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21.
22.
N-Ethylmaleimide (NEM) reducing enzyme was purified to homogeneity from cell-free extracts of Candida lipolytica by chromatography techniques. The molecular weight of the native enzyme was estimated to be about 43,000 by gel filtration using Superose 12 and to be 47,000 by SDS-PAGE. This enzyme can use both NADPH and NADH as an electron donor, and catalyzes the reduction of the carbon-carbon double bond of five membered ring compounds which have two conjugated carbonyl groups on both sides of a double bond. 相似文献
23.
M Tagaya T Yagami T Noumi M Futai F Kishi A Nakazawa T Fukui 《The Journal of biological chemistry》1989,264(2):990-994
Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis. The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli. The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme. The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively. In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme. These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates. Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates. Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme. Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin. It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding. 相似文献
24.
Nakazawa Miki; Kakihana Junko; Hisabori Toru; Manabe Katsushi 《Plant & cell physiology》1990,31(6):789-796
Procedures for the purification of native phytochrome from etiolatedpea seedlings without the use of immuno-purification techniquesare described. Phytochrome (in the PFR form) was purified bypolyethyleneglycol fractionation, adsorption to pentyl agaroseand batch elution, chromatography on DEAE-Sepharose, adsorptionto phenyl Toyopearl and batch elution, and chromatography onRed Toyopearl. The resulting phytochrome had specific absorbanceratios (SAR = A666/A280 of PR) that ranged from 0.55 to 0.6.The subsequent chromatography on Sephacryl S-300 yielded verypure phytochrome with a SAR of 0.98. PR and PFR peaks in thedifference spectrum of the phytochrome were centered at 665and 730 nm, respectively. The spectral change ratio (Ar/Afr)of the difference spectrum was unchanged after the chromatographyon phenyl Toyopearl, and the value was 1.051.08, indicatingthat the spectral properties of this preparation were intact.The absorption spectra indicated that the peak absorbance ofPFR was at 728730 nm and that of PR was at 666667nm. These peak positions were essentially same as those obtainedwith the undegraded oat phytochrome. Incubation of the samplepurified on Sephacryl S-300 at 25?C for 5 h in either the PRor PFR form did not result in degradation of the molecule. Therate of dark reversion of PFR observed with the purified peaphytochrome was similar to that observed in vivo. The additionof dithionite had no effect on the reversion rate.
2Present address: Fuji-Gotenba, Research Lab. of Chugai PharmaceuticalCo. Ltd., Gotenba, Shizuoka, 412 Japan (Received February 22, 1990; Accepted May 28, 1990) 相似文献
25.
26.
Kohtaro Asayama Kazushige Dobashi Yasusuke Kawada Takaya Nakane Akira Kawaoi Shinpei Nakazawa 《The Histochemical journal》1996,28(1):63-71
Summary To quantitate the developmental changes in selenium-dependent cellular glutathione peroxidase during the perinatal period,
tissue sections from foetal (day 12 to day 22) and neonatal (day 6) rats were stained immunohistochemically using specific
polyclonal antiserum. The intensity of the staining was quantified by fluorescence microscopy image analysis. There was a
general trend of enriched glutathione peroxidase in the epithelial linings and metabolically active sites. Significant fluorescence
was detected in cardiomyocytes, hepatocytes, renal tubular epithelium, bronchiolar epithelium and intestinal epithelium at
day 15. The intensity increased in a stepwise manner therafter. The overall increase in the intensity of staining in the heart,
liver, kidneys, lungs and intestine was 1.5-, 2.3-, 1.6-, 1.7- and 3.0-fold, respectively. The phase of most rapid increase
occurred during the foetal period in the liver, intestine and heart. In the kidneys and lungs, glutathione peroxidase increased
significantly during foetal life, and to a similar extent postnatally. These results suggest that the intracellular H2O2-scavenging system develops during the foetal period as an essential mechanism for living under atmospheric oxygen conditions.
The late development observed in the kidneys and lungs is consistent with the relative biological immaturity of these organs
in full-term neonates. 相似文献
27.
28.
Tetsuya Matsumoto Mitsuo Kaku Kazuhiro Tateda Nobuhiko Furuya Yoichi Hirakata Keizo Yamaguchi 《Microbiology and immunology》1994,38(4):287-293
We evaluated antibody-coated bacteria (ACB) in expectorated sputum to discriminate contaminating or colonizing organisms from true pathogens. We examined 60 expectorated sputum samples from 51 patients with lower respiratory infections (chronic obstructive pulmonary disease 25, pneumonia 20, purulent tracheobronchitis 6). All samples were examined with quantitative culture and immunofluorescent demonstration of ACB. From the results of quantitative culture, we divided specimens into pathogen-isolated and pathogen-free samples. Among pathogen-isolated samples, in which we isolated accepted pathogenic organisms at ≥ 107 colony-forming units per ml, 16 of 23 samples were ACB-positive (69.5%). In contrast, among pathogen-free samples, in which we isolated accepted pathogens at < 107 colony forming units per ml or only upper respiratory flora, only 3 of 37 samples were ACB-positive (8.1%). The ACB-positive rate was significantly higher in pathogen-isolated than in pathogen-free samples (P < 0.001). Consequently, detecting ACB in expectorated sputum shows good potential as another criterion for distinguishing contaminating or colonizing organisms from true pathogens. 相似文献
29.
Nakazawa Miki; Hayashi Hidenori; Yoshida Yuhji; Manabe Katsushi 《Plant & cell physiology》1993,34(1):83-91
Peptide fragments were obtained by limited proteolysis withtrypsin and Staphylococcus aureus V 8 protease from either thePR or the PFR form of 121-kDa phytochrome purified from etiolatedpea (Pisum sativum L.) shoots. Patterns of bands after polyacrylamidegel electrophoresis in the presence of SDS of the digests weredifferent, with some bands appearing preferentially when thedigestions were carried out with the PR or the PFR form. Amino-terminalsequences of the fragments were analyzed to determine the exactlocations of the amino-termini of the fragments within the aminoacid sequence of the apoprotein of pea phytochrome. The aminoacid compositions of some of the sequenced fragments were determinedin order to confirm the carboxy-terminal amino acids. Threecleavage regions were identified as kinetically favored sitesof cleavage of PFR (Arg-746 to Lys-752, around Glu-877 and aroundArg-1010), whereas only one was identified for PR (Glu-38 toArg-62). Regions of Glu-255, Arg-383, Arg-583 to Glu-620 andLys-1093 to Glu-1115 were also identified as potential sitesof proteolytic cleavage in both forms of the phytochrome. Othercleavage sites, the specificities of which have not yet beendetermined, are Glu-404, Glu-695 and Lys-1045. Surface-exposed parts of phytochrome in the PR and PFR formsare discussed. (Received June 13, 1992; Accepted October 27, 1992) 相似文献
30.
Vesselina Gadzheva Kohji Ichimori Hiroe Nakazawa Zahary Raikov 《Free radical research》1994,21(3):177-186
Superoxide scavenging activities (SSA) of newly synthesized spin-labeled nitrosourea and triazene derivatives, and their precursor nitroxides were investigated by the ESR/spin-trapping method using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and hypoxanthine/xanthine oxidase as the superoxide-generating system. The spin-labeled nitrosoureas, triazenes and their precursor nitroxides exhibited excellent SSA, whereas clinically used nitrosourea and triazene, which do not contain the nitroxide moiety, did not show any SSA. Furthermore, it was deduced that these nitroxides scavenge superoxide by redox cycling between nitroxide and corresponding hydroxylamine. 相似文献