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51.
The ppp(A2'p)nA-dependent endoribonucleases from a number of different mammalian sources have been investigated. The enzyme from reticulocyte lysates shows optimal activity of 50-150 mM KCl and requires the presence of Mg2+. Whilst the enzyme is inactivated after passage of reticulocyte lysates through Sephadex columns in the absence of ATP, it retains full activity provided ATP is included in the column buffer. The activity of the partially purified nuclease was unaffected by the addition of reticulocyte RNase inhibitor, which, in contrast, effectively inhibited other endogenous endonucleases. The ppp(A2'p)nA-dependent Rnase co-purified with a ppp(A2'p)nA-binding protein and with a protein which could be specifically covalently labelled with an oxidised radioactive analogue of ppp(A2'p)nA. This covalent labelling could be carried out either with the partially purified RNase or in crude extracts from rabbit reticulocytes, mouse Krebs and Ehrlich ascites tumour cells and human lymphoblastoid (Daudi) or HeLa cells. In each case the affinity labelled protein migrated to a position corresponding to a apparent molecular weight of about 85 000 on electrophoresis on dodecylsulphate/polyacrylamide gels. In all cases labelling could be prevented by the addition of an excess of unlabelled ppp(A2'p)nA but not, for example, by a similar excess of the biologically inactive dimer ppp(A2'p)'A. It is concluded that the RNase and ppp(A2'p)nA binding activities are likely to reside in the same molecule.  相似文献   
52.
We previously identified a cluster of hematopoietic serine protease genes on chromosome 14 at band q11.2. This cluster contains the cathepsin G gene and the two related cathepsin G-like genes CGL-1 and CGL-2. The CGL-1 gene is identical with the cytotoxic T cell serine protease CSP-B (also called SECT, and in mice, CCP1, granzyme B, or CTLA-1). In this report, we determined that CGL-2 is identical with a recently described gene called h-CCPX. The coding sequences of CG, CGL-1, and CGL-2 are 65-75% identical at the DNA level. The intervening sequences are much less conserved, except for introns 3 of the CGL-1 and CGL-2 genes, which are 93% identical. Each of the genes has the same overall organization, with 5 exons and 4 introns, very short 5' untranslated regions, and identical splice phases for all of the introns. Cathepsin G is expressed at high levels in promyelocytes/promonocytes, and CGL-1/CSP-B is expressed at high levels in activated cytolytic T cells, lymphokine-activated killer (LAK), and natural killer (NK) cells. CGL-2/h-CCPX is expressed at much lower levels in activated peripheral blood lymphocytes, LAK and NK cells. To begin to define the regulatory elements that target expression of each of these genes to their specific lineages at specific times, the 5' flanking region of each gene was sequenced. The 5' flanking regions are minimally related and have few conserved consensus elements. Further experiments will be required to determine the critical cis-acting regulatory sequences required for tissue- and development-specific expression of each of these genes.  相似文献   
53.
Blattella germanica (L.) that were fed hydramethylnon bait produced residues that were toxic to exposed conspecifics. Insecticidal activity was traced to the feces of treated insects by feeding radiolabeled material, where approximately 50% of the recovered radioactivity was unmetabolized parent compound. Ingestion of toxicant-laden feces by all life stages was evident, but the effect of this behavior was greatest on early instar nymphs. Baits containing toxicants with delayed activity, such as hydramethylnon, probably affect cockroach field populations indirectly through coprophagy.  相似文献   
54.
55.
We have identified 45 mutants of Caenorhabditis elegans that show ectopic surface binding of the lectins wheat germ agglutinin (WGA) and soybean agglutinin (SBA). These mutations are all recessive and define six genes: srf-2, srf-3, srf-4, srf-5, srf-8 and srf-9. Mutations in these genes fall into two phenotypic classes: srf-2, -3, -5 mutants are grossly wild-type, except for their lectin-binding phenotype; srf-4, -8, -9 mutants have a suite of defects, including uncoordinated movement, abnormal egg laying, and defective copulatory bursae morphogenesis. Characterization of these pleiotropic mutants at the cellular level reveals defects in the migration of the gonadal distal tip cell and in axon morphology. Unexpectedly, the pleiotropic mutations also interact with mutations in the lin-12 gene, which encodes a putative cell surface receptor involved in the control of cell fate. We propose that the underlying defect in the pleiotropic mutations may be in the general processing or secretion of extracellular proteins.  相似文献   
56.
