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961.
962.
Generation of mutant murine cytomegalovirus strains from overlapping cosmid and plasmid clones 下载免费PDF全文
Ehsani ME Abraha TW Netherland-Snell C Mueller N Taylor MM Holwerda B 《Journal of virology》2000,74(19):8972-8979
We have developed a cosmid and plasmid system to generate mutant strains of murine cytomegalovirus (MCMV). The system is based on a series of seven overlapping cosmid clones that regenerate MCMV when cotransfected into mouse cells. The unaltered cosmids produce MCMV that is indistinguishable from wild-type MCMV based on restriction enzyme digest patterns of virus DNA and growth rates both in vitro and in vivo. Analysis of viral DNA from plaque-purified recombinant isolates taken from in vitro and in vivo stocks indicated that regeneration did not introduce novel mutations in the recombinant viral genomes. Isolation of specific genes and subsequent generation of specific mutant MCMVs was accomplished by replacement of cosmids with overlapping plasmid subclones. A new vector, PmeSUB, featuring a multiple cloning site and a stringent origin of replication, was constructed to make large subclones for use with smaller subclones containing the gene of interest. The utility of this system was demonstrated by the generation of two different mutant MCMVs from different combinations of overlapping plasmid subclones of one cosmid. The advantages of this system are that (i) target genes are maintained as small clones making them amenable to standard in vitro mutagenesis manipulations and that (ii) no reporter or selection genes are necessary to identify mutants. 相似文献
963.
The role of cell-mediated cytotoxicity in the pathogenesis of ulcerative colitis and Crohn's disease has been controversial since reports indicating either a decreased, or an increased, activity of cytotoxic T cells in active stages of inflammatory bowel disease exist. Some of these discrepancies may be attributed to the fact that so far mostly peripheral blood lymphocytes rather than intestinal T cells have been examined. To overcome some of these limitations we performed in situ hybridizations for the detection of perforin and granzyme A mRNA expressing cells, i.e. of cytotoxic cells activated in situ, in the affected intestinal mucosa. These studies revealed increased frequencies of activated, cytotoxic T cells in active stages of ulcerative colitis and Crohn's disease. Interestingly, activated perforin mRNA expressing T cells are present both in the CD4 and in the CD8 T cell subsets. In the latter T cell subset up to 60% of the mucosal T cells isolated from the affected sites express perforin mRNA at detectable levels. The elevated frequency of activated cytotoxic cells and their histological distribution also in close proximity to the epithelial cells may thus indicate an important role for cytotoxic cells in the pathogenesis of inflammatory bowel disease since activated cytotoxic T cells may further exacerbate the inflammatory process through the production of pro-inflammatory cytokines such as interferon-gamma or tumor necrosis factor-alpha, but also through the release of pro-inflammatory cytokines and chemokines upon lysis of epithelial cells and the increased influx of luminal antigens at the site of epithelial erosions. 相似文献
964.
Disruption of epithelial tight junctions is prevented by cyclic nucleotide-dependent protein kinase inhibitors 总被引:5,自引:0,他引:5
Klingler C Kniesel U Bamforth SD Wolburg H Engelhardt B Risau W 《Histochemistry and cell biology》2000,113(5):349-361
Tight junctions (TJs), the most apical of the intercellular junctions, prevent the passage of ions and molecules through the paracellular pathway. Intracellular signalling molecules are likely to be involved in the regulation of TJ integrity. In order to specifically investigate the role of protein kinase A (PKA) in the maintenance of epithelial TJ integrity, calcium-switch experiments were performed, in which calcium was removed from EpH4 and MDCK culture medium, in the absence or presence of the PKA inhibitors H-89 or HA-1004. Removal of calcium from the culture media of the epithelial cells resulted in disruption of the TJs, characterised by a loss of membrane association of the TJ-associated proteins occludin, ZO-1 and ZO-2, by a loss of TJ strands, by a marked decrease in the transepithelial electrical resistance and by a dramatic increase in the transepithelial permeability to tracers. The association of occludin, ZO-1 and ZO-2 with the actin cytoskeleton is not affected. In contrast, when the removal of calcium was performed in the presence of either the PKA inhibitor H-89 or HA-1004, all barrier characteristics were preserved. Our data indicate that following the removal of calcium from the culture medium of epithelial cells in vitro, PKA is activated and subsequently is involved in the disruption of TJs. 相似文献
965.
