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991.
Dragovich PS Blazel JK Ellis DA Han Q Kamran R Kissinger CR LeBrun LA Li LS Murphy DE Noble M Patel RA Ruebsam F Sergeeva MV Shah AM Showalter RE Tran CV Tsan M Webber SE Kirkovsky L Zhou Y 《Bioorganic & medicinal chemistry letters》2008,18(20):5635-5639
The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed. 相似文献
992.
Kuduk SD Chang RK Dipardo RM Di Marco CN Murphy KL Ransom RW Reiss DR Tang C Prueksaritanont T Pettibone DJ Bock MG 《Bioorganic & medicinal chemistry letters》2008,18(18):5107-5110
A series of carbo- and heterocyclic alpha-hydroxy amide-derived bradykinin B1 antagonists was prepared and evaluated. A 4,4-difluorocyclohexyl alpha-hydroxy amide was incorporated along with a 2-methyl tetrazole in lieu of an oxadiazole to afford a suitable compound with good pharmacokinetic properties, CNS penetration, and clearance by multiple metabolic pathways. 相似文献
993.
A systematic review of nitric oxide donors and L-arginine in experimental stroke; effects on infarct size and cerebral blood flow. 总被引:1,自引:0,他引:1
BACKGROUND: Nitric oxide (NO) is a candidate treatment for acute ischaemic stroke, however published studies in experimental stroke have given conflicting results. METHODS: We performed a systematic review of published controlled studies of L-arginine (the precursor for NO) and NO donors in experimental stroke. Data were analysed using the Cochrane Collaboration Review Manager software. Standardised mean difference (SMD) and 95% confidence intervals (95% CI) were calculated. RESULTS: Altogether, 25 studies(s) were identified. L-Arginine and NO donors reduced total cerebral infarct volume in permanent (SMD -1.21, 95% CI -1.69 to -0.73, p < 0.01, s = 10) and transient models of ischaemia (SMD -0.78, 95% CI -1.21 to -0.35, p < 0.01, s = 7). Drug administration increased cortical CBF in permanent (SMD +0.86, 95% CI 0.52-1.21, p < 0.01, s = 8) but not transient models (SMD +0.34, 95% CI -0.02 to 0.70, p = 0.07, s = 4). CONCLUSIONS: Administration of NO in experimental stroke reduces stroke lesion volume in permanent and transient models. This may be mediated, in part, by increased cerebral perfusion in permanent models. These data support clinical trials in stroke patients, although the presence of a narrow therapeutic time window may be a limiting factor. 相似文献
994.
995.
Lionel Guittat Patrizia Alberti Dennis Gomez Anne De Cian Gaëlle Pennarun Thibault Lemarteleur Chafke Belmokhtar Rajaa Paterski Hamid Morjani Chantal Trentesaux Eliane Mandine François Boussin Patrick Mailliet Laurent Lacroix Jean-François Riou Jean-Louis Mergny 《Cytotechnology》2004,45(1-2):75-90
The enzyme telomerase is involved in the replication of telomeres, specialized structures that cap and protect the ends of chromosomes. Its activity is required for maintenance of telomeres and for unlimited lifespan, a hallmark of cancer cells. Telomerase is overexpressed in the vast majority of human cancer cells and therefore represents an attractive target for therapy. Several approaches have been developed to inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 3′-telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small diffusable molecules, both natural and synthetic. This review presents an update of recent investigations pertaining to these agents and discusses their biological properties in the context of the initial paradigm that the exposure of cancer cells to these agents should lead to progressive telomere shortening followed by a delayed growth arrest response. 相似文献
996.
de Lambert B Chaix C Charreyre MT Martin T Aigoui A Perrin-Rubens A Pichot C Mandrand B 《Analytical biochemistry》2008,373(2):229-238
Polymer-oligonucleotide conjugates were synthesized from the amphiphilic block copolymer poly(tert-butylacrylamide-b-(N-acryloylmorpholine-co-N-acryloxysuccinimide)) using an original solid-phase DNA synthesis strategy. This method provided conjugates highly functionalized with oligonucleotides throughout the polymer chain. After purification, block copolymer-oligonucleotide conjugates were spotted on a multidetection microarray system developed by Apibio using a standard nanodroplet piezo inkjet spotting technique to develop the oligosorbent assay (OLISA). Two genotyping models (HLA-DQB1 and platelet glycoproteins [GPs]), which are particularly difficult to study with standard systems, were evaluated. For both models, block copolymer-oligonucleotide conjugates used as capture probes amplified the responses of in vitro diagnostic assays. The detection limit reached by using conjugates was estimated at 15 pM for a 219-bp DNA target (HLA-DQB1 model). Moreover, single nucleotide polymorphism was detected in the platelet GPs genotyping model. The use of polymer conjugates led to a significant improvement in both sensitivity and specificity of standard hybridization assays even when applied to complex biological models. 相似文献
997.
