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21.
Shiroishi M Tsujimoto H Makyio H Asada H Yurugi-Kobayashi T Shimamura T Murata T Nomura N Haga T Iwata S Kobayashi T 《Microbial cell factories》2012,11(1):78
ABSTRACT: BACKGROUND: Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. RESULTS: We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. CONCLUSIONS: We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography. 相似文献
22.
Yamachika E Tsujigiwa H Matsubara M Hirata Y Kita K Takabatake K Mizukawa N Kaneda Y Nagatsuka H Iida S 《Journal of molecular histology》2012,43(2):223-233
Some progress has been made in development of methods to regenerate bone from cultured cells, however no method is put to
practical use. Here, we developed methods to isolate, purify, and expand mesenchymal stem cells (MSCs) from mouse compact
bone that may be used to regenerate bone in vivo. These cells were maintained in long-term culture and were capable of differentiating
along multiple lineages, including chondrocyte, osteocyte, and adipocyte trajectories. We used standard cell isolation and
culture methods to establish cell cultures from mouse compact bone and bone marrow. Cultures were grown in four distinct media
to determine the optimal composition of culture medium for bone-derived MSCs. Putative MSCs were subjected to flow cytometry,
alkaline phosphatase assays, immunohistochemical staining, and several differentiation assays to assess cell identity, protein
expression, and developmental potential. Finally, we used an in vivo bone formation assay to determine whether putative MSCs
were capable of regenerating bone. We found that compact bone of mice was a better source of MCSs than the bone marrow, that
growth in plastic flasks served to purify MSCs from hematopoietic cells, and that MSCs grown in basic fibroblast growth factor
(bFGF)-conditioned medium were, based on multiple criteria, superior to those grown in leukemia inhibitory factor-conditioned
medium. Moreover, we found that the MSCs isolated from compact bone and grown in bFGF-conditioned medium were capable of supporting
bone formation in vivo. The methods and results described here have implications for understanding MSC biology and for clinical
purpose. 相似文献
23.
Hiroki Nishimura Gen Komaki Tetsuya Ando Toshihiro Nakahara Takakazu Oka Keisuke Kawai Toshihiko Nagata Aya Nishizono Yuri Okamoto Kenjiro Okabe Masanori Koide Chikara Yamaguchi Satoshi Saito Kazuyoshi Ohkuma Katsutaro Nagata Tetsuro Naruo Masato Takii Nobuo Kiriike Toshio Ishikawa 《BioPsychoSocial medicine》2008,2(1):1-8
Background
Over the last five to ten years there has been an increase in psychosomatic complaints (PSC) in Swedish children. The objective of the study was to examine the relation between PSC and sense of coherence (SOC). 相似文献24.
Hifumi E Morihara F Hatiuchi K Okuda T Nishizono A Uda T 《The Journal of biological chemistry》2008,283(2):899-907
We have successfully developed a catalytic antibody capable of degrading the active site of the urease of Helicobacter pylori and eradicating the bacterial infection in a mouse stomach. This monoclonal antibody UA15 was generated using a designed recombinant protein UreB, which contained the crucial region of the H. pylori urease beta-subunit active site, for immunization. The light chain of this antibody (UA15-L) by itself showed a proteolytic activity to substantially degrade both UreB and the intact urease. Oral administration of UA15-L also significantly reduced the number of H. pylori in a mouse stomach. This is the first example of a monoclonal catalytic antibody capable of functioning in vivo, and such an antibody may have a therapeutic utility in the future. 相似文献
25.
Kishida T Asada H Kubo K Sato YT Shin-Ya M Imanishi J Yoshikawa K Mazda O 《Journal of biotechnology》2008,133(2):201-207
26.
