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111.
Protein-tyrosine dephosphorylation is a major mechanism in cellular regulation. A large number of protein-tyrosine phosphatases
is known from Eukarya, and more recently bacterial homologues have also been identified. By employing conserved sequence patterns
from both eukaryotic and bacterial protein-tyrosine phosphatases, we have identified three homologous sequences in two of
the four complete archaeal genomes. Two hypothetical open reading frames in the genome of Methanococcus jannaschii (MJ0215 and MJECL20) and one in the genome of Pyrococcus horikoshii (PH1732) clearly bear all the conserved residues of this family. No homologues were found in the genomes of Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum. This is the first report of protein-tyrosine phosphatase sequences in Archaea.
Received: 29 April 1998 / Accepted: 27 November 1998 相似文献
112.
Kallinteri P Fatouros D Klepetsanis P Antimisiaris SG 《Journal of liposome research》2004,14(1-2):27-38
The use of arsenic-containing compounds in cancer therapy is currently being re-considered, after the recent approval of arsenic trioxide (Trisenox) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy-dispersive X-ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37 degrees C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO-encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature. 相似文献
113.
Soergel DG Georgakopoulos D Stull LB Kass DA Murphy AM 《American journal of physiology. Heart and circulatory physiology》2004,286(5):H1785-H1792
Myocardial stunning is a form of acute reversible cardiac dysfunction that occurs after brief periods of ischemia and reperfusion. In several animal models, stunning is associated with proteolytic truncation of troponin I (TnI). Mice expressing the same proteolytic TnI fragment [TnI-(1-193)] demonstrate cardiac depression with a decreased maximal calcium-activated tension. We therefore hypothesized preferential improvement in mice expressing TnI-(1-193) treated with the calcium-sensitizing drug EMD-57033. TnI-(1-193) and nontransgenic myofibrils exhibited significant sensitization to calcium in Mg-ATPase assays after EMD-57033 exposure. However, only transgenic myofibrils exhibited an increase in maximal activity (P = 0.023). EMD-57033 also increased maximal calcium-activated force in TnI-(1-193) muscle, such that it was comparable to nontransgenic cardiac muscle. EMD-57033 enhanced in vivo systolic function modestly in controls but had a marked effect in transgenic mice, with an almost threefold greater leftward shift of the end-systolic pressure-volume relation (P = 0.0005). These data indicate a targeted efficacy of EMD-57033 in offsetting the contractile defect in TnI-(1-193) mice, and this may have therapeutic implications in models displaying this myofilament defect. 相似文献
114.
Etiopathogenesis of mucosal inflammation in inflammatory bowel disease remains a complex and enigmatic field; various factors (genetic, environmental and microbial) trigger an event that activates intestinal immune and nonimmune systems culminating in inflammation and tissue injury. Specifically, both innate and adaptive immune systems seem to play important roles in the pathophysiology of this disease. Cyclosporine A represents a macrolide immune modulator with primary inhibitory effects on T helper lymphocyte production of interleukin-2, and other cytokines leading to altered T-lymphocyte and B-lymphocyte function. The diversity of its therapeutic outcome reported in inflammatory bowel disease may be due to the intricate immuno-pathogenic profile of the disease and the variety of the applied dose-dependent courses of therapy. Cyclosporine A exerts additional actions on other components of the inflammatory infiltrate, including neutrophils and mast cells, thereby appearing to be a multi-dynamic therapeutic approach, although with potential drawbacks, that may be applied alone or combined with other immunomodulatory agents in inflammatory bowel disease patients. Because cyclosporine A induces apoptosis of T-lymphocytes responsible for perpetuation of the chronic inflammatory process in the disease with potential tumorigenic effect, it may exert a further inhibitory effect on cancer development in inflammatory bowel disease patients, and can be combined with other relative agents, such as rapamycin, which also promotes T-lymphocyte apoptosis. Therefore, recently established multifactorial action of cyclosporine A in relation to the pathogenesis of the disease can open new horizons for prospective, controlled trials in large cohorts, aiming to emphasize cyclosporine A's potential. 相似文献
115.