Isoprostanes F(2) are biologically active prostaglandin F(2)-like compounds formed by free radical-catalyzed oxidation of arachidonic acid (C20:4). Here, we show that a series of dinor isoprostanes F(1), which we term phytoprostanes F(1) (PPF(1)s), are formed by nonenzymatic oxidation of linolenate (C18:3) in plants. Identification and quantification of PPF(1)s were achieved by a negative ion chemical ionization gas chromatography-mass spectrometry method using oxygen 18-labeled PPF(1)s as internal standards. PPF(1)s were found in leaves, flowers, and roots of taxonomically distinct plant species at concentrations ranging from 43 to 1380 ng/g of dry weight. In addition, esterified PPF(1)s were found at 10- to 150-fold higher concentrations. During the drying and storage of various plant organs, endogenous PPF(1) levels increased dramatically by 15- to 263-fold. Because the structurally related prostaglandin F(2alpha) and isoprostanes F(2) exert potent biological activities (i.e., broncho- and vasoconstriction) in the nanomolar range, PPF(1)s could potentially exert similar biological activities. Notably, fresh birch pollen, which can easily be inhaled, contains exceedingly high concentrations (32,440 ng/g) of free PPF(1)s. 相似文献
966.
Rose MR Mueller LD 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2000,355(1403):1657-1662
The concept of the force of natural selection was developed to explain the evolution of ageing. After ageing, however, comes a period in which mortality rates plateau and some individual organisms could, in theory, live forever. This late-life immortality has no presently agreed upon explanation. Two main theories have been offered. The first is heterogeneity within ageing cohorts, such that only extremely robust individuals survive ageing. This theory can be tested by comparisons of more and less robust cohorts. It can also be tested by fitting survival data to its models. The second theory is that late-life plateaus in mortality reflect the inevitable late-life plateau in the force of natural selection. This theory can be tested by changing the force of natural selection in evolving laboratory populations, particularly the age at which the force plateaus. This area of research has great potential for elucidating the overall structure of life-history evolution, particularly the interrelationship between the three life-history phases of development, ageing and immortality. 相似文献
967.
968.
969.
Growth and differentiation factor 5 (GDF-5), a member of the TGF-beta superfamily, is involved in many developmental processes, like chondrogenesis and joint formation. Mutations in GDF-5 lead to diseases, e.g. chondrodysplasias like Hunter-Thompson, Grebe and DuPan syndromes and brachydactyly. Similar to other TGF-beta superfamily members, GDF-5 transmits signals through binding to two different types of membrane-bound serine-/threonine-kinase receptors termed type I and type II. In contrast to the large number of ligands, only seven type I and five type II receptors have been identified to date, implicating a limited promiscuity in ligand-receptor interaction. However, in contrast to other members of the TGF-beta superfamily, GDF-5 shows a pronounced specificity in type I receptor interaction in cross-link experiments binding only to BMP receptor IB (BMPR-IB). In mice, deletion of either GDF-5 or BMPR-IB results in a similar phenotype, indicating that GDF-5 signaling is highly dependent on BMPR-IB. Here, we demonstrate by biosensor analysis that GDF-5 also binds to BMP receptor IA (BMPR-IA) but with approximately 12-fold lower affinity. Structural and mutational analyses revealed a single residue of GDF-5, Arg57 located in the pre-helix loop, being solely responsible for the high binding specificity to BMPR-IB. In contrast to wild-type GDF-5, variant GDF-5R57A interacts with BMPR-IA and BMPR-IB with a comparable high binding affinity. These results provide important insights into how receptor-binding specificity is generated at the molecular level and might be useful for the generation of receptor subtype specific activators or inhibitors. 相似文献
970.
Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality 总被引:30,自引:6,他引:24
Elmén J Thonberg H Ljungberg K Frieden M Westergaard M Xu Y Wahren B Liang Z Ørum H Koch T Wahlestedt C 《Nucleic acids research》2005,33(1):439-447
Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here, we show that incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform. 相似文献