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Christoph Syldatk Vera Lehmensiek Gaby Ulrichs Ulrich Bilitewski Karsten Krohn Hartmut Höke Fritz Wagner 《Biotechnology letters》1992,14(2):99-104
Summary Resting cells of a mutant ofArthrobacter sp. (DSM 3747) were used for the bioconversion of D,L-5-benzylhydantoin and related compounds to the corresponding L-amino acids. After optimization of the reaction conditions in shake flask experiments, bioconversions were performed in a preparative scale in a 2-l-bioreactor under nitrogen atmosphere. Specific productivities of 0.4 (p-NO2-L-phenylalanine) up to 3.9 mM amino acid x g cell dry mass–1 x h–1 (p-Cl-L-phenylalanine) were obtained. D,L-5-p-COOH-Benzylhydantoin, D,L-5-phenylhydantoin and D,L-5-p-OH-phenylhydantoin were not accepted as substrates. 相似文献
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Understanding the structure and function of protein complexes and multi‐domain proteins is highly important in biology, although the in vitro characterization of these systems is often complicated by their size or the transient nature of protein/protein interactions. To assist in the characterization of such protein complexes, we have developed a modular approach to fusion protein generation that relies upon S ortase‐mediated and Na tive chemical ligation using synthetic Pe ptide linkers (SNaPe) to link two separately expressed proteins. In this approach, we utilize two separate linking steps – sortase‐mediated and native chemical ligation – together with a library of peptide linkers to generate libraries of fusion proteins. We have demonstrated the viability of SNaPe to generate libraries from fusion protein constructs taken from the biosynthetic enzymes responsible for late stage aglycone assembly during glycopeptide antibiotic biosynthesis. Crucially, SNaPe was able to generate fusion proteins that are inaccessible via direct expression of the fusion construct itself. This highlights the advantages of SNaPe to not only access fusion proteins that have been previously unavailable for biochemical and structural characterization but also to do so in a manner that enables the linker itself to be controlled as an experimental parameter of fusion protein generation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
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Giant protoplasts of Saccharomyces cerevisiae of 10-35 µm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and “patchability” of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66 ± 0.07 µF/cm2) and conductance (23-44 µS/cm2) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels. 相似文献
6.
Debarati Paul Neha Rastogi Ulrich Krauss Michael Schlomann Gunjan Pandey Janmejay Pandey Anuradha Ghosh Rakesh K. Jain 《Indian journal of microbiology》2008,48(2):279-286
Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism
of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic
machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of
the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated
the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils.
The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed
wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained
in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the
taxonomic ranks of the host microorganisms. 相似文献
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Karl Welzenbach Ulrich Hommel Gabriele Weitz-Schmidt 《The Journal of biological chemistry》2002,277(12):10590-10598
The beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) is a conformationally flexible alpha/beta heterodimeric receptor, which is expressed on the surface of all leukocytes. LFA-1 mediates cell adhesion crucial for normal immune and inflammatory responses. Intracellular signals or cations are required to convert LFA-1 from a nonligand binding to a ligand binding state. Here we investigated the effect of small molecule inhibitors on LFA-1 by monitoring the binding of monoclonal antibodies mapped to different receptor domains. The inhibitors were found to not only induce epitope changes in the I domain of the alpha(L) chain but also in the I-like domain of the beta(2) chain depending on the individual chemical structure of the inhibitor and its binding site. For the first time, we provide strong evidence that the I-like domain represents a target for allosteric LFA-1 inhibition similar to the well established regulatory L-site on the I domain of LFA-1. Moreover, the antibody binding patterns observed in the presence of the various inhibitors establish a conformational interaction between the LFA-1 I domain and the I-like domain in the native receptor that is formed upon activation. Differentially targeting the binding sites of the inhibitors, the L-site and the I-like domain, may open new avenues for highly specific therapeutic intervention in diseases where integrins play a pathophysiological role. 相似文献
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Luttge Ulrich; Osmond C. Barry; Ball Erika; Brinckmann Enno; Kinze Gabriele 《Plant & cell physiology》1972,13(3):505-514
Bisulfite compounds are shown to be nonspecific inhibitors ofphotosynthetic processes and of ion transport in green tissues.CO2 fixation and light-dependent transient changes in externalpH are inhibited about 50% by 5x104 M glyoxal-Na-bisulfite.Chloride uptake in the light and in the dark is inhibited tothe same extent at this concentration. At 5x103 M theinhibitor reduces ATP levels in the light and in the dark, andeffects on glycolate oxidase and PEP carboxylase are observed.The extent of inhibition is dependent on time of treatment withglyoxal-Na-bisulfite and freshly prepared NaHSO3 is equallyas effective as the addition compound. Possible explanations of the bisulfite effects and the relationshipsto SO2 effects on photosynthesis are discussed. (Received September 1, 1971; ) 相似文献