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141.
Escherichia coli alkaline phosphatase: X-ray structural studies of a mutant enzyme (His-412-->Asn) at one of the catalytically important zinc binding sites. 下载免费PDF全文
L. Ma T. T. Tibbitts E. R. Kantrowitz 《Protein science : a publication of the Protein Society》1995,4(8):1498-1506
The X-ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His-412 was replaced by Asn has been determined at both low (-Zn) and high (+Zn) concentrations of zinc. In the wild-type structure, His-412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn1) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269:31614-31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild-type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N (+Zn) enzyme was refined to an R-factor of 0.174 at 2.2 A resolution, and the structure of the H412N(-Zn) enzyme was refined to an R-factor of 0.166 at a resolution of 2.6 A. Both indicated that the Asn residue substituted for His-412 did not coordinate well to Zn1. In the H412N(-Zn) structure, the Zn1 site had very low occupancy and the phosphate was shifted by 1.8 A from its position in the wild-type structure. The Mg binding site was also affected by the substitution of Asn for His-412. Both structures of the H412N enzyme also revealed a surface-accessible cavity near the Zn1 site that may serve as a binding site for Tris.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
142.
143.
在海拔3700m高原选择50名男性青年的体重、立定跳远、引体向上、仰卧起坐、60m和2000m跑速度6项指标进行高原移居青年体制综合评价方法的研究。通过用百分位法制定各项指标评分标准,以各项指标评分相加作为体质总分,并用各项指标的简单相关系数与多元逐步回归方程的标准化偏回归系数的乘积(贡献率)分配“权重”,建立综合评价总分的计算式:Y=0.05X1+0.16(X4+X6)+0.21(X2+X3+X 相似文献
144.
Michael Ma Shuenn-Jue Wu Maureen Howard Alexej B. Borkovec 《In vitro cellular & developmental biology. Plant》1984,20(9):739-742
Summary We report here that the use of murine thymoma cell EL-4 conditioned medium enhances hybridoma yield in a low-antigen dose
in vitro immunization protocol. This improved protocol allowed the production of a panel of monoclonal antibodies toDrosophila yolk proteins using less than 1 nanomole of antigen. We believe this refinement will be valuable for the application of hybridoma
technology to biologically active materials that are hard to isolate and purify due to their low concentration in the biological
fluids.
This research was supported by the College of Agriculture and Life Sciences, University of Maryland, USDA-University of Maryland
Cooperative
Editor's Statement This observation should simplify in vitro immunization approaches and shed new light on the factors required
for the in vitro immune response. Wallace L. McKeehan 相似文献
145.
146.
Identification and DNA sequence of fixZ, a nifB-like gene from Rhizobium leguminosarum. 总被引:12,自引:2,他引:10 下载免费PDF全文
Previously, several mutants which nodulated peas but which failed to fix nitrogen were isolated following Tn5 mutagenesis of pRL 1JI, a symbiotic plasmid of Rhizobium leguminosarum. Two of these alleles, fix52::Tn5 and fix137::Tn5 were in a region of pRL 1JI which hybridized to a probe that contained the nifA gene and the amino-terminal region of the nifB gene of Klebsiella pneumoniae. The nitrogen fixation defect of the fix52::Tn5 mutant strain was corrected by a 2.0kb fragment of the corresponding wild-type DNA cloned in a wide host-range plasmid. The DNA sequence of this region revealed an open reading frame corresponding to the gene within which the fix52::Tn5 allele was located. The polypeptide corresponding to this open reading frame had a deduced molecular weight of 39,936 and the gene was termed fixZ. The deduced amino acid sequence of the fixZ gene product contained two clusters of cysteine residues, suggesting that the protein may contain an iron-sulphur cluster. The sequence of the fixZ polypeptide was very similar to the sequence of the K. pneumoniae nifB gene (provided by W. Arnold and A. Pühler) which is required for the synthesis of the FeMo-cofactor of nitrogenase. It was shown that the previously observed hybridization was due to homology between the amino terminal regions of fixZ and nifB. Upstream from fixZ was found another open reading frame whose 5' terminus was not established, but within which was located the fix137::Tn5 allele. This gene was termed fixY. The deduced amino acid sequence of the sequenced part of fixY showed similarity to that of the regulatory nifA gene of K. pneumoniae (provided by W. J. Buikema and F. M. Ausubel). Thus in R. leguminoarum the fix genes that correspond to the nifA and nifB genes are in the same relative orientation as in K. pneumoniae. 相似文献
147.
Qing-Sheng Ma Andrew W. B. Johnston Gerd Hombrecher J. Allan Downie 《Molecular & general genetics : MGG》1982,187(1):166-171
Summary Mutants of Rhizobium leguminosarum which failed to fix nitrogen within nodules on peas were isolated following the insertion of the transposon Tn5 into pRL1JI, a Rhizobium plasmid known to carry the genes for nitrogenase. The sites of the Tn5 insertions were identified by restriction endonuclease mapping of cloned fragments of DNA from the mutant strains. One group of mutants was located within 4 kilobases of the structural genes for nitrogenase and another was located about 30 kilobases from this region. Two mutants from the first group, one of which appeared to be affected in a nitrogenase gene, induced nodules that contained bacterioids, but the number of plant cells containing bacteroids was less than in a normal nodule. Another group of mutants, which was located about 30 kilobases from the nitrogenase genes failed to form bacterioids. Electron microscopy of the nodules induced by these mutants indicated that there was a defect in their release from infection threads. 相似文献
148.
149.
Marie Kselíková Tomáš Mařík Bedřich Bíbr Jaroslav Lener 《Biological trace element research》1980,2(1):57-64
This study describes the interaction of molybdenum with blood components. Molybdenum-99 was added to blood, and after four
washings, 3% of the total radioactivity was found in red cells. More specifically, the radioactivity was determined to be
associated with the cell membrane.
Molybdenum-99 in the +VI form did not interact with the human erythrocyte membrane; however, Mo(V) forms did interact. Of
five different compounds, the highes uptake was observed with a brown Mo(V)-ascorbate complex generated from Mo(VI) and ascorbic
acid in the molar ratio 1∶20. A membrane suspension of Mo-ascorbate-treated human erythrocytes was prepared and the solubilized
proteins were separated on a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS). Molybdenum-99 binding to
spectrin was demonstrated, as well as some minor interactions with membrane hemoglobin and bands 6 and 8. 相似文献
150.
Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between and . When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to .In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments.The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern. 相似文献