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991.
Predominance of HLA-Restricted Cytotoxic T-Lymphocyte Responses to Serotype-Cross-Reactive Epitopes on Nonstructural Proteins following Natural Secondary Dengue Virus Infection 总被引:5,自引:1,他引:4 下载免费PDF全文
Anuja Mathew Ichiro Kurane Sharone Green Henry A. F. Stephens David W. Vaughn Siripen Kalayanarooj Saroj Suntayakorn Dasnayanee Chandanayingyong Francis A. Ennis Alan L. Rothman 《Journal of virology》1998,72(5):3999-4004
We examined the memory cytotoxic T-lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue virus infection. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue virus proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for NS1.2a (NS1 and NS2a collectively) and NS3 proteins, respectively. All CTL lines derived from both patients were cross-reactive with other serotypes of dengue virus. The CD8+ NS1.2a-specific lines from patient KPP94-037 were HLA B57 restricted, and the CD8+ NS3-specific lines from patient KPP94-024 were HLA B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA DR7 restricted. A majority of the CD8+ CTLs isolated from patient KPP94-024 were found to recognize amino acids 221 to 232 on NS3. These results demonstrate that in Thai patients after symptomatic secondary natural dengue infections, CTLs are mainly directed against nonstructural proteins and are broadly cross-reactive. 相似文献
992.
Minimal Requirement for a Lentivirus Vector Based on Human Immunodeficiency Virus Type 1 总被引:8,自引:1,他引:7 下载免费PDF全文
V. Narry Kim Kyriacos Mitrophanous Susan M. Kingsman Alan J. Kingsman 《Journal of virology》1998,72(1):811-816
The use of human immunodeficiency virus vectors for gene therapy is hampered by concern over their safety. This concern might be ameliorated, in part, if the viral accessory genes and proteins could be eliminated from the vector genomes and particles. Here we describe a minimal vector system that is capable of transducing nondividing cells and which does not contain tat, vif, vpr, vpu, and nef. 相似文献
993.
Role of Potassium Channels in Amyloid-Induced Cell Death 总被引:20,自引:1,他引:19
Luis V. Colom Maria E. Diaz David R. Beers Alan Neely Wen-jie Xie Stanley H. Appel 《Journal of neurochemistry》1998,70(5):1925-1934
Abstract: Basal forebrain cholinergic neurons are severely depleted early in Alzheimer's disease and appear particularly susceptible to amyloid β-peptide (Aβ) toxicity in vivo. To model this effect in vitro, a cholinergic septal cell line (SN56) was exposed to Aβ. SN56 cells exhibited a tetraethylammonium (TEA)-sensitive outward K+ current with delayed rectifier characteristics. Increases of 64% (±19; p < 0.02) and 44% (±12; p < 0.02) in K+ current density were noted 6–12 and 12–18 h following the addition of Aβ to SN56 cell cultures, respectively. Morphological observation and staining for cell viability showed that 25 ± 4 and 39 ± 4% of SN56 cells were dead after 48- and 96-h exposures to Aβ, respectively. Perfusion of SN56 cells with 10–20 mM TEA blocked 71 ± 6 to 92 ± 2% of the outward currents, widened action potentials, elevated [Ca2+]i, and inhibited 89 ± 14 and 68 ± 14% of the Aβ toxicity. High [K+]o, which depolarizes cell membranes and increases [Ca2+]i, also protected SN56 cells from Aβ toxicity. This effect appeared specific since glucose deprivation of SN56 cells did not alter K+ current density and TEA did not protect these cells from hypoglycemic cell death. Furthermore, Aβ was toxic to a dopaminergic cell line (MES23.5) that expressed a K+ current with delayed rectifier characteristics; K+ current density was not altered by Aβ and MES23.5 cells were not protected by TEA from Aβ toxicity. In contrast, a noncholinergic septal cell line (SN48) that shows minimal outward K+ currents was resistant to the toxicity of Aβ. These data suggest that a K+ channel with delayed rectifier characteristics may play an important role in Aβ-mediated toxicity for septal cholinergic cells. 相似文献
994.
