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991.
Responses to Potato virus A (PVA, genus Potyvirus) segregate to three phenotypic groups in a diploid cross between Solanum tuberosum subsp. andigena and a highly interspecific potato hybrid. The aim of this study was to compare gene expression between the progeny genotypes which react with hypersensitive response (HR) to PVA, allow PVA accumulation in inoculated leaves but restrict PVA infection to the inoculated leaf by blocking systemic movement [non-necrotic resistance (nnr)], or are susceptible (S) and systemically infected with PVA. Expression levels of ca 10 000 genes were compared using probes arranged in a microarray format, and real-time RT-PCR was applied for quantitative comparison of the expression of selected defense-related genes (DRGs). Results showed that a few DRGs were autoactivated in HR genotypes at an early stage of plant growth in the absence of PVA infection, which was not observed in the two other phenotypic groups (nnr and S). More detailed studies on the DRGs encoding a beta-1,3-glucanase, a chitinase and a basic PR-1b protein showed that autoactivation of the genes was not evident in vitro and up to 2 weeks of growth in soil in a controlled growth cabinet but was apparent 2 weeks later. Hence, autoinduction of these DRGs in the HR genotypes could be associated with growth stage, environmental factors or both. Furthermore, a number of other DRGs were induced in the inoculated leaves of HR genotypes as a response to infection with PVA, which was not observed in nnr and S genotypes. These results provide some novel information about factors underpinning the higher levels of virus resistance realised in potato genotypes carrying virus-specific R genes and suggest that part of the resistance is attributable to additional ‘minor’ genes functioning simultaneously, hence adding to the overall responsiveness and level of resistance against infection. These results also imply that some genotypes might be more responsive to chemical induction of pathogen and pest resistance, which could be considered in screening of progenies in plant-breeding programs.  相似文献   
992.
993.
We found the 2′,5′-oligoadenylate synthetase-like (OASL) gene to be significantly elevated by high virus loads in human liver infected with hepatitis C virus (HCV). Here, we determined whether OASL inhibited HCV replication using an in vitro system. We constructed three expression vectors of OASL to produce isoform a (OASLa), isoform b (OASLb), and the C-terminal ubiquitin-like domain of isoform a (Ub). When Huh7 JFH-1 HCV replicon cells were separately transfected with these three vectors, colony formation of HCV-replicating cells was inhibited by 95%, 94%, and 65%, respectively. Both OASLa and OASLb were also inhibitory for cells as well as the virus because colony formation of OASL-producing cells was reduced to 41% and 8%, respectively. Stable Huh7 clones producing each of the three OASLs were established and assessed for their inhibition of HCV replication using luciferase reporter gene-containing JFH-1 replicon RNA. HCV replication was inhibited by 50-90% in several stable OASL clones. Association analysis in six Ub clones expressing different levels of Ub mRNA showed that the degree of inhibition of HCV replication was significantly associated with the amount of Ub present. In conclusion, OASL possesses two domains with HCV inhibitory activity. The N-terminal OAS-homology domain without OAS activity is inhibitory for cell growth as well as HCV replication, whereas C-terminal Ub is inhibitory only for HCV replication. Therefore, OASLa, a major isoform of this molecule induced in human liver, may mediate anti-HCV activity through two different domains.  相似文献   
994.
The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGN-related (DC-SIGNR) molecules on the cell surface are known to enhance human immunodeficiency virus type 1 (HIV-1) infection by capturing the virions and transmitting them to CD4+ T-cell, a process termed trans-infection. The neck region and carbohydrate recognition domain of the two proteins are important for efficient binding to the HIV-1 envelope protein. DC-SIGNR is polymorphic in Exons 4 and 5 that encode the neck region and carbohydrate recognition domain, respectively; the former contains a variable number of tandem repeats, and the latter the SNP (rs2277998). Since it remains unclear whether the DC-SIGNR polymorphism is related to the risk of HIV-1 infection, we tested possible effects of the polymorphism on HIV-1 trans-infection efficiency, by constructing six kinds of cDNAs encoding DC-SIGNR variants with various numbers of repeat units and various SNP. We were able to express the variants on the surface of Raji cells, a human B cell line. Flow cytometry showed that all the tested DC-SIGNR molecules were efficiently expressed on the cell surface at various levels; the assay for HIV trans-infection efficacy showed that all the tested variants had that activity with different efficacy levels. We found a correlation between the HIV trans-infection efficiency and the mean fluorescent intensity of DC-SIGNR expression (R2 = 0.95). Thus, our results suggest that the variation of the tested DC-SIGNR genotypes affects the efficacy of trans-infection by affecting the amounts of the protein expressed on the cell surface.  相似文献   
995.
The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.  相似文献   
996.
The Slit family of guidance cues binds to Roundabout (Robo) receptors to modulate neuronal, leukocytic, and endothelial migration. Slit-Robo signaling had been reported to function as chemoattractive signal for vascular endothelial cells during angiogenesis. In this study, we found that Robo1 was expressed in lymphatic endothelial cells to mediate the migration and tube formation of these cells upon Slit2 stimulation, which were specifically inhibited by the function-blocking antibody R5 to Slit2/Robo1 interaction. To further explore the lymphangiogenic effect and significance mediated by Slit-Robo signaling, we intercrossed Slit2 transgenic mice with a non-metastatic RIP1-Tag2 mouse tumor model, and found that transgenic overexpression of Slit2 significantly enhanced tumor lymphangiogenesis and subsequently promoted mesenteric lymph node metastasis of pancreatic islet tumors. Taken together, our findings reveal that through interacting with Robo1, Slit2 is a novel and potent lymphangiogenic factor and contributes to tumor lymphatic metastasis.  相似文献   
997.
