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131.
Changes in structure and dynamics of the Fv fragment of a catalytic antibody upon binding of inhibitor
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Kroon GJ Mo H Martinez-Yamout MA Dyson HJ Wright PE 《Protein science : a publication of the Protein Society》2003,12(7):1386-1394
Binding of the product inhibitor p-nitrophenol to the monoclonal esterolytic antibody NPN43C9 has been investigated by performing NMR spectroscopy of the heterodimeric variable-domain fragment (Fv) of the antibody in the presence and absence of inhibitor. Structural information from changes in chemical shift upon binding has been related to the changes in local dynamics in the active site of the catalytic antibody using NMR relaxation measurements. Significant changes in the chemical shifts of the backbone resonances upon binding extend beyond the immediate vicinity of the antigen binding site into the interface between the two associated polypeptides that form the Fv heterodimer, a possible indication that the binding of ligand causes a change in the relative orientations of the component light (V(L)) and heavy (V(H)) chain polypeptides. Significant differences in backbone dynamics were observed between the free Fv and the complex with p-nitrophenol. A number of resonances, including almost all of the third hypervariable loop of the light chain (L3), were greatly broadened in the free form of the protein. Other residues in the antigen-binding site showed less broadening of resonances, but still required exchange terms (R(ex)) in the model-free dynamics analysis, consistent with motion on a slow timescale in the active site region of the free Fv. Binding of p-nitrophenol caused these resonances to sharpen, but some R(ex) terms are still required in the analysis of the backbone dynamics. We conclude that the slow timescale motions in the antigen-binding site are very different in the bound and free forms of the Fv, presumably due to the damping of large-amplitude motions by the bound inhibitor. 相似文献
132.
Mo RR Eisenbraun JK Sonstein J Craig RA Curtis JL Stoolman LM Chen J Yung RL 《Journal of immunology (Baltimore, Md. : 1950)》2003,171(2):745-753
D10.G4.1 (D10) cells, a murine conalbumin-reactive Th2 cell line, made to overexpress the beta(2) integrin LFA-1 by pharmacological manipulation or by transfection become autoreactive and are capable of inducing in vivo autoimmunity. However, whether this is specific to LFA-1 and whether overexpression of other T cell integrin molecules has the same effect are unknown. We examined the functional consequences of T cell CD49d (alpha(4) integrin) overexpression by transfecting murine CD49d cDNA into D10 cells. Similar to the LFA-1-transfected cells, the CD49d-overexpressing T cells are autoreactive and proliferate in response to APCs in an MHC class II-dependent manner in the absence of nominal Ag. Additionally, CD49d overexpression is associated with increased in vitro adhesion to endothelial cells and increased in vivo splenic homing. However, in contrast to LFA-1 overexpression, increased T cell CD49d expression is not associated with autoreactive cytotoxicity or the ability to induce in vivo autoimmunity. In addition to the novel observation that CD49d overexpression is sufficient to induce T cell autoreactivity, our results also support the hypothesis that the ability to induce in vivo autoimmunity is related to T cell cytotoxicity and not to T cell proliferation function in the D10 murine adoptive transfer model of autoimmunity. 相似文献
133.
134.
Yang J Si T Ling Y Ruan Y Han Y Wang X Zhou M Zhang D Zhang H Kong Q Liu C Li X Yu Y Liu S Shu L Ma D Wei J Zhang D 《Life sciences》2003,72(26):3017-3021
An increasing amount of evidence suggests that the pathophysiology of schizophrenia is associated with the abnormal immune system, and cytokines may be important in schizophrenia. Among these cytokines, interleukin-1beta may play a role in the pathogenesis of the disease. In the present study, we investigated the genetic association between a TaqI polymorphism in interleukin-1beta gene (IL-1beta) and schizophrenia by restriction fragment length polymorphism (RFLP) analysis among 132 Chinese families of Han descent. The transmission disequilibrium test (TDT) did not demonstrate an allelic association with schizophrenia. Our results suggested that the TaqI polymorphism in IL-1beta gene might not confer increased susceptibility for schizophrenia. 相似文献
135.