Identification and structure of a putative Ca2+-binding domain at the C terminus of AQP1 总被引:2,自引:0,他引:2
Fotiadis D Suda K Tittmann P Jenö P Philippsen A Müller DJ Gross H Engel A 《Journal of molecular biology》2002,318(5):1381-1394
Aquaporin-1 (AQP1) is the first functionally identified aquaporin of a growing family of membrane water channels found in all forms of life. Recently, a possible secondary function as a cyclic guanosine monophosphate (cGMP) gated ion channel was attributed to AQP1. We have reconstituted purified protein from bovine and human red blood cell membranes into highly ordered 2D crystals. The topography of both AQP1s was determined by electron microscopy from freeze-dried, unidirectionally metal-shadowed 2D crystals as well as from surface topographs of native crystals recorded in buffer solution with the atomic force microscope (AFM). In spite of the high level of sequence homology between bovine and human AQP1, the surfaces showed distinct differences. Alignment of both sequences and comparison of the acquired surface topographies with the atomic model of human AQP1 revealed the topographic changes on the surface of bovine AQP1 to be induced by a few amino acid substitutions. A striking degree of sequence homology was found between the carboxyl-terminal domains of AQP1s from different organisms and EF-hands from Ca2+-binding proteins belonging to the calmodulin superfamily, suggesting the existence of a Ca2+-binding site at the C terminus of AQP1 instead of the putative cGMP-binding site reported previously. To unveil its position on the acquired surface topographies, 2D crystals of AQP1 were digested with carboxypeptidase Y, which cleaves off the intracellular C terminus. Difference maps of AFM topographs between the native and the peptidase-treated AQP1s showed the carboxylic tail to be close to the 4-fold symmetry axis of the tetramer. SDS-PAGE and matrix-assisted laser desorption/ionisation mass spectrometry of native and decarboxylated bovine and human AQP1 revealed that the EF-hand motif found at the C terminus of AQP1 was partially resistant to peptidase digestion. The importance of the C-terminal domain is implicated by structural instability of decarboxylated AQP1. A possible role of the C terminus and calcium in translocation of AQP1 in cholangiocytes from intracellular vesicles to the plasma membrane and in triggering its fusion is discussed. Functional studies are now required to identify the physiological role of the Ca2+-binding site. 相似文献
116.
The synergistic interaction between xanthan and glucomannan in solution and in the gel phase has been studied by circular dichroism spectroscopy and differential scanning calorimetry. The study in solution of the polysaccharidic mixture indicates a preferred stoichiometry of the interaction corresponding to a weight fraction of xanthan around 0.55. This finding is in reasonable agreement with the differential scanning calorimetry measurements carried out on the gel phase. Models from conformational analysis based on these results were formulated in terms of 1:1 and 2:1 Konjac glucomannan/xanthan molecular assemblies. The experimental and calculation results clearly indicate the involvement of the side chains of xanthan and suggest that the ordered portions of the macromolecular complex in solution act in the gel phase as junction zones. 相似文献
117.
Barbouti A Doulias PT Nousis L Tenopoulou M Galaris D 《Free radical biology & medicine》2002,33(5):691-702
Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H(2)O(2) by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H(2)O(2) was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 microM H(2)O(2). As the H(2)O(2) was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 microl growth medium (containing 1.5 x 10(5) cells) generated 2.0 +/- 0.2 microM H(2)O(2) per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H(2)O(2) were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H(2)O(2) itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H(2)O(2), it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis. 相似文献
118.
MOTIVATION: This paper studies the problem of discovering subsequences, known as motifs, that are common to a given collection of related biosequences, by proposing a greedy algorithm for learning a mixture of motifs model through likelihood maximization. The approach adds sequentially a new motif to a mixture model by performing a combined scheme of global and local search for appropriately initializing its parameters. In addition, a hierarchical partitioning scheme based on kd-trees is presented for partitioning the input dataset in order to speed-up the global searching procedure. The proposed method compares favorably over the well-known MEME approach and treats successfully several drawbacks of MEME. RESULTS: Experimental results indicate that the algorithm is advantageous in identifying larger groups of motifs characteristic of biological families with significant conservation. In addition, it offers better diagnostic capabilities by building more powerful statistical motif-models with improved classification accuracy. 相似文献
119.
