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181.
Mutagenesis of the Neisseria gonorrhoeae porin reduces invasion in epithelial cells and enhances phagocyte responsiveness 总被引:3,自引:0,他引:3
Porin (PorB), the major outer membrane protein of Neisseria gonorrhoeae, has been implicated in pathogenesis previously. However, the fact that porin deletion mutants are not viable has complicated investigations. Here, we describe a method of manipulating the porin gene site-specifically. N. gonorrhoeae MS11, which harbours the porB1B (P.1B) porin allele, was used to generate mutants carrying deletions in the surface loops 1 and 5. An 11-amino-acid deletion in loop 1 impaired Opa50-dependent invasion into human Chang epithelial cells, whereas loop 5 deletion exhibited no apparent phenotype. In a second approach, the complete gonococcal porB1B was replaced by the porBNia gene of Neisseria lactamica. Such mutants were unable to induce efficient uptake by epithelial cells but induced an enhanced respiratory response in HL60 phagocytic cells. The increased respiratory burst was accompanied by an enhanced phagocytic uptake of the mutant compared with the wild-type strain. Our data extend previous evidence for multiple central functions of PorB in the infection process. 相似文献
182.
C. A. Stella C. Korch E. H. Ramos A. Bauer R. Kölling J. R. Mattoon 《Molecular genetics and genomics : MGG》1999,262(2):332-341
Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine. A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize l-leucine as sole nitrogen source and very low levels of [14C]l-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [14C]l-leucine uptake. Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine. The complementing gene, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation. The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin mutant containing the allele act1-1. Sac3p has been previously shown to be localized in the nucleus. When a lep1 mutant was crossed with a sac3 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic. 相似文献
183.
The mitochondrial TIM22 preprotein translocase is highly conserved throughout the eukaryotic kingdom
Bauer MF Rothbauer U Mühlenbein N Smith RJ Gerbitz K Neupert W Brunner M Hofmann S 《FEBS letters》1999,464(1-2):41-47
The Mohr-Tranebjaerg syndrome (MTS), a neurodegenerative syndrome characterized by progressive sensorineural hearing loss, dystonia, mental retardation and blindness, is a mitochondrial disease caused by mutations in the deafness/dystonia peptide 1 (DDP1) gene. DDP1 shows similarity to the yeast proteins Tim9, Tim10 and Tim12, components of the mitochondrial import machinery for carrier proteins. Here, we show that DDP1 belongs to a large family of evolutionarily conserved proteins. We report the identification, chromosomal localization and expressional analysis of six human family members which represent further candidate genes for neurodegenerative diseases. 相似文献
184.
Phospholipase D stimulation by receptor tyrosine kinases mediated by protein kinase C and a Ras/Ral signaling cascade 总被引:6,自引:0,他引:6
Voss M Weernink PA Haupenthal S Möller U Cool RH Bauer B Camonis JH Jakobs KH Schmidt M 《The Journal of biological chemistry》1999,274(49):34691-34698
Stimulation of phospholipase D (PLD) in HEK-293 cells expressing the M(3) muscarinic receptor by phorbol ester-activated protein kinase C (PKC) apparently involves Ral GTPases. We report here that PKC, but not muscarinic receptor-induced PLD stimulation in these cells, is strongly and specifically reduced by expression of dominant-negative RalA, G26A RalA, as well as dominant-negative Ras, S17N Ras. In contrast, overexpression of the Ras-activated Ral-specific guanine nucleotide exchange factor, Ral-GDS, specifically enhanced PKC-induced PLD stimulation. Moreover, recombinant Ral-GDS potentiated Ral-dependent PKC-induced PLD stimulation in membranes. Epidermal growth factor, platelet-derived growth factor, and insulin, ligands for receptor tyrosine kinases (RTKs) endogenously expressed in HEK-293 cells, apparently use the PKC- and Ras/Ral-dependent pathway for PLD stimulation. First, PLD stimulation by the RTK agonists was prevented by PKC inhibition and PKC down-regulation. Second, expression of dominant-negative RalA and Ras mutants strongly reduced RTK-induced PLD stimulation. Third, overexpression of Ral-GDS largely potentiated PLD stimulation by the RTK agonists. Finally, using the Ral binding domain of the Ral effector RLIP as an activation-specific probe for Ral proteins, it is demonstrated that endogenous RalA is activated by phorbol ester and RTK agonists. Taken together, strong evidence is provided that RTK-induced PLD stimulation in HEK-293 cells is mediated by PKC and a Ras/Ral signaling cascade. 相似文献
185.