Antipeptide antibodies were used to detect, purify, and characterize nonfilament F-pilin in the cell envelope of an F'tra+ strain of Escherichia coli. Affinity-purified goat antibodies raised against a peptide corresponding to the amino-terminal 14 amino acids of F-pilin detected F-pilin in immuno-overlay ("Western") blots of electrophoretically separated inner and outer membrane proteins. As expected, the molecule was absent from inner membrane preparations of F- or F'traA[Am] strains. Immunoreactive material was purified from inner membrane fractions and shown to be F-pilin by amino acid analysis. The anti-peptide antibodies also detected membrane forms of F-pilin produced by cells containing plasmid pTG801 (Grossman, T. & Silverman, P. (1989) J. Bacteriol. 171, 650-656). Most cell envelope pilin was in the inner membrane fraction, but a significant quantity fractionated with the outer membrane as well. The hydropathy profile of F-pilin suggested that the molecule is an integral membrane protein with two membrane-spanning domains. In confirmation, F-pilin and pTG801 pilins in inner membrane preparations were solubilized by a single extraction with the nonionic detergents Nonidet P-40 (2%) or Triton X-100 (2%), but not by 2 M KCl or 0.1 M NaOH. Moreover, analysis of traA'-'phoA constructs indicated that both the amino and carboxyl termini of F-pilin face the periplasm. The periplasmic location of the amino terminus was confirmed by immunoelectron microscopy of spheroplasts from F' and pTG801 strains, using a monoclonal antibody that recognizes an amino-terminal epitope. These data suggest a specific structure for membrane F-pilin. We discuss that structure in relation to the probable structure of filament F-pilin.  相似文献   
57.
We have examined transmembrane signaling events via the TCR/CD3 complex (TCR/CD3) at various stages of T cell development for evidence of developmental regulation. Engagement of TCR/CD3 induced defective activation of phospholipase C (PLC) in thymocytes relative to peripheral blood T lymphocytes. The defect in PLC activation via TCR/CD3 was restricted to immature thymocytes (CD3low, CD4+CD8+). Mature thymocytes (CD3high, CD4+CD8-/CD8+CD4-) were similar to PBL in signaling via TCR/CD3. Both immature and mature thymocytes expressed a similar profile of PLC isoenzyme mRNA species, indicating that the defect in signaling in immature thymocytes was not due to altered expression of PLC isoenzymes. Activation of tyrosine phosphorylation pathways implicated in the coupling of TCR/CD3 to PLC was impaired in immature thymocytes, as evidenced by depressed phosphorylation of CD3 zeta subunit after stimulation with anti TCR/CD3 mAb. This was associated with lower levels of p59fyn tyrosine kinase and minimal or undetectable stimulus-induced kinase activation in immature thymocytes relative to mature thymocytes. We conclude that the capacity to signal via TCR/CD3 is regulated during T cell development by mechanisms acting at the level of TCR/CD3-associated tyrosine phosphorylation pathways.  相似文献   
58.
59.
Sulfate reduction and S-oxidation in a moorland pool sediment   总被引:3,自引:2,他引:1  
In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO4 2– reduction rates in the sediment, (2) depletion of SO4 2– in the overlying water column and (3) release of35S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO4 2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core35SO4 2– injection. Rates of SO4 2– consumption in the overlying water were estimated by changes in SO4 2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m–2 d–1. Rates of SO4 2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m–2 d–1 (November) to 0.43–1.81 mmol m–2 d–1 (July, August and April). Maximum rates of oxidation to SO4 2– in July 1990 estimated by combination of SO4 2– reduction rates and rates of in situ SO4 2– uptake in the enclosed water column were 10.3 and 10.5 mmol m–2 d–1 at an organic rich and at a sandy site respectively.Experiments with35S2– and35SO4 2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO4 2– and (2) the presence of mainly non SO4 2–-S derived from reduced S transported from the sediment into the overlying water. A35S2– tracer experiment showed that about 7% of35S2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author  相似文献   
60.
Among the seven known isozymes of carbonic anhydrase in higher vertebrates, isozyme III is the least efficient in catalytic hydration of CO2 and the least susceptible to inhibition by sulfonamides. We have investigated the role of two basic residues near the active site of human carbonic anhydrase III (HCA III), lysine 64 and arginine 67, to determine whether they can account for some of the unique properties of this isozyme. Site-directed mutagenesis was used to replace these residues with histidine 64 and asparagine 67, the amino acids present at the corresponding positions of HCA II, the most efficient of the carbonic anhydrase isozymes. Catalysis by wild-type HCA III and mutants was determined from the initial velocity of hydration of CO2 at steady state by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and water at chemical equilibrium by mass spectrometry. We have shown that histidine 64 functions as a proton shuttle in carbonic anhydrase by substituting histidine for lysine 64 in HCA III. The enhanced CO2 hydration activity and pH profile of the resulting mutant support this role for histidine 64 in the catalytic mechanism and suggest an approach that may be useful in investigating the mechanistic roles of active-site residues in other isozyme groups. Replacing arginine 67 in HCA III by asparagine enhanced catalysis of CO2 hydration 3-fold compared with that of wild-type HCA III, and the pH profile of the resulting mutant was consistent with a proton transfer role for lysine 64. Neither replacement enhanced the weak inhibition of HCA III by acetazolamide or the catalytic hydrolysis of 4-nitrophenyl acetate.  相似文献   
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