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51.
Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue 总被引:4,自引:0,他引:4
Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway. 相似文献
52.
53.
This study reports the unique compartmentalization of cortisol and 11-deoxycortisol biosynthesis in vitro from [(3)H]17alpha-hydroxyprogesterone (17P) in testicular tissues of groupers after sex inversion induced by 17alpha-methyltestosterone (MT). Before MT implantation, the ovarian tissues produced only nonpolar metabolites. Following sex inversion some 6 months later, synthesis of these nonpolar metabolites was not detectable. Instead, cortisol and 11-deoxycortisol, with yields of about 3% and 14%, respectively, were synthesized together with two other polar metabolites. The corticosteroids and polar metabolites were distinctly nondetectable in ovarian tissues of the control fish throughout the experiment. While the significance of this testicular synthesis of corticosteroids is presently unclear, it could be related to the increased energy demands arising from the reorganization of gonadal tissues during sex inversion. 相似文献
54.
Udo H Lam CK Mori S Inouye M Inouye S 《Journal of molecular microbiology and biotechnology》2000,2(4):557-563
Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases. 相似文献
55.
Turk E Kim O le Coutre J Whitelegge JP Eskandari S Lam JT Kreman M Zampighi G Faull KF Wright EM 《The Journal of biological chemistry》2000,275(33):25711-25716
The Na(+)/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni(2+) affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na(+)-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mm (Na(+)) and 158 microm (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters. 相似文献
56.
Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns 总被引:2,自引:0,他引:2
We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research. 相似文献
57.
Isolation and characterization of a platelet membrane protein related to the vitronectin receptor 总被引:7,自引:0,他引:7
S C Lam E F Plow S E D'Souza D A Cheresh A L Frelinger M H Ginsberg 《The Journal of biological chemistry》1989,264(7):3742-3749
Glycoprotein IIb-IIIa is the most prominent Arg-Gly-Asp (RGD)-binding adhesion receptor on platelets. By affinity chromatography on an immobilized RGD peptide, we have investigated the possible existence of other platelet-associated adhesion receptors that bind RGD peptides. When an octyl glucoside extract of surface-radioiodinated platelets was applied to an affinity matrix of KYGRGDS-coupled Sepharose 4B, a 160-kDa-labeled protein (P160) and GPIIb-IIIa bound and were specifically eluted by soluble GRGDSP peptide, but not by the variant GRGESP peptide. Furthermore, a dodecapeptide corresponding to fibrinogen gamma 400-411 eluted only GPIIb-IIIa but not P160 from the RGD affinity matrix. Characterization of P160 by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the O'Farrell gel electrophoresis system indicated that P160 is a component of platelet GPIc. GoH3, a monoclonal antibody recognizing the alpha subunit of the very late antigen-6, failed to immunoprecipitate P160 from the RGD eluate, indicating that it did not contain the very late antigen-6 alpha subunit. In immunoblots, P160 reacted specifically with a polyclonal anti-peptide antibody recognizing the alpha subunit of the vitronectin receptor (VnR), but not with the monoclonal anti-GPIIb antibody PMI-1, suggesting that P160 is the alpha subunit of platelet VnR. This possibility was further substantiated by the complete identity between the determined amino-terminal sequence of P160 and the known sequence of the VnR alpha subunit. Moreover, direct association of P160 with a beta subunit having an apparent molecular weight similar to that of GPIIIa was demonstrated by immunoprecipitation with LM609, an anti-VnR complex monoclonal antibody. These results indicate that the VnR complex is present on platelets and may play a functional role in platelet adhesive reactions. 相似文献
58.
Stone KL Bjornson RD Blasko GG Bruce C Cofrancesco R Carriero NJ Colangelo CM Crawford JK Crawford JM daSilva NC Deluca JD Elliott JI Elliott MM Flory PJ Folta-Stogniew EJ Gulcicek E Kong Y Lam TT Lee JY Lin A LoPresti MB Mane SM McMurray WJ Tikhonova IR Westman S Williams NA Wu TL Hongyu Z Williams KR 《The Yale journal of biology and medicine》2007,80(4):195-211
59.
Madureira PA Matos P Soeiro I Dixon LK Simas JP Lam EW 《The Journal of biological chemistry》2005,280(45):37310-37318
The MHV-68 latent protein, M2, does not have homology to any known viral or cellular proteins, and its function is unclear. To define the role played by M2 during MHV-68 latency as well as the molecular mechanism involved, we used M2 as bait to screen a yeast two-hybrid mouse B-cell cDNA library. Vav1 was identified as an M2-interacting protein in two independent screenings. Subsequent yeast two-hybrid interaction studies showed that M2 also binds to Vav2, but not Vav3, and that three "PXXP" motifs located at the C terminus of M2 are important for this interaction. The interactions between M2 and Vav proteins were also confirmed in vivo in 293T and WEHI-231 B-cells by co-immunoprecipitation assays. Rac1/GST-PAK "pull-down" experiments and Western blot analysis using a phospho-Vav antibody demonstrated that expression of M2 in WEHI-231 cells enhances Vav activity. We further showed in WEHI-231 cells that M2 expression promotes proliferation and survival and is associated with enhanced cyclin D2 and repressed p27(Kip1), p130, and Bim expression. Taken together, these experiments suggest that M2 might have an important role in disseminating the latent virus during the establishment and maintenance of latency by modulating B-cell receptor-mediated signaling events through Vav to promote B-cell activation, proliferation, and survival. 相似文献
60.
Beatty ME Stone A Fitzsimons DW Hanna JN Lam SK Vong S Guzman MG Mendez-Galvan JF Halstead SB Letson GW Kuritsky J Mahoney R Margolis HS;Asia-Pacific Americas Dengue Prevention Boards Surveillance Working Group 《PLoS neglected tropical diseases》2010,4(11):e890