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51.
52.
Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component. 相似文献
53.
G. Gradl P. Grandison E. Lindridge Y. Wang J. Watson F. Rudert 《Apoptosis : an international journal on programmed cell death》1996,1(2):131-140
Different CD95 (Fas/APO-1) isoforms and phosphory lated CD95 species were identified in human T and B cell lines. We had shown
previously that the CD95 intracellular domain (IC), expressed as a glutathione S-transferase (GST) fusion protein in murine
L929 fibroblasts, was phosphorylatedin vivo. GST-CD95IC was phosphorylatedin vitro by a kinase present in extracts from the human lymphocytic cell lines Jurkat and MP-1 and from murine L929 cells. Phosphoamino
acid analysis indicated that phosphorylation occurred at multiple threonine residues and also at tyrosine (Tyr232 and Tyr291)
and serine. Amino acids 191 to 275 of CD95 were sufficient for phosphorylation at threonine, tyrosine and serine and also
mediated interaction with a 35 kDa cellular protein. Immuno-precipitation of CD95 and chemical cross-linking revealed CD95-associated
proteins of approximately 35, 45 and 75 kDa. GST-CD95IC affinity chromatography detected binding of the 35 and 75 kDa protein
species. The 75 kDa species may correspond to the CD95-associated proteins RIP or FAF1 and the 35 kDa protein may represent
a TRADD analogue. These data indicate that several cellular proteins interact with CD95, possibly in a multi-protein complex,
and that a kinase activity is associated with CD95 not onlyin vitro but alsoin vivo. Therefore, receptor phosphorylation may play a role in CD95 signal transduction.
This work was in part supported by a grant from the Health Research Council of New Zealand (to JW). 相似文献
54.
V. P. Upelniek A. Yu. Novoselskaya J. Sutka G. Galiba E. V. Metakovsky 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1995,90(3-4):372-379
Electrophoretic patterns of seed storage proteins, the high-molecular-weight glutenins and gliadins, were studied in 468 plants of the common wheat cultivar Chinese Spring regenerated from callus culture of immature embryos, in 115 plants grown from seeds treated with nitrosoethylurea and in 260 control plants. From 5 to 21 single grains were analysed from each plant. In these three groups, the frequency of inherited mutations causing the loss of all proteins controlled by a locus (null-mutations, probably caused by a chromosomal deficiency) was 0.69%, 2.07%, and 0.05% per locus (the differences were statistically significant), respectively, while that of mutations causing the loss of a single protein band was 0.11%, 0.33%, and 0.05%, respectively. The loss of all of the gliadins controlled by Gli-B1 or GH-B2 (mutations were probably caused by a deletion of satellites of the corresponding chromosomes), was significantly higher than the loss of gliadins controlled by genomes A and D. Gene mutations altering the electrophoretic mobility of a single protein band in the pattern were found only in the second group of plants (0.44%). Therefore, chemical mutagenesis which produced not only more mutations than cultivation of immature wheat embryos in vitro, but also a higher ratio of mutations that altered DNA sequences, can be considered as an easier and comparatively more promising way for obtaining new improved variants of loci controlling biochemical characteristics in wheat. Somaclonal variation, on the other hand, was probably mainly caused by chromosomal abnormalities and could therefore hardly be considered as a useful tool in wheat breeding. 相似文献
55.
Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P2 ). Its activity is inhibited by Ca2+ and Zn2+ and is activated by Mg2+ , polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed. 相似文献
56.
57.
The ability of aras protein to associate with proteins present in rat brain cytosolin vitro was investigated using chemical cross-linking agents and the125I-labelled v-H-ras protein. Two iodinated protein complexes with apparent molecular weights of 40 and 85 kDa were observed when a mixture of rat brain cytosol and [125I]ras was treated with the cross-linking agent disuccinimidyl suberate and subjected to SDS-PAGE. Formation of the [125I] 85 kDa complex was enhanced by a high concentration of EDTA while generation of the 40 kDa species was abolished by this treatment. Formation of the [125I] 85 kDa complex was inhibited by unlabelledras protein, GTP, GTPS, and GDP but not by ATPS and GMP.Chromatography of the cross-linked brain cytosol-[125I]ras mixture on DEAE cellulose partially resolved the [125I] 85 kDa complex from the [125I]ras protein. The [125I] 85 kDa complex (formed using ethyleneglycolbis (succinimidylsuccinate) as the cross-linking agent) could be immunoprecipitated using a rabbit anti-ras polyclonal antibody. Treatment of the immunoprecipitate with hydroxylamine to cleave the cross-link yielded [125I]-labelledras. A substantial enrichment of the proportion of the [125I] 85 kDa complex in the cross-linked extract was achieved by preparative SDS-PAGE. It is concluded that thein vitro chemical cross-linking approach employed here has detected tworas binding proteins in rat brain cytosol: a 65 kDa heat-sensitive and a 20 kDa heat-stable protein. The possibility that the 65 kDaras binding protein is aras regulatory orras effector protein which has not so far been characterised is briefly discussed.Abbreviations DSS
disuccinimidyl suberate
- EGS
ethyleneglycolbis (succinimidylsuccinate)
- GTPS
guanosine 5-[-thio] triphosphate
- ATPS
adenosine 5-[-thio] triphosphate 相似文献
58.
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
59.
Six species of Pratylenchus indigenous to Great Britain, P. crenatus, P. fallax, P. neglectus, P. penetrans, P. pinguicaudatus and P. thornei were analysed by slab gel electrophoresis to compare protein patterns and isoenzyme phenotypes of esterase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, phosphoglucose isomerase and phosphoglucomutase. Multiple electromorphs were obtained from all enzymes examined. The results demonstrated that isoenzyme phenotypes are useful to supplement the morphological characterisation of these nematode species. Pair-wise comparisons of the six species were performed giving coefficients of similarity in the range 11–41%. A dendrogram of the six species, generated from the five enzyme banding patterns, gave two groups: group 1 contained P. pinguicaudatus, P. fallax and P. thornei and group 2 contained P. penetrans, P. neglectus and P. crenatus. 相似文献
60.
Jesper Hald Niels Rasmussen Mogens H. Claesson 《Cancer immunology, immunotherapy : CII》1995,41(4):243-250
Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour. 相似文献