Structural Requirements for Ligand Interactions at the Benzodiazepine Recognition Site of the GABAA Receptor 总被引:2,自引:2,他引:0
Abstract: His101 of the GABAA receptor α1 subunit is an important determinant of benzodiazepine recognition and a major site of photolabeling by [3 H]flunitrazepam. To investigate further the chemical specificity of the residue in this position, we substituted it with phenylalanine, tyrosine, lysine, glutamate, glutamine, or cysteine. The mutant α subunits were coexpressed with the rat β2 and γ2 subunits in TSA201 cells, and the effects of the substitutions on the binding of benzodiazepine site ligands were examined. [3 H]Ro 15-4513 bound to all mutant receptors with equal or greater affinity than to the wild-type receptor. However, flunitrazepam and ZK93423 recognition was adversely affected by substitutions of the amino acid in this position. The binding of the antagonists, Ro 15-1788 and ZK93426, was also sensitive to the mutations, with the largest decreases in affinity occurring with the tyrosine, lysine, and glutamate substitutions. In all mutants that recognized flunitrazepam, GABA potentiated the binding of this ligand to a similar extent, suggesting that it is a full agonist at these receptors. The effects of GABA on the binding of Ro 15-1788 and Ro 15-4513 suggest that their efficacies may have been changed by some of the substitutions. This study further emphasizes the importance of the residue at position 101 in both ligand recognition and pharmacological effect. 相似文献
995.
In this study bovine pulmonary artery endothelial cells (BPAEC) were used as a model system to investigate the effects of the hypoxanthine–xanthine oxidase (HXXO) oxygen radical donor system on ET-1 secretion into pulmonary vasculature. Incubation of BPAEC with HXXO for 4 h caused a significant reduction in ET-1 secretion, which was significantly offset by allopurinol or catalase, but not by Cu/Zn superoxide dismutase (SOD). ET-1 secretion was also reduced by H2O2, and this effect was again significantly offset by catalase. XO alone also reduced ET-1 secretion, but to a significantly lesser degree than did HXXO, and this effect was not offset by allopurinol, catalase, or SOD. None of the oxidant treatments were associated with a loss of immunoreactive ET-1 from endothelial cell medium containing synthetic peptide. The HXXO- and H2O2-mediated reductions in ET-1 secretion were accompanied by evidence of reduced cell viability. This loss of viability was absent when cells were treated with HXXO + catalase, allopurinol, or mercaptopropionyl glycine, but not when SOD was present. We conclude that under conditions of oxidative stress, the pulmonary vascular endothelium responds by secreting less ET-1. This may be relevant to its vasodilator functions in the pulmonary vasculature, which would therefore be compromised when the endothelium is exposed to oxidant stress. 相似文献
996.
Alexander F. Arendsen Jonathan Hadden Graeme Card Alan S. McAlpine Susan Bailey Vjacheslav Zaitsev Elizabeth H. M. Duke P. F. Lindley Monika Kröckel Alfred X. Trautwein Martinus C. Feiters John M. Charnock C. David Garner Sophie J. Marritt Andrew J. Thomson Ingeborg M. Kooter Michael K. Johnson Willy A. M. van den Berg Walter M. A. M. van Dongen W. R. Hagen 《Journal of biological inorganic chemistry》1998,3(1):81-95
The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ2-sulfido bridges, two μ2-oxo bridges, and a partially occupied, unidentified μ2 bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]1+ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all FeIII to three FeII plus one FeIII. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen. 相似文献
997.
998.
dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics 总被引:19,自引:9,他引:10
Michael M. Neff Joseph D. Neff Joanne Chory Alan E. Pepper 《The Plant journal : for cell and molecular biology》1998,14(3):387-392
PCR-based detection of single nucleotide polymorphisms is a powerful tool for the plant geneticist. Cleaved amplified polymorphic sequence analysis is the most widely used approach for the detection of single nucleotide polymorphisms. However, this technique is limited to mutations which create or disrupt a restriction enzyme recognition site. This paper presents a modification of this technique where mismatches in a PCR primer are used to create a polymorphism based on the target mutation. This technique is useful for following known mutations in segregating populations and genetic mapping of isolated DNAs used for positional based cloning of new genes. In addition, a computer program has been developed that facilitates the design of these PCR primers. 相似文献
999.
The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intra-cellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.© Kluwer Academic Publishers 相似文献
1000.
Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation 总被引:1,自引:0,他引:1
Mullen Jeffrey Adam Gerhard Blowers Alan Earle Elizabeth 《Molecular breeding : new strategies in plant improvement》1998,4(5):449-457
To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resistant to kanamycin (300 mg/L) were recovered for each size of YAC tested. Two of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also contained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert region of the 80 kb YAC confirmed that one of the two double-resistant lines had integrated a fully intact single copy of the YAC DNA while the other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorporated relatively small portions of the YAC insert DNA distal to the selectable marker. Our data suggest genomic DNA cloned in artificial chromosomes up to 150 kb in size have a reasonable likelihood of being transferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolation of agronomically useful plant genes using map-based cloning strategies. 相似文献