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71.
The interactions of benzoquinones with the reduced forms ofthe bound plastoquinone acceptors, QA and QB, were studied withoxygen-evolving photosystem II (PS II) particles from the thermophiliccyanobacterium Synechococcus elongatus, which largely lack poolplastoquinone molecules [Takahashi and Katoh (1986) Biochim.Biophys. Acta 845: 183]. Oxygen evolution in the presence ofvarious electron acceptors was determined and flash-inducedchanges in absorbance in the blue region were analyzed in termsof difference spectra, dependence on the concentration of benzoquinoneand on temperature, and sensitivity to 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU). The more hydrophobic the quinone molecule, the higherwas the rate of oxygen evolution, and the maximum rate of 3,000µmoles O2.(mg chlorophyll)1.h1 was recordedin the presence of phenyl- and dichloro-p-benzoquinones. DCMUinhibited oxygen evolution by more than 95%. However, spectrophotometricstudies revealed that, even though electrons were transferredto benzoquinones predominantly via the direct oxidation of by added benzoquinones occurred in such a way as to indicate thatabout 40% of PS II reaction centers were not associated withfunctional QB sites. was very stable in the presence of ferricyanide. However, benzoquinonesinduced the slow oxidation of . The characteristics of the benzoquinone reductioin in thePS II preparation is discussed.
1Present address: Department of Life Sciences, Faculty of Science,Himeji Institute of Technology, Shosha 2167, Himejishi, Hyogo-ken,671-22 Japan (Received May 8, 1990; Accepted August 14, 1990) 相似文献
72.
Two dynorphin-degrading cysteine proteases, I and II, were extracted with Triton X-100 from neuroblastoma cell membrane, isolated from accompanying dynorphin-degrading trypsin-like enzyme by affinity chromatography on columns of soybean trypsin inhibitor-immobilized Sepharose and p-mercuribenzoate-Sepharose, and separated by ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose and TSK gel DEAE-5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes, were inhibited by p-chloromercuribenzoate, N-ethylmaleimide, and high-molecular-weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E-64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1-17) at the Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1-17) at the Lys11-Leu12 bond and the Leu12-Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg-Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme. 相似文献
73.
Summary Maximum production of alkaline serine protease by Bacillus alcalophilus subsp. halodurans KP 1239 was achieved after 24 h cultivation, at an initial pH of 7.6, on a medium containing 1.0% sodium citrate, 0.3% yeast extract, and 0.3% KH2PO4. The enzyme was purified to crystalline form from culture broth. The enzyme was most active at 60° C and at pH 11.5. The molecular weight, isoelectric point and sedimentation coefficient in water at 20° C were estimated as 29 000, 8.8 and 3.3S, respectively. The N-terminal amino acid sequence was Ala-Gln-Ser-Val-Pro-Trp-Gly-Ile-Ser-Arg-Val-Gln-Ala-Pro-Ala-Ala-His-Asn-Arg-Gly-. The enzyme shared its antigenic determinants with B. alcalophilus ATCC 21522 serine protease, but not with the subtilisins Carlsberg and BPN.
Offprint requests to: Yuzuru Suzuki 相似文献
74.
Yuichiro Arai Se KyungKim Hiroyasu Kinemuchi Takeshi Tadano Shinetsu Satoh Nobunori Satoh Katsuyuki Oyama Kensuke Kisara 《Neurochemistry international》1990,17(4):587-592
The present study was carried out mainly to clarify whether the two amphetamine metabolites, p-hydroxyamphetamine (P-OHA) and p-hydroxynorephedrine (p-OHN) are taken up by mouse brain 5-hydroxytryptamine (5-HT) nerve terminals to inhibit type A monoamine oxidase (MAO-A) and then potentiate the abnormal behavior, head-twitch. Of the two metabolites, only intracerebroventricular p-OHA, at 80 μg/mouse, sufficient to cause a head-twitch response (HTR), appreciably inhibited MAO-A activity without affecting MAO-B activity in homogenates of the mouse striatum, hypothalamus and the rest of the forebrain; and p-OHN did not inhibit either type of MAO at the dose tested. Estimation of intra- and extrasynaptosomal MAO-A activity showed that both metabolites significantly inhibited only the intrasynaptosomal deamination of 5-HT by MAO-A with p-OHA being more potent. Taken together with our previous findings, these present results clearly indicate that p-OHA may accumulate in the 5-HT nerve terminals through the uptake system, and concomitantly inhibit MAO-A activity. These actions of p-OHA may increase intraneuronal 5-HT levels and then potentiate 5-HT release to cause interaction with the post-synaptic 5-HT receptors. 相似文献
75.
Mitsuo Satoh Shinji Hosoi Seiji Sato 《In vitro cellular & developmental biology. Plant》1990,26(11):1101-1104
Summary The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or
aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells
increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular
proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide,
was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium.
In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although
amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases.
This work was supported by the management of the Research Association for Biotechnology as a part of the R&D of Basic Technology
for Future Industries sponsored by NEDO (New Energy and Industrial Technology Development Organization). 相似文献
76.
Y. Satoh K. Hatakeyama K. Kohama M. Kobayashi Y. Kurusu H. Yukawa 《Journal of industrial microbiology & biotechnology》1990,5(2-3):159-165
Summary Electroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per g DNA was 5×104 when the cell suspension was pulsed at a cell density of 1×1010/ml and at a DNA amount of 1.0g. 相似文献
77.
T Mio T Yabe M Sudoh Y Satoh T Nakajima M Arisawa H Yamada-Okabe 《Journal of bacteriology》1996,178(8):2416-2419
The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2 delta amd chs3 delta null mutants. 相似文献
78.
Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease. 总被引:9,自引:7,他引:2
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Y Shimizu K Yamaji Y Masuho T Yokota H Inoue K Sudo S Satoh K Shimotohno 《Journal of virology》1996,70(1):127-132
In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein. To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction. We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A. The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3. The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40. Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity. Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity. In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40. This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation. 相似文献
79.
The ascidian egg contains muscle and endoderm determinants that play critical roles in the specification of muscle and endoderm cells, respectively. Endoderm cells of the ascidian embryo express alkaline phosphatase (AP) as a tissue-specific enzyme. We obtained egg fragments from the unfertilized eggs of Ciona savignyi by means of centrifugal force. The largest fragment (red fragments) contained the egg nucleus while other small fragments (black, clear and brown fragments) were anucleate. When inseminated, only red fragments developed into partial embryos, which showed only epidermis cell differentiation and, very rarely, AP activity. When red fragments were fused with other fragments, only black fragments promoted AP expression, suggesting that endoderm determinants were concentrated in the black fragments. A lower dose (1500 J/m2 ) of ultraviolet (UV) light did not eliminate the AP-promoting ability of black fragments, while this dose significantly repressed the ability to promote the expression of the muscle-marker. A higher dose (4500 J/m2 ) of UV light markedly reduced the AP-promoting activity of black fragments. These results suggest that factors for endodermal AP development are inactivated by UV irradiation, but are more resistant than muscle determinants. 相似文献
80.