Moënne-Loccoz Y Tichy HV O'Donnell A Simon R O'Gara F 《Applied and environmental microbiology》2001,67(8):3418-3425
The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and L-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant. 相似文献
136.
Many biocontrol fluorescent pseudomonads can protect plants from soilborne fungal pathogens through production of the antifungal secondary metabolite 2,4-diacetylphloroglucinol (Phl). One of the phl biosynthetic genes, phlD, encodes a polyketide synthase similar to plant chalcone synthases. Here, restriction analysis of phlD from 39 Phl+ biocontrol fluorescent pseudomonads yielded seven different banding patterns. The gene was sequenced in seven strains, representing the different restriction patterns. Cluster analysis of phlD restriction data or phlD sequences indicated that phlD polymorphism was high, and two main clusters were obtained when predicted PhlD sequences were compared. When the seven PhlD sequences were studied with those of other procaryotic polyketide synthases (gram-positive bacteria) and plant chalcone synthases, however, Phl+ pseudomonads, gram-positive bacteria, and plants clustered separately. Yet, sequence analysis of active site regions for PhlD and plant chalcone synthases revealed that PhlD can be considered a member of the chalcone synthase family, which may be interpreted as convergent evolution of key enzymes involved in secondary metabolism. For the 39 Phl+ pseudomonads, a relationship was found among phlD restriction patterns, phylogenetic groups defined by 16S rDNA restriction analysis (confirmed by 16S rDNA sequencing), and production levels of Phl in vitro. 相似文献
137.
Faure M Moënnoz D Montigon F Fay LB Breuillé D Finot PA Ballèvre O Boza J 《Analytical biochemistry》2002,307(2):244-251
The intestinal mucoprotein synthesis rate was measured in vivo for the first time. For this, a rapid, reproducible, and convenient method to purify mucoproteins from large numbers of intestinal samples at the same time was developed. The method takes advantage of both the high mucin resistance to protease activities due to their extensive glycosylations and the high mucin molecular size. Intestinal homogenates were partially digested with Flavourzyme. Nonprotected proteins partially degraded were easily separated from mucoproteins by small gel filtration chromatography using Sepharose CL-4B. Electrophoretically pure mucins were obtained. Their amino acid composition was typical of purified intestinal epithelial mucins. The mucoprotein synthesis rate was determined in vivo in rats using the flooding dose method with the stable isotope L-[1-13C]valine. Free L-[1-13C]valine enrichments in the intracellular pool were determined by GC-MS. L-[1-13C]valine enrichments into purified mucoproteins or intestinal mucosal proteins were measured by gas chromatography-combustion-isotope ratio mass spectrometry. In rats, we found that the gut mucosa protein synthesis rate (%/day) decreased regularly from duodenum (122%/day) to colon (43%/day). In contrast, mucoprotein fractional synthesis rates were in the same range along the digestive tract, between 112%/day (colon) and 138%/day (ileum). 相似文献
138.
Katagiri K Zhang-Hoover J Mo JS Stein-Streilein J Streilein JW 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(1):84-89
Anterior chamber-associated immune deviation (ACAID), a manifestation of ocular immune privilege, prevents Th1-dependent delayed hypersensitivity from developing in response to eye-derived Ags, thereby preserving vision. Since Th2-type cells have recently been shown to mediate destructive inflammation of the cornea, we wondered whether pre-emptive induction of ACAID could inhibit Th2 responses. Using a murine model of OVA -specific, Th2-dependent pulmonary inflammation, we pretreated susceptible mice by injecting OVA alone into the anterior chamber, or by injecting OVA-pulsed, TGF-beta2-treated peritoneal exudate cells i.v. These mice were then immunized with OVA plus alum strategy that generates Th2-mediated OVA-specific pulmonary pathology. When pretreated mice were challenged intratracheally with OVA, their bronchoalveolar lavage fluids contained far fewer eosinophils and significantly less IL-4, IL-5, and IL-13 compared with that of positive, nonpretreated controls. Similarly, lung-draining lymph node cells of pretreated mice secreted significantly less IL-4, IL-5, and IL-13 when challenged in vitro with OVA. Moreover, sera from pretreated mice contained much lower titers of OVA-specific IgE Abs. We conclude that Ags injected into the anterior chamber of the eye impair both Th1 and Th2 responses. These results reduce the likelihood that ACAID regulates Th1 responses via a Th2-like mechanism. Thus, immune privilege of the eye regulates inflammation secondary to both Th1- and Th2-type immune responses. 相似文献
139.