Bauer Paul J.; Schauf Heike; Schwarzer Andreas; Brown Joel E. 《American journal of physiology. Cell physiology》1999,276(3):C558
Na+/Ca2+exchange has been investigated in squid(Loligopealei) rhabdomeric membranes.Ca2+-containing vesicles have beenprepared from purified rhabdomeric membranes by extrusion throughpolycarbonate filters of 1-µm pore size. After removal of externalCa2+, up to 90% of the entrappedCa2+ could be specificallyreleased by the addition of Na+;this finding indicates that most of the vesicles containedNa+/Ca2+exchanger. The Na+-inducedCa2+ efflux had a half-maximumvalue (K1/2) of~44 mM and a Hill coefficient of ~1.7. The maximalNa+-inducedCa2+ efflux was ~0.6 nmolCa2+ · s1 · mgprotein1. SimilarNa+-inducedCa2+ effluxes were measured ifK+ was replaced withLi+ orCs+. Vesicles loaded withCa2+ byNa+/Ca2+exchange also released this Ca2+byNa+/Ca2+exchange, suggesting thatNa+/Ca2+exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate ofCa2+ efflux enhanced byapproximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin,Na+/Ca2+exchange was characterized by aK1/2 of ~25 mMand a Hill coefficient of 1.6. For these vesicles, the maximalNa+-inducedCa2+ efflux was about twice asgreat as in control vesicles. We conclude thatNa+/Ca2+exchange proteins localized in rhabdomeric membranes mediate Ca2+ extrusion in squid photoreceptors. 相似文献
186.
A protein with a relative molecular mass of 31 kDa was specifically extracted by EGTA from a detergent-insoluble fraction of Giardia lamblia. N-terminal sequencing showed this protein to be identical to alpha 1-giardin, a component of the ventral disc which, based on its predicted amino acid sequence, has been classified as annexin XIX. Purified alpha 1-giardin associated with multilamellar phosphatidyl serine-containing vesicles in a Ca(2+)-dependent manner, confirming that it is a functional annexin. Molecular modelling of the amino acid sequence of the giardial annexin into the X-ray structure of annexin V suggests that the Ca(2+)-binding sites, which, as in other annexins, are all located on the convex surface of the molecule, are of the low-affinity type III. 相似文献
187.
188.
Bauer N Leljak-Levanic D Jelaska S 《Zeitschrift für Naturforschung. C, Journal of biosciences》2004,59(7-8):554-560
Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days. 相似文献
189.
Three pumpkin embryogenic lines were initiated on wounded zygotic embryos cultured on medium with or without 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryo development was controlled by the availability of various compounds in the medium: presence/absence of 2,4-D, nitrogen sources. The highest rate of DNA methylation was in the early embryo stages, predominantly on MSC medium with 2,4-D and on auxin-free medium supplemented with 1.0 mM NH4Cl. DNA methylation was correlated with early embryo development in a manner that was not exclusively dependent on the presence/absence of exogenous auxin. DNA methylation decreased during embryo maturation on auxin-free MSC medium and on auxin-free MSC supplemented with 12.3 M 5-azacytidine (5-azaC). The embryogenic features of the pumpkin tissue were preserved, even after a 2-month treatment with 5-azaC.Abbreviations 5-azaC
5-Azacytidine
- CRED-RA
Coupled restriction enzyme digestion and random amplification
- 2,4-D
2,4-Dichlorophenoxyacetic acid
- DNMRT
Duncans new multiple range test
- IAA
Indole-3-acetic acid
- 5-mC
5-Methylcytosine 相似文献
190.
Micic Z Hahn V Bauer E Schön CC Knapp SJ Tang S Melchinger AE 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2004,109(7):1474-1484
In many sunflower-growing regions of the world, Sclerotinia sclerotiorum (Lib.) de Bary is the major disease of sunflower (Helianthus annuus L.). In this study, we mapped and characterized quantitative trait loci (QTL) involved in resistance to S. sclerotiorum midstalk rot and two morphological traits. A total of 351 F3 families developed from a cross between a resistant inbred line from the germplasm pool NDBLOS and the susceptible line CM625 were assayed for their parental F2 genotype at 117 codominant simple sequence repeat markers. Disease resistance of the F3 families was screened under artificial infection in field experiments across two sowing times in 1999. For the three resistance traits (leaf lesion, stem lesion, and speed of fungal growth) and the two morphological traits, genotypic variances were highly significant. Heritabilities were moderate to high (h2=0.55–0.89). Genotypic correlations between resistance traits were highly significant (P<0.01) but moderate. QTL were detected for all three resistance traits, but estimated effects at most QTL were small. Simultaneously, they explained between 24.4% and 33.7% of the genotypic variance for resistance against S. sclerotiorum. Five of the 15 genomic regions carrying a QTL for either of the three resistance traits also carried a QTL for one of the two morphological traits. The prospects of marker-assisted selection (MAS) for resistance to S. sclerotiorum are limited due to the complex genetic architecture of the trait. MAS can be superior to classical phenotypic selection only with low marker costs and fast selection cycles. 相似文献