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Summary Plant hairy root cultures of Lithospermum erythrorhizon were carried out to produce shikonin derivatives by employing in situ extraction with n-hexadecane in a shake flask and a bubble column bioreactor. Over 95 % shikonin produced was recovered in the n-hexadecane layer. In flask cultures the maximum concentration of shikonin with n-hexadecane extraction was 3 times higher than that obtained without extraction. In the two phase bubble column reactor, 572.6 mg/L of shikonin and 15.6 g/L of dry cell mass were obtained after 54 days. Shikonin was produced at a constant level of 10.6 mg/L day during this period. 相似文献
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Abstract: The α subunit of Gz (αz) harbors two N-terminal serine residues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the α subunit of Gt1 provide binding surfaces for the β1 subunit. We used three serine-to-alanine mutants of αz to investigate the functional importance of the two N-terminal serine residues. Wild-type or mutant αz was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist-dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous α subunits of Gi were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of αz to interact with δ-opioid, dopamine D2, or adenosine A1 receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant αz subunits was not impaired because they transduced βγ-mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four αz subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hormone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of adenylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of αz has no apparent effect on receptor-mediated responses, but these serine residues may be essential for ensuring transition of αz into the active conformation. 相似文献
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The human class I alcohol dehydrogenase isozyme beta 2 beta 2 was used as the antigen to raise monoclonal antibodies. Altogether seven lines of hybridomas secreting monoclonal antibodies were obtained. None of the antibodies was isozyme specific and all of them exhibited a similar affinity against all isozymes of the human class I ADH. Five out of the seven monoclonal antibodies had no effect on beta 2 beta 2 activity. Antibody G3 acted as a non-competitive inhibitor with a KI of 3 micrograms/ml at pH 7.5. Increasing pH was effective in reducing the level of inhibition. On the other hand, antibody 1D4 exhibited a pH-dependent activation of ADH activity. In the presence of this antibody, the pH optimum of beta 2 beta 2 was shifted from 9 to 8.5 and total activity was increased by 70% at this optimal pH. Kinetic analysis indicates that 1D4 probably acts as a non-competitive activator and may exert its action by interacting with the coenzyme binding site. 相似文献
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Stabilization of Z-DNA by demethylation of thymine bases: 1.3-A single-crystal structure of d(m5CGUAm5CG) 总被引:1,自引:0,他引:1
Methylation of cytosine bases at the C5 position has been known to stabilize Z-DNA. We had previously predicted from calculations of solvent-accessible surfaces that the methyl group at the same position of thymine has a destabilizing effect on Z-DNA. In the current studies, the sequence d(m5CGUAm5CG) has been crystallized and its structure solved as Z-DNA to 1.3-A resolution. A well-defined octahedral hexaaquomagnesium complex was observed to bridge the O4 oxygens of the adjacent uridine bases at the major groove surface, and four well-structured water molecules were found in the minor groove crevice at the d(UA) dinucleotide. These solvent interactions were not observed in the previously published Z-DNA structure of the analogous d(m5CGTAm5CG) sequence. A comparison of the thymine and uridine structures supports our prediction that demethylation of thymine bases helps to stabilize Z-DNA. A comparison of this d(UA)-containing Z-DNA structure with the analogous d(TA) structure shows that access of the O4 position is hindered by the C5 methyl of thymine due to steric and hydrophobic inhibition. In the absence of the methyl group, a magnesium-water complex binds to and slightly affects the structure of the Z-DNA major groove surface. This perturbation of the solvent structure at the major groove surface is translated into a much larger 1.41-A widening of the minor groove crevice, thereby allowing the specific binding of two water molecules at well-defined sites of each internal d(UA) base pair. Possible mechanisms by which modifications at the major groove surface of Z-DNA can affect the solvent properties of the minor groove crevice are discussed. 相似文献
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Apyrase (ATP-diphosphohydrolase, EC 3.6.1.5) and inorganic pyrophosphatase (EC 3.6.1.1) were partially purified fromS. aureofaciens RIA 57 and characterized. Apyrase degrades, in addition to ATP, other nucleoside triphosphates and nucleoside diphosphates,
diphosphate, thiamine diphosphate, phosphoenolpyruvate and oligophosphates of chain lengthn ≦ 90. The apyrase activity was detected in the membrane and supernatant fractions. Its properties (substrate specificity, effect
of inhibitors, pH optimum and effect of Mg2+ ions) were similar in both fractions except for the effect of oligomycin that inhibited only the membrane fraction. Pyrophosphatase
exhibited a strict substrate specificity, substrates other than diphosphate being degraded relatively slowly. Of other enzymes
exhibiting the phosphatase activity acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1), trimetaphosphatase
(EC 3.6.1.2) and exopolyphosphatase (EC 3.6.1.11) degrading oligophosphates of chain lengthn = 15, 40 and 60, were detected. 相似文献
29.
Measurements of proton translocation in CF1-depleted, N, N′-dicyclohexylcarbodiimide-resealed broken chloroplasts were made under different light intensities. Kinetic analysis of the data shows that the outward leakage of accumulated protons through CF0 is still dependent on light intensity with a first-order rate constant equal to mR0, where R0 is the initial rate of proton uptake which normally increases with light intensity and m is a characteristic constant which is independent of proton gradient and light intensity. Measurements of proton translocation in these modified chloroplasts cross-linked with glutaraldehyde under illumination and in the dark respectively suggest that the light-dependent proton leakage through CF0 is regulated by conformation change in the membrane. It is proposed that the ovserved regulation of proton leakage through the CF1.CF0 complex in native chloroplasts is for optimizing the steady state synthesis of ATP under different light intensities. 相似文献
30.