The polycation liposome (PCL), a recently developed gene transfer system, is simply prepared by a modification of liposomes with cetylated polyethylenimine (PEI), and shows remarkable transgene efficiency with low cytotoxicity. In the present study, we investigated the applicability of PCLs for in vivo gene transfer, since the PCL-mediated transgene efficiency was found to be maintained in the presence of serum. PCLs composed of dioleoylphosphatidylethanolamine (DOPE) with 5 mol% cetyl PEI (PEI average mr. wt. 1800), were superior for transfection to those of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (2:1 as molar ratio) with 5 mol% cetyl PEI in vitro, although the latter PCLs were more efficient for gene transfer in vivo. PCL-DNA complexes were injected into mice via a tail or the portal vein, with the DNA being a plasmid encoding green fluorescent protein (GFP) or luciferase; and the expression was monitored qualitatively or quantitatively, respectively. Tail vein injection resulted in high expression of both GFP and luciferase genes in lung, and portal vein injection resulted in high expression of both genes in the liver. Concerning the gene delivery efficiency, the PCL was found to be superior to PEI or cetyl PEI alone. The optimal conditions for in vivo transfection with PCLs were also examined. 相似文献
27.
Atsuhiro Matsumoto Takanori Kanai Yohei Mikami Po–Sung Chu Nobuhiro Nakamoto Hirotoshi Ebinuma Hidetsugu Saito Toshiro Sato Hideo Yagita Toshifumi Hibi 《PloS one》2013,8(4)
Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt−/−) mice and RAG-2 and RORγt double deficient (RAG-2−/− × RORγt−/−) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl4), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4+ T cells, NKT cells, and lineage marker-negative SCA-1+Thy1high ILCs, of the liver of wild type (WT) mice. CCl4-treated RORγt−/− mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2−/− × RORγt−/− mice that lacked T and NKT cells. Surprisingly, RAG-2−/− × RORγt−/− mice developed significantly severer CCl4-induced hepatitis compared with RAG-2−/− mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2−/− mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice. 相似文献
28.
Yuki Kanase Takafumi Kitada Hidetsugu Tabata Kosho Makino Tetsuta Oshitari Hiromi Ohashi Takashi Yoshinaga Hideaki Natsugari Hideyo Takahashi 《Bioorganic & medicinal chemistry》2018,26(9):2508-2513
The physicochemical properties of 4-substituted carbamazepine derivatives were investigated. It was elucidated that the 4-substitution is not effective in reducing the rotations (E/Z) about the N—C1′ axes around the outer carbamoyl moiety. However, the atropisomers were isolated with high stereochemical stability, meaning that the 4-substitution reduced the butterfly motion of the tricyclic ring system efficiently. The Cl/CH3-substituted carbamazepine derivatives showed greater inhibitory effects on hNav1.2 channel currents compared with carbamazepine, although no difference in the activity between enantiomers was observed. 相似文献
29.
The purpose of this study was to measure the changes of supination and pronation in the ankle joint at landing to quantify the influence of shock attenuation during landing. The subjects did two different motions, jumping down on the force platform from posterior and lateral views. The rear view of single foot contact in a jump from height of 30 and 60 cm showed a landing on the inside of the rear part of the foot (pronation) followed after about 0.03 sec by a rolling outward of the foot (supination). The variables describing changes in three angles of the ankle joint indicated that the standing position was more sensitive on the pronation and supination during ground contact. 相似文献
30.
Lee S Kamide T Tabata H Takahashi H Shiro M Natsugari H 《Bioorganic & medicinal chemistry》2008,16(21):9519-9523
The pyrimido[1,2-a][1,4]benzodiazepines (1a-c) and the 8-membered analogues (diazocines 2a and 2b) were separated into their atropisomers with HPLC on a chiral column. High stereochemical stability was observed in the atropisomer of the 8-membered derivatives (2a and 2b), and the 1,4-benzodiazepine (1c) with 2'-chloro at the pendant phenyl showed a lower energy barrier for the conversion between the atropisomers compared with that with the unsubstituted pendant phenyl (1a). The aR isomer of 1a-c was revealed to be the eutomer in GABA(A) receptor binding, and the eutomer 1c-R showed extremely potent activity with an IC(50) value of 1.5 nM. 相似文献