Factors involved in transition from the immunotolerant to immunoactive phase in chronic hepatitis B virus (HBV) infection remain unclear. We investigated viral mutations occurring during transition and elucidated their virological and immunological significance. Full-length HBV DNA sequences were serially determined in a chronic HBV carrier from the immunotolerant to immunoactive phase. Viral replicative competence was examined by transfection analysis. HBV-specific CD8+ T cell response was evaluated by coculture of CD8+ T cells with autologous dendritic cells followed by interferon-γ Elispot assay. Eleven point mutations and two deletions appeared around the onset of the immunoactive phase. Viral replicative competence declined significantly after the onset of active hepatitis. Examination of the CD8+ T cell response against two putative T-cell epitopes, which contained substituted amino acids from the immunotolerant to immunoactive phase, showed that mutant HBV epitopes gave a lesser T cell response than wild-type HBV ones. In summary, point mutations and deletions may occur prior to or concurrent with the onset of the immunoactive phase during chronic HBV infection. These mutations may result in a significant decrease in both viral replicative competence and HBV-specific CD8+ T cell response, suggesting a possible adaptation for the maintenance of viral persistence.  相似文献   
998.
The production rate of dengue viruses (DENVs), especially low-passage virus isolates, is low, and, therefore, the isolates are generally used only after several passages. However, in vitro passages could induce mutation(s). In this study, we established a system for the characterization of low-passage viral isolates using an infectious cDNA clone. We used R05-624, a plaque derived from type 2 (DENV-2) Thai strain, for the construction of the cDNA clone, named pmMW/R05-624. We found that transfection of both of mammalian Vero cells and mosquito C6/36 cells with viral RNA derived from the cDNA clone produced a significant amount of progeny virus: 3.2 × 106 focus-forming units (FFU) production per ml of cultured fluid only 3 days after transfection with 2 μg RNA. Conversely, no detectable level of viruses was produced by conventional methods using a single cell line, Vero or C6/36. When this system was applied for the characterization of eight low-passage clinical viral isolates by placing their 5′-half or 3′-half in the above cDNA clone, we found that all the isolates, except for L04-225, produced similar levels of progeny virus. Among a total of eight cDNA clones reconstructed with the NS4A-3′NCR region derived from L04-225, one clone carried an insertion and produced a low level of progeny virus. Thus, our system to efficiently rescue clinical samples or low-passage viral isolates could be useful for assessing the virological and molecular characteristics of DENV that could be related to disease pathogenesis.  相似文献   
999.
VAPB is a highly conserved integral membrane protein that is ubiquitously expressed in all eukaryotic organisms and located within the membranes of the endoplasmic reticulum (ER). The P56S missense mutation of the VAPB protein is linked to a hereditary form of amyotrophic lateral sclerosis (ALS8), and the pathogenesis of ALS8 has remained enigmatic. We report the cloning of five novel splice variants of the human VAPB gene, all of which are expressed at the mRNA level in the human nervous system. When transfected into human HEK293 or SH-SY5Y cells, two of these variants (VAPB-2 and VAPB-4,5) were readily detectable by immunoblotting whereas two variants (VAPB-3 and VAPB-3,4) became detectable after proteasomal inhibition, a condition commonly found in neurodegenerative diseases. Interestingly, one of these novel VAPB variants, VAPB-2, co-immunoprecipitated with wt-VAPB. However, so far none of these splice variants could be detected by immunoblotting of lysates from selected human tissues, suggesting that in vivo, the proteins translated from the variant VAPB mRNAs are quickly degraded or, alternatively, the expressed proteins are below detection limit of the available antibodies. We speculate that under conditions of proteasomal inhibition, as encountered in many neurodegenerative diseases including ALS, variant VAPB proteins might accumulate in affected cells and contribute to ALS pathogenesis.  相似文献   
1000.
The worldwide outbreak of the swine-origin 2009 H1N1 influenza A virus (IAV) and an increasing number of influenza cases caused by a highly pathogenic avian influenza (HPAI) H5N1 have accelerated the need to develop vaccines and antiviral agents against IAVs. Among various antivirals, neutralizing monoclonal antibodies (mAbs) are considered important passive therapeutics having an immediate effect against viral pathogens. Here we report a pseudovirus neutralization assay for rapid screening of neutralizing mAbs targeting hemagglutinin (HA) of H5N1 and H1N1 IAV. In this study, we generated six pseudoviruses with an HIV-1 backbone, respectively, expressing HA of four clades of H5N1 IAV and the 2009 epidemic H1N1 IAV. The resulting pseudoviruses were able to infect a variety of human and non-human cells, with 293T cells from human kidney as the most susceptible target cells. Using the established pseudovirus neutralization assay, we showed that three of ten selected mAbs specific to HA could potently neutralize infection of a pseudovirus bearing HA from the homologous IAV A/VietNam/1194/2004(H5N1) strain. This was highly consistent with the result of a microneutralization assay testing the same strain of a live IAV. Since the pseudovirus neutralization assay does not involve an infectious virus and can be performed without the requirement of a biosafety-3 laboratory, it may be applied for safe and rapid screening of neutralizing mAbs and antiviral agents targeting HA of IAVs.  相似文献   
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