashi N Nomura W Narumi T Lewin NE Itotani K Blumberg PM Tamamura H 《Bioconjugate chemistry》2011,22(1):82-87
Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity. 相似文献
150.
Rahman S Ishizuka-Katsura Y Arai S Saijo S Yamato I Toyama M Ohsawa N Inoue M Honda K Terada T Shirouzu M Yokoyama S Iwata S Murata T 《Protein expression and purification》2011,78(2):181-188
The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase. 相似文献