Rauh-Adelmann C Moskow JM Graham JR Yen LG Boucher JI Murphy CE Nadler TK Gordon NF Radding JA 《Analytical biochemistry》2008,375(2):255-264
Aberrant epidermal growth factor receptor (EGFR, ErbB1) signaling is implicated in cell transformation, motility, and invasion in a variety of cell types, and EGFR is the target of several anticancer drugs. However, the kinetics of EGFR signaling and the individual contributions of site-specific phosphorylation events remain largely unknown. A peptide-based, multiplex immunoassay approach was developed to simultaneously measure both total and phosphorylated protein in a single sample. The approach involves the proteolytic digestion of proteins prior to the isolation and quantitation of site-specific phosphorylation events within an individual protein. Quantitation of phosphorylated and total proteins, in picomolar to nanomolar concentrations, were interpolated from standard curves generated with synthetic peptides that correspond to the peptide targets used in the immunoassays. In this study, a bead-based, nine-plex immunoassay measuring total and phosphorylated protein was constructed to measure temporal, site-specific phosphorylation of key members of the EGFR pathway (ErbB1 receptor, MEK1, MEK2, ERK1, and ERK2) in A431 cells stimulated with epidermal growth factor. The effect of MEK inhibition on this pathway was determined using a known MEK kinase inhibitor, SL327. The results reported herein are the first quantitative measurements of site-specific phosphorylation events and total proteins in a single sample, at the same time representing a new paradigm for standardized protein and phosphorylation analysis using multiplexed, peptide-based, sandwich immunoassays. 相似文献
998.
We cloned, expressed, and purified a chimeric fusion between a soluble green fluorescent protein (smGFP) and the calmodulin binding protein calspermin. We have shown that the fusion protein, labeled smGN, has a K(i) in the calmodulin-dependent cyclic nucleotide phosphodiesterase activity assay of 1.97 nM, i.e., 3800 times smaller than that of the commonly used calmodulin inhibitor W7. Association and dissociation rate constants (k(a) and k(d)) and the dissociation equilibrium constant (K(D)) of smGN for calmodulin were determined using surface plasmon resonance (SPR). The k(a)=1.24 x 10(6)M(-1)s(-1), the k(d)=5.49 x 10(-3)s(-1), and the K(D)=4.42 x 10(-9)M. We also found that the GFP moiety was important for successfully binding calspermin to the surface of the CM5 flow cell at a sufficiently high concentration for SPR, and that this procedure may be used for SPR analysis of other acidic polypeptides, whose pI< or =4. To determine whether smGN might also bind to other calmodulin-like proteins in a heterologous system, we purified proteins from a plant total cell extract or a plant total protein extract by affinity chromatography against smGN. The purified proteins were identified as calmodulins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry, indicating a high level of specificity. We conclude that the high affinity and specific binding between smGN and calmodulin make it an easily localized recombinant alternative to chemical calmodulin inhibitors. 相似文献
999.
Ramarao MK Shen MW Murphy EA Duan W Zhao Y McKew J Lee KL Thakker P Behnke ML Clark JD 《Analytical biochemistry》2008,383(2):217-225
Cytosolic phospholipase A2 alpha (cPLA2α, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA2α inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA2α and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA2α inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 °C and pH 7.4. The compounds tested bound cPLA2α with a 1:1 stoichiometry, and the dissociation constant Kd of the inhibitors calculated from the ITC experiments correlated well with the IC50 values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA2α was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA2α. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA2α was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics. 相似文献
1000.
Condensin is a 5 subunit complex that plays an important role in the structure of chromosomes during mitosis. It is known that phosphorylation of condensin subunits by cdc2/cyclin B at the beginning of mitosis is important for condensin activity, but the sites of these phosphorylation events have not been identified nor has their role in regulating condensin function. Here we identify two threonine residues in the CAP-G subunit of condensin, threonines 308 and 332, that are targets of cdc2/cyclin B phosphorylation. Mutation of these threonines to alanines results in defects in CAP-G localization with chromosomes during mitosis. These results are the first to identify phosphorylation sites within the condensin complex that regulate condensin localization with chromosomal DNA. 相似文献