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31.
A simple and efficient procedure for the generation of random GC to AT transition mutations in a specific DNA segment is described. A restriction fragment is inserted in each orientation into an M13 vector, single-stranded virion DNA from each recombinant phage is treated with methoxylamine, and, after reannealing of the mutagenized strands, a double-stranded restriction fragment is obtained. This methoxylamine-derivatized DNA segment is then joined with linearized M13 RF DNA, competent E. coli is transfected, and mutations are directly identified by sequencing of the phage DNA. Using this technique, single and double nucleotide substitutions were generated at a frequency greater than 50% in a 56-base pair segment of the signal codons of the TEM beta-lactamase. 相似文献
32.
Hydrogenase activity in Azospirillum brasilense is inhibited by nitrite, nitric oxide, carbon monoxide, and acetylene. 总被引:3,自引:0,他引:3 下载免费PDF全文
Nitrite, NO, CO, and C2H2 inhibited O2-dependent H2 uptake (H3H oxidation) in denitrifying Azospirillum brasilense Sp7 grown anaerobically on N2O or NO3-. The apparent Ki values for inhibition of O2-dependent H2 uptake were 20 microM for NO2-, 0.4 microM for NO, 28 microM for CO, and 88 microM for C2H2. These inhibitors also affected methylene blue-dependent H2 uptake, presumably by acting directly on the hydrogenase. Nitrite and NO inhibited H2 uptake irreversibly, whereas inhibition due to CO was easily reversed by repeatedly evacuating and backfilling with N2. The C2H2 inhibition was not readily reversed, partly due to difficulty in removing the last traces of this gas from solution. The NO2- inhibition of malate-dependent respiration was readily reversed by repeatedly washing the cells, in contrast to the effect of NO2- on H2-dependent respiration. These results suggest that the low hydrogenase activities observed in NO3(-)-grown cultures of A. brasilense may be due to the irreversible inhibition of hydrogenase by NO2- and NO produced by NO3- reduction. 相似文献
33.
Meria E. Penttilä K. M. Helena Nevalainen Alain Raynal Jonathan K. C. Knowles 《Molecular & general genetics : MGG》1984,194(3):494-499
Summary The possibility of cloning filamentous fungal genes by expression in the yeast Saccharomyces cerevisiae has been studied. A genome bank of Aspergillus niger was made in E. coli using a yeast cosmid shuttle vector and over 10,000 different cosmid clones were individually isolated. Yeast transformants carrying Aspergillus DNA were screened for the expression of the genes for fungal secreted glycoproteins, -galactosidase, -glucosidase, and amyloglucosidase, and for the expression of fungal genes complementing yeast ura3 and leu2 mutations.Of the five Aspergillus genes studied, only one, -glucosidase, was found to be expressed in yeast, and this at a low level. This suggests that there are essential differences between the genes of yeast and filamentous fungi. 相似文献
34.
Quantification of the importance of individual steps in the control of aromatic amino acid metabolism. 总被引:7,自引:4,他引:3 下载免费PDF全文
The quantitative importance of the individual steps of aromatic amino acid metabolism in rat liver was determined by calculation of the respective Control Coefficients (Strengths). The Control Coefficient of tryptophan 2,3-dioxygenase for tryptophan degradation was determined in a variety of physiological conditions and with a range of activities of tryptophan 2,3-dioxygenase. The Control Coefficient varied from 0.75 with basal enzyme activity to 0.25 after maximal induction of the enzyme by dexamethasone. The remainder of the control for tryptophan degradation was associated with the transport of the amino acid across the plasma membrane, with only very small contributions from kynureninase and kynurenine hydroxylase. The Control Coefficients of tyrosine aminotransferase for tyrosine degradation were approx. 0.70 and 0.20 with basal and dexamethasone-induced tyrosine aminotransferase activities respectively; the Control Coefficients of the transport of the amino acid into the cell were 0.22 and 0.58 respectively. Phenylalanine hydroxylase was found to have a Control Coefficient for the degradation of phenylalanine of approx. 0.50 under conditions of basal enzyme activity; after maximal activation by glucagon, the Control Coefficient decreased to 0.12. The transport of phenylalanine was responsible for the remaining control in the pathway. These results have important implications, directly for the regulation of aromatic amino acid metabolism in the liver, and indirectly for the regulation of neuroamine synthesis in the brain. 相似文献
35.
