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991.
992.
The ubiquitous Na+/H+ exchanger NHE1 is regulated by protein phosphorylation events, but the mechanisms involved are incompletely understood. We
recently cloned NHE1 from the red blood cells of the winter flounder, Pleuronectes americanus (paNHE1), and demonstrated its activation by osmotic cell shrinkage, β-adrenergic stimuli, and the Ser/Thr protein phosphatase
PP1 and PP2A inhibitor calyculin A (CLA) (Pedersen et al. [2003] Am. J. Physiol.
284, C1561–C1576). Here, we investigate the mechanisms involved in paNHE1 activation by these stimuli. Osmotic shrinkage and
CLA were only partially additive in their effects on paNHE1 activity, and CLA-mediated paNHE1 activation was inhibited by
osmotic cell swelling. Activation by the β-adrenergic agonist isoproterenol (IP) was fully additive to activation by osmotic
shrinkage or CLA. IP-mediated, but neither shrinkage-nor CLA-mediated paNHE1 activation were associated with an increase in
cellular cyclic adenosine monophosphate (cAMP) level. IP-mediated activation was partially blocked by the protein kinase A
(PKA) inhibitor H89 (10μM), wherease shrinkage- and CLA-mediated activation were unaffected. All three stimuli activated paNHE1 in a manner unaffected
by inhibitors of protein kinase C (calphostin C, 5 μM) and protein kinase G (KT5823, 10 μM) as well as of myosin light chain kinase (ML-7, 10 μM). IP-mediated, but not shrinkage-mediated, paNHE1 activation was associated with an increase in serine phosphorylation of
the paNHE1 protein. It is suggested that paNHE1 activation by osmotic shrinkage and by PP1/PP2A inhibition involves partially
convergent signaling pathways, whereas activation of paNHE1 by β-adrenergic stimuli is mediated by a separate pathway. 相似文献
993.
Moisan AM Fortin J Dumont M Samson C Bessette P Chiquette J Laframboise R Lépine J Lespérance B Pichette R Plante M Provencher L Voyer P Goldgar D Bridge P Simard J 《Genetic testing》2006,10(2):104-115
The discovery of deleterious mutations in the breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, has facilitated the identification of individuals at particularly high risk of these diseases. There is a wide variation between populations in the prevalence and related risks of various types of BRCA1/2 mutations, so estimates cannot be extrapolated to Canadians, especially not founder populations such as French- Canadians. Polymerase chain reaction (PCR)-based methods were used to detect the majority of these mutations. These approaches usually failed to detect large DNA rearrangements, which have been claimed to be involved in other populations in 5% to up to 36% of BRCA1-positive families. There is very little information about the contribution of this type of mutation in BRCA2-positive families. To investigate if our available mutation spectrum of BRCA1 and BRCA2 in high-risk French-Canadian breast/ovarian cancer families has been biased by PCR-based direct sequencing methods, we first used Southern blot analysis to test DNA samples from 61 affected/obligate carrier individuals from 58 families in which no BRCA1/2 deleterious mutation was found. Finally, 154 individuals from 135 BRCA1/2 nonconclusive families, including all those tested previously by Southern blot analysis, were tested with the new multiplex ligation probe amplification (MLPA) technique. These approaches failed to detect any rearrangement. Moreover, if the frequency of MLPA-detectable rearrangements in our cohort of 135 BRCA1/2 nonconclusive families was 2.2% or higher, we would have had a 95% or greater chance of observing at least one such rearrangement. As no rearrangements were identified, such large rearrangements must be quite rare in our population. 相似文献
994.
995.
Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis. 相似文献
996.
Dozot M Boigegrain RA Delrue RM Hallez R Ouahrani-Bettache S Danese I Letesson JJ De Bolle X Köhler S 《Cellular microbiology》2006,8(11):1791-1802
Physiological adaptation of intracellular bacteria is critical for timely interaction with eukaryotic host cells. One mechanism of adaptation, the stringent response, is induced by nutrient stress via its effector molecule (p)ppGpp, synthesized by the action of RelA/SpoT homologues. The intracellular pathogen Brucella spp., causative agent of brucellosis, possesses a gene homologous to relA/spoT, named rsh, encoding a (p)ppGpp synthetase as confirmed by heterologous complementation of a relA mutant of Sinorhizobium meliloti. The Rsh deletion mutants in Brucella suis and Brucella melitensis were characterized by altered morphology, and by reduced survival under starvation conditions and in cellular and murine models of infection. Most interestingly, we evidenced that expression of virB, encoding the type IV secretion system, a major virulence factor of Brucella, was Rsh-dependent. All mutant phenotypes, including lack of VirB proteins, were complemented with the rsh gene of Brucella. In addition, RelA of S. meliloti functionally replaced Brucella Rsh, describing the capacity of a gene from a plant symbiont to restore virulence in a mammalian pathogen. We therefore concluded that in the intramacrophagic environment encountered by Brucella, Rsh might participate in the adaptation of the pathogen to low-nutrient environments, and indirectly in the VirB-mediated formation of the final replicative niche. 相似文献
997.
998.
Bettina Couderc Marie Penary Mustapha Tohfe Anne Pradines Antoine Casteignau Danièle Berg Gilles Favre 《BMC biotechnology》2006,6(1):26-11
Background
The use of integrating viral vectors in Gene therapy clinical trials has pointed out the problem of the deleterous effect of the integration of the ectopic gene to the cellular genome and the safety of this strategy. We proposed here a way to induce the death of gene modified cells upon request by acting on a pro-apoptotic protein cellular localization and on the activation of its apoptotic function. 相似文献999.
Patrick Martin Olivier Albagli Marie Christine Poggi Kim E Boulukos Philippe Pognonec 《BMC biotechnology》2006,6(1):4-9
Background
Internal Ribosome Entry Site (IRES)-based bicistronic vectors are important tools in today's cell biology. Among applications, the expression of two proteins under the control of a unique promoter permits the monitoring of expression of a protein whose biological function is being investigated through the observation of an easily detectable tracer, such as Green Fluorescent Protein (GFP). However, analysis of published results making use of bicistronic vectors indicates that the efficiency of the IRES-controlled expression can vary widely from one vector to another, despite their apparent identical IRES sequences. We investigated the molecular basis for these discrepancies. 相似文献1000.
Magdalena Ietswaart Marie Johnston Chris H Dijkerman Clare L Scott Sara A Joice Steven Hamilton Ronald S MacWalter 《BMC neurology》2006,6(1):39-7