Characterization of a highly pathogenic H5N1 avian influenza A virus isolated from duck meat 总被引:11,自引:0,他引:11
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Tumpey TM Suarez DL Perkins LE Senne DA Lee JG Lee YJ Mo IP Sung HW Swayne DE 《Journal of virology》2002,76(12):6344-6355
Since the 1997 H5N1 influenza virus outbreak in humans and poultry in Hong Kong, the emergence of closely related viruses in poultry has raised concerns that additional zoonotic transmissions of influenza viruses from poultry to humans may occur. In May 2001, an avian H5N1 influenza A virus was isolated from duck meat that had been imported to South Korea from China. Phylogenetic analysis of the hemagglutinin (HA) gene of A/Duck/Anyang/AVL-1/01 showed that the virus clustered with the H5 Goose/Guandong/1/96 lineage and 1997 Hong Kong human isolates and possessed an HA cleavage site sequence identical to these isolates. Following intravenous or intranasal inoculation, this virus was highly pathogenic and replicated to high titers in chickens. The pathogenesis of DK/Anyang/AVL-1/01 virus in Pekin ducks was further characterized and compared with a recent H5N1 isolate, A/Chicken/Hong Kong/317.5/01, and an H5N1 1997 chicken isolate, A/Chicken/Hong Kong/220/97. Although no clinical signs of disease were observed in H5N1 virus-inoculated ducks, infectious virus could be detected in lung tissue, cloacal, and oropharyngeal swabs. The DK/Anyang/AVL-1/01 virus was unique among the H5N1 isolates in that infectious virus and viral antigen could also be detected in muscle and brain tissue of ducks. The pathogenesis of DK/Anyang/AVL-1/01 virus was characterized in BALB/c mice and compared with the other H5N1 isolates. All viruses replicated in mice, but in contrast to the highly lethal CK/HK/220/97 virus, DK/Anyang/AVL-1/01 and CK/HK/317.5/01 viruses remained localized to the respiratory tract. DK/Anyang/AVL-1/01 virus caused weight loss and resulted in 22 to 33% mortality, whereas CK/HK/317.5/01-infected mice exhibited no morbidity or mortality. The isolation of a highly pathogenic H5N1 influenza virus from poultry indicates that such viruses are still circulating in China and may present a risk for transmission of the virus to humans. 相似文献
140.
Yang JM Bell J Huang Y Tirado M Thomas D Forster AH Haigis RW Swanson PD Wallace RB Martinsons B Krihak M 《Biosensors & bioelectronics》2002,17(6-7):605-618
An integrated, stacked microlaboratory for performing automated electric-field-driven immunoassays and DNA hybridization assays was developed. The stacked microlaboratory was fabricated by orderly laminating several different functional layers (all 76 x 76 mm(2)) including a patterned polyimide layer with a flip-chip bonded CMOS chip, a pressure sensitive acrylic adhesive (PSA) layer with a fluidic cutout, an optically transparent polymethyl methacrylate (PMMA) film, a PSA layer with a via, a patterned polyimide layer with a flip-chip bonded silicon chip, a PSA layer with a fluidic cutout, and a glass cover plate layer. Versatility of the stacked microlaboratory was demonstrated by various automated assays. Escherichia coli bacteria and Alexa-labeled protein toxin staphylococcal enterotoxin B (SEB) were detected by electric-field-driven immunoassays on a single chip with a specific-to-nonspecific signal ratios of 4.2:1 and 3.0:1, respectively. Furthermore, by integrating the microlaboratory with a module for strand displacement amplification (SDA), the identification of the Shiga-like toxin gene (SLT1) from E. coli was accomplished within 2.5 h starting from a dielectrophoretic concentration of intact E. coli bacteria and finishing with an electric-field-driven DNA hybridization assay, detected by fluorescently labeled DNA reporter probes. The integrated microlaboratory can be potentially used in a wide range of applications including detection of bacteria and biowarfare agents, and genetic identification. 相似文献