Tsikas D Schubert B Gutzki FM Sandmann J Frölich JC 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,798(1):87-99
Asymmetric dimethylarginine (ADMA; N(G),N(G)-dimethyl-L-arginine) is the most important endogenous inhibitor of nitric oxide synthase and a potential risk factor for cardiovascular diseases. This article describes a gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate quantification of ADMA in human plasma or serum and urine using de novo synthesized [2H(3)]-methyl ester ADMA (d(3)Me-ADMA) as the internal standard. Aliquots (100 microl) of plasma/serum ultrafiltrate or native urine and of aqueous solutions of synthetic ADMA (1 microM for plasma and serum; 20 microM for urine) are evaporated to dryness. The residue from plasma/serum ultrafiltrate or urine is treated with a 100 microl aliquot of 2M HCl in methanol, whereas the residue of the ADMA solution is treated with a 100 microl aliquot of 2M HCl in tetradeuterated methanol. Methyl esters are prepared by heating for 60 min at 80 degrees C. After cooling to room temperature, the plasma or urine sample is combined with the d(3)Me-ADMA sample, the mixture is evaporated to dryness, the residue treated with a solution of pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v) and the sample is incubated for 30 min at 65 degrees C. Solvent and reagents are evaporated under a stream of nitrogen gas, the residue is treated with a 200 microl aliquot of 0.4M borate buffer, pH 8.5, and toluene (0.2 ml for plasma, 1 ml for urine). Reaction products are extracted by vortexing for 1 min, the toluene phase is decanted, and a 1 microl aliquot is injected into the GC-tandem MS instrument. Quantitation is performed by selected reaction monitoring (SRM) of the common product ion at m/z 378 which is produced by collision-induced dissociation of the ions at m/z 634 for endogenous ADMA and m/z 637 for d(3)Me-ADMA. In plasma and urine of healthy humans ADMA was measured at concentrations of 0.39+/-0.06 microM (n=12) and 3.4+/-1.1 micromol/mmol creatinine (n=9), respectively. The limits of detection and quantitation of the method are approximately 10 amol and 320 pM of d(3)Me-ADMA, respectively. 相似文献
120.
Tsikas D Schwedhelm E Stutzer FK Gutzki FM Rode I Mehls C Frölich JC 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,784(1):77-90
Measurement of 3-nitro-L-tyrosine (NO(2)Tyr) and protein-related 3-nitro-L-tyrosine in human plasma is associated with numerous methodological problems which result in highly divergent basal plasma levels often ranging within two orders of magnitude. Recently, we have described an interference-free GC-tandem MS-based method for NO(2)Tyr which yielded the lowest basal plasma NO(2)Tyr levels reported thus far. This method was extended to quantify protein-associated 3-nitrotyrosine and in particular 3-nitrotyrosinated albumin (NO(2)TyrALB) in human plasma. NO(2)TyrALB and albumin (ALB) were extracted from plasma by affinity column extraction and digested enzymatically at neutral pH. 3-Nitro- L-[2H(3)]tyrosine was used as internal standard. In plasma of 18 healthy young volunteers the molar ratio of NO(2)TyrALB to albumin-derived tyrosine (TyrALB), i.e. NO(2)TyrALB/TyrALB, was determined to be 1.55+/-0.54x1:10(6) (mean+/-SD). The plasma concentration of NO(2)TyrALB was estimated as 24+/-4 nM. The NO(2)Tyr plasma levels in these volunteers were determined to be 0.73+/-0.53 nM. In the same volunteers, NO(2)TyrALB/TyrALB, NO(2)TyrALB and NO(2)Tyr were measured 15 days later and the corresponding values were determined to be 1.25+/-0.58x1:10(6), 25+/-6 nM and 0.69+/-0.16 nM. For comparison, NO(2)Tyr and NO(2)TyrALB were measured in six plasma samples from healthy volunteers by GC-MS and GC-tandem MS. Different values were found for NO(2)Tyr, i.e. 5.4+/-2.8 versus 2.7+/-1.5 nM, and comparable values for NO(2)TyrALB/TyrALB, i.e. 0.5+/-0.2x1:10(6) versus 0.4+/-0.1x1:10(6), by these methods. The ratio of the values measured by GC-MS to those measured by GC-tandem MS were 2.9+/-3.1 for NO(2)Tyr and 1.2+/-0.2 for NO(2)TyrALB/TyrALB. The present GC-tandem MS method provides accurate values of NO(2)Tyr and NO(2)TyrALB in human plasma. 相似文献