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131.
We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M (r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always <2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases. 相似文献
132.
The major damage to DNA caused by alkylating agents involves the formation of O(6)-methylguanine (O(6)-meG). Almost all species possess O(6)-methylguanine-DNA-methyltransferase (Ogt) to repair such damage. Ogt repairs O(6)-meG lesions in DNA by stoichiometric transfer of the methyl group to a cysteine residue in its active site (PCHR). Thermus thermophilus HB8 has an Ogt homologue, TTHA1564, but in this case an alanine residue replaces cysteine in the putative active site. To reveal the possible function of TTHA1564 in processing O(6)-meG-containing DNA, we characterized the biochemical properties of TTHA1564. No methyltransferase activity for synthetic O(6)-meG-containing DNA could be detected, indicating TTHA1564 is an alkyltransferase-like protein. Nevertheless, gel shift assays showed that TTHA1564 can bind to DNA containing O(6)-meG with higher affinity (9-fold) than normal (unmethylated) DNA. Experiments using a fluorescent oligonucleotide suggested that TTHA1564 recognizes O(6)-meG in DNA using the same mechanism as other Ogts. We then investigated whether TTHA1564 functions as a damage sensor. Pull-down assays identified 20 proteins, including a nucleotide excision repair protein UvrA, which interacts with TTHA1564. Interaction of TTHA1564 with UvrA was confirmed using a surface plasmon resonance assay. These results suggest the possible involvement of TTHA1564 in DNA repair pathways. 相似文献
133.
134.
Kosuke Anan Moriyasu Masui Shinichiro Hara Miho Ohara Masaharu Kume Shoichi Yamamoto Shunji Shinohara Hiroki Tsuji Shinji Shimada Shigenori Yagi Nobuyoshi Hasebe Hiroyuki Kai 《Bioorganic & medicinal chemistry letters》2017,27(17):4194-4198
NR2B subunit containing N-methyl-d-aspartate (NMDA) receptor is an attractive target for chronic pain due to its involvement in disease states and its limited distribution in the central nervous system. Cyclohexanol-based leads 6a and 6c were identified as potent NR2B-selective NMDA antagonists utilizing a scaffold hopping approach. Further optimization of this series through replacement of the amide in the leads with an isoxazole and efforts to optimize the pharmacokinetic profiles led to the discovery of orally available brain penetrants 7k and 7l, which demonstrated analgesic activity in the mouse formalin test at early and late phases. 相似文献
135.
Okanishi Hiroki Kim Kwang Masui Ryoji Kuramitsu Seiki 《Extremophiles : life under extreme conditions》2017,21(2):283-296
Extremophiles - Recent studies have revealed the physiological significance of post-translational lysine acylations such as acetylation in the regulation of various cellular processes. Here, we... 相似文献
136.
Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domain 总被引:2,自引:0,他引:2
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Atsushi Yamagata Ryoji Masui Yoshimitsu Kakuta Seiki Kuramitsu Keiichi Fukuyama 《Nucleic acids research》2001,29(22):4617-4624
A recJ homolog was cloned from the extremely thermophilic bacterium Thermus themophilus HB8. It encodes a 527 amino acid protein that has 33% identity to Escherichia coli RecJ protein and includes the characteristic motifs conserved among RecJ homologs. Although T.thermophilus RecJ protein (ttRecJ) was expressed as an inclusion body, it was purified in soluble form through denaturation with urea and subsequent refolding steps. Limited proteolysis showed that ttRecJ has a protease-resistant core domain, which includes all the conserved motifs. We constructed a truncated ttRecJ gene that corresponds to the core domain (cd-ttRecJ). cd-ttRecJ was overexpressed in soluble form and purified. ttRecJ and cd-ttRecJ were stable up to 60°C. Size exclusion chromatography indicated that ttRecJ exists in several oligomeric states, whereas cd-ttRecJ is monomeric in solution. Both proteins have 5′→3′ exonuclease activity, which was enhanced by increasing the temperature to 50°C. Mg2+, Mn2+ or Co2+ ions were required to activate both proteins, whereas Ca2+ and Zn2+ had no effects. 相似文献
137.
Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes 总被引:1,自引:0,他引:1
Futaki S Masui Y Nakase I Sugiura Y Nakamura T Kogure K Harashima H 《The journal of gene medicine》2005,7(11):1450-1458
BACKGROUND: One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application. METHODS: Using representative commercially available cationic liposomes (Lipofectin, Lipofectamine, and Lipofectamine 2000), the effects of GALA on transfection efficiency were studied by luciferase assay and confocal microscopic observations. RESULTS: A concentration-dependent increase in the transfection efficiency was observed for GALA. Addition of 0.1 microM GALA to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of Lipofectin, but higher concentration of GALA decreased transfection efficiency. Successful reduction in the liposomal dosage was attained by employing GALA while maintaining a high transfection efficiency. Interestingly, although the transfection efficiency was higher in the presence of GALA, a lower amount of the plasmid DNA was taken up by the cells. Confocal microscopic observations of the rhodamine-labeled plasmid did not show a significant difference in the cellular localization among cells incubated in the presence or absence of GALA, suggesting that a slight increase in GALA-induced release of the plasmid to the cytosol may cause a significant change in the transfection efficiency. CONCLUSION: The unique features of GALA to mediate improved transfection efficiencies were identified. 相似文献
138.
N.N. Mendona D.C. Masui J.C. McNamara F.A. Leone R. P.M. Furriel 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2007,146(4):534
The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg− 1 and K0.5 = 1.31 ± 0.05 mmol L− 1. Stimulation of K+-phosphatase activity by magnesium (Vmax = 125.3 ± 7.5 U mg− 1; K0.5 = 2.09 ± 0.06 mmol L− 1), potassium (Vmax = 134.2 ± 6.7 U mg− 1; K0.5 = 1.33 ± 0.06 mmol L− 1) and ammonium ions (Vmax = 130.1 ± 5.9 U mg− 1; K0.5 = 11.4 ± 0.5 mmol L− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (KI = 304.9 ± 18.3 μmol L− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K+-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na+,K+)-ATPase was stimulated by high salinity acclimation. 相似文献
139.
Silva EC Masui DC Furriel RP Mantelatto FL McNamara JC Barrabin H Leone FA Scofano HM Fontes CF 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2008,149(4):622-629
Euryhaline crustaceans rarely hyporegulates and employ the driving force of the Na,K-ATPase, located at the basal surface of the gill epithelium, to maintain their hemolymph osmolality within a range compatible with cell function during hyper-regulation. Since polyamine levels increase during the adaptation of crustaceans to hyperosmotic media, we investigate the effect of exogenous polyamines on Na,K-ATPase activity in the posterior gills of Callinectes danae, a euryhaline swimming crab. Polyamine inhibition was dependent on cation concentration, charge and size in the following order: spermine > spermidine > putrescine. Spermidine affected K0.5 values for Na+ with minor alterations in K0.5 values for K+ and NH4+, causing a decrease in maximal velocities under saturating Na+, K+ and NH4+ concentrations. Phosphorylation measurements in the presence of 20 µM ATP revealed that the Na,K-ATPase possesses a high affinity site for this substrate. In the presence of 10 mM Na+, both spermidine and spermine inhibited formation of the phosphoenzyme; however, in the presence of 100 mM Na+, the addition of these polyamines allowed accumulation of the phosphoenzyme. The polyamines inhibited pumping activity, both by competing with Na+ at the Na+-binding site, and by inhibiting enzyme dephosphorylation. These findings suggest that polyamine-induced inhibition of Na,K-ATPase activity may be physiologically relevant during migration to fully marine environments. 相似文献
140.