36.
In the plasma membranes from several mammalian tissues (including normal and tumor tissues), a Mg2+ (or Ca2+)-dependent ATP phosphohydrolase activity is present in much greater amount than the (Na+ + K+)-ATPase. The ouabain-insensitive activity can be attributed to at least two enzymes, an ATPase (EC 3.6.1.3) and an ATP diphosphohydrolase (EC 3.6.1.5). The ATPase hydrolyzes ATP and other nucleoside triphosphates and is not inhibited by azide. The ATP diphosphohydrolase hydrolyzes both ATP and ADP (and other nucleoside tri- and diphosphates) and the hydrolysis of adenine nucleotides is strongly inhibited by 10 mM azide. The ratios of these two enzymes in the various membranes (as determined by the extent of azide inhibition) vary widely. The ATP diphosphohydrolase accounts for most of the Mg2+ (or Ca2+)-dependent ATP hydrolysis activity of the plasma membranes of liver (mouse), kidney (dog), two mouse sarcomas, and a human astrocytoma (xenograft in athymic mice). The ATPase is more dominant in the plasma membranes from mouse brain and human oat cell carcinoma. The widespread presence of the ATP diphosphohydrolase in plasma membrane from various types of tissues is demonstrated for the first time and is of particular interest in view of its relatively high activity in the plasma membranes of two sarcomas. The membrane-bound ATP diphosphohydrolase is characterized with respect to its metal ion activators, substrates, and inhibitors. These results should facilitate the distinction of this enzyme from other ATP hydrolyzing enzymes of plasma membranes in future investigations. 相似文献
37.
Isolation and growth of a Pseudomonas species that utilizes cyanide as a source of nitrogen 总被引:3,自引:0,他引:3
A simple method of isolating bacteria that utilize cyanide as a source of nitrogen for growth has been developed. This involved supplying hydrogen cyanide as a vapour to glucose-containing minimal-salts agar plates. The bacteria isolated were Gram-negative, oxidase-positive rods producing a fluorescent green pigment and were tentatively identified as strains of Pseudomonas fluorescens. Three organisms were studied further and shown to be P. fluorescens biotype II. One of these (NCIB 11764) was grown in a glucose-containing fed-batch culture with either NH4Cl or KCN as the limiting nutrient. Cyanide-grown bacteria produced stoichiometric amounts of ammonia from cyanide when pulsed with cyanide under aerobic conditions. Stimulation of oxygen uptake was seen on addition of cyanide to suspensions of cyanide-grown but not ammonia-grown bacteria. 相似文献
38.
M van de Rijn P G Lerch R W Knowles C Terhorst 《Journal of immunology (Baltimore, Md. : 1950)》1983,131(2):851-855
The human beta 2-microglobulin (beta-2m)-associated human thymocyte differentiation antigens T6 and M241 were compared using biochemical techniques. T6 and M241 antigens reside on different molecules with apparent m.w. of 49,000 and 43,000, respectively. Here we show that both proteins have a protein backbone m.w. of 33,000. In addition, T6 and M241 have a large portion of their peptides in common. When we compared the protein backbone m.w. of T6 and M241 with the murine beta-2m-associated thymus leukemia (TL) antigens, we found a considerable difference in size, suggesting that T6 and M241 may not be human homologues of TL antigens and constitute a novel type of major histocompatibility (MHC) class I antigens. 相似文献
39.
40.
A specific rabbit antibody against the 4-azido-2-nitrophenyl determinant was photo-labelled by the homologous hapten ε-(4-azido-2-nitrophenyl)-l-lysine, and by the close structural isomer ε-(5-azido-2-nitrophenyl)-l-lysine. The extents of covalent labelling of the antibody-binding site were assessed by using radioactive haptens and exhaustive displacement dialysis, which leaves the unlabelled sites empty but largely intact. A single photolysis of hapten–antibody complex suffices to label those sites that are capable of being labelled. Although there is considerable overlap among sub-populations of antibody that will bind the two haptens non-covalently, sites that can be covalently labelled by one reagent cannot be labelled by the other. 相似文献