全文获取类型
收费全文 | 2478篇 |
免费 | 246篇 |
国内免费 | 1篇 |
专业分类
2725篇 |
出版年
2022年 | 15篇 |
2021年 | 26篇 |
2020年 | 20篇 |
2019年 | 24篇 |
2018年 | 24篇 |
2017年 | 27篇 |
2016年 | 55篇 |
2015年 | 84篇 |
2014年 | 94篇 |
2013年 | 127篇 |
2012年 | 151篇 |
2011年 | 169篇 |
2010年 | 145篇 |
2009年 | 120篇 |
2008年 | 125篇 |
2007年 | 158篇 |
2006年 | 125篇 |
2005年 | 141篇 |
2004年 | 124篇 |
2003年 | 116篇 |
2002年 | 144篇 |
2001年 | 35篇 |
2000年 | 41篇 |
1999年 | 55篇 |
1998年 | 50篇 |
1997年 | 33篇 |
1996年 | 32篇 |
1995年 | 23篇 |
1994年 | 27篇 |
1993年 | 23篇 |
1992年 | 35篇 |
1991年 | 19篇 |
1990年 | 12篇 |
1989年 | 21篇 |
1988年 | 19篇 |
1987年 | 19篇 |
1986年 | 18篇 |
1985年 | 20篇 |
1984年 | 18篇 |
1983年 | 14篇 |
1982年 | 15篇 |
1981年 | 20篇 |
1980年 | 15篇 |
1977年 | 11篇 |
1976年 | 9篇 |
1975年 | 9篇 |
1974年 | 12篇 |
1973年 | 11篇 |
1971年 | 11篇 |
1967年 | 8篇 |
排序方式: 共有2725条查询结果,搜索用时 15 毫秒
911.
Two crystal structures of the C123S mutant of 2-methylisocitrate lyase have been determined, one with the bound reaction products, Mg(2+)-pyruvate and succinate, and the second with a bound Mg(2+)-(2R,3S)-isocitrate inhibitor. Comparison with the structure of the wild-type enzyme in the unbound state reveals that the enzyme undergoes a conformational transition that sequesters the ligand from solvent, as previously observed for two other enzyme superfamily members, isocitrate lyase and phosphoenolpyruvate mutase. The binding modes reveal the determinants of substrate specificity and stereoselectivity, and the stringent specificity is verified in solution using various potential substrates. A model of bound 2-methylisocitrate has been developed based on the experimentally determined structures. We propose a catalytic mechanism involving an alpha-carboxy-carbanion intermediate/transition state, which is consistent with previous stereochemical experiments showing inversion of configuration at the C(3) of 2-methylisocitrate. Structure-based sequence analysis and phylogenic tree construction reveal determinants of substrate specificity, highlight nodes of divergence of families, and predict enzyme families with new functions. 相似文献
912.
913.
Debra L. Rateri Jessica J. Moorleghen Victoria Knight Anju Balakrishnan Deborah A. Howatt Lisa A. Cassis Alan Daugherty 《PloS one》2012,7(12)
Background
Whole body genetic deletion of AT1a receptors in mice uniformly reduces hypercholesterolemia and angiotensin II-(AngII) induced atherosclerosis and abdominal aortic aneurysms (AAAs). However, the role of AT1a receptor stimulation of principal cell types resident in the arterial wall remains undefined. Therefore, the aim of this study was to determine whether deletion of AT1a receptors in either endothelial cells or smooth muscle cells influences the development of atherosclerosis and AAAs.Methodology/Principal Findings
AT1a receptor floxed mice were developed in an LDL receptor −/− background. To generate endothelial or smooth muscle cell specific deficiency, AT1a receptor floxed mice were bred with mice expressing Cre under the control of either Tie2 or SM22, respectively. Groups of males and females were fed a saturated fat-enriched diet for 3 months to determine effects on atherosclerosis. Deletion of AT1a receptors in either endothelial or smooth muscle cells had no discernible effect on the size of atherosclerotic lesions. We also determined the effect of cell-specific AT1a receptor deficiency on atherosclerosis and AAAs using male mice fed a saturated fat-enriched diet and infused with AngII (1,000 ng/kg/min). Again, deletion of AT1a receptors in either endothelial or smooth muscle cells had no discernible effects on either AngII-induced atherosclerotic lesions or AAAs.Conclusions
Although previous studies have demonstrated whole body AT1a receptor deficiency diminishes atherosclerosis and AAAs, depletion of AT1a receptors in either endothelial or smooth muscle cells did not affect either of these vascular pathologies. 相似文献914.
Joseph Hughes Richard C. Allen Marc Baguelin Katie Hampson Gregory J. Baillie Debra Elton J. Richard Newton Paul Kellam James L. N. Wood Edward C. Holmes Pablo R. Murcia 《PLoS pathogens》2012,8(12)
The ability of influenza A viruses (IAVs) to cross species barriers and evade host immunity is a major public health concern. Studies on the phylodynamics of IAVs across different scales – from the individual to the population – are essential for devising effective measures to predict, prevent or contain influenza emergence. Understanding how IAVs spread and evolve during outbreaks is critical for the management of epidemics. Reconstructing the transmission network during a single outbreak by sampling viral genetic data in time and space can generate insights about these processes. Here, we obtained intra-host viral sequence data from horses infected with equine influenza virus (EIV) to reconstruct the spread of EIV during a large outbreak. To this end, we analyzed within-host viral populations from sequences covering 90% of the infected yards. By combining gene sequence analyses with epidemiological data, we inferred a plausible transmission network, in turn enabling the comparison of transmission patterns during the course of the outbreak and revealing important epidemiological features that were not apparent using either approach alone. The EIV populations displayed high levels of genetic diversity, and in many cases we observed distinct viral populations containing a dominant variant and a number of related minor variants that were transmitted between infectious horses. In addition, we found evidence of frequent mixed infections and loose transmission bottlenecks in these naturally occurring populations. These frequent mixed infections likely influence the size of epidemics. 相似文献
915.
Sodium-1,2-C Acetate Incorporation in Roots of Frost-hardy and Less Hardy Alfalfa Varieties under Hardening Conditions 下载免费PDF全文
When the temperature of incorporation of sodium acetate-1, 2-14C into lipids of alfalfa (Medicago media Pers. var. Rambler and Medicago sativa L. var. Caliverde) roots was lowered from 22 C to 1 C, elongation and desaturation of fatty acids and the labeling of phosphatidylcholine were strongly stimulated. 相似文献
916.
Quansheng Zhu Liyo Kao Rustam Azimov Natalia Abuladze Debra Newman Alexander Pushkin Weixin Liu Connie Chang Ira Kurtz 《The Journal of biological chemistry》2010,285(48):37178-37187
NBCe1-A and AE1 both belong to the SLC4 HCO3− transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala800–Lys967. Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met858 accessible to both biotin maleimide and TAMRA and Thr926–Ala929 only to TAMRA labeling. The intracellular surface contains a highly exposed (Met813–Gly828) region and a cryptic (Met887–Arg904) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp960. Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro868–Leu967 (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1. 相似文献
917.
It is well established that electrostatic interactions play a vital role in enzyme catalysis. In this work, we report theory-guided mutation experiments that identified strong electrostatic contributions of a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase catalysis. The Glu232Asp mutant was found to bind the substrate analogue 4-methylbenzoyl-CoA more tightly than does the wild-type dehalogenase. In contrast, the kcat for 4-chlorobenzoyl-CoA conversion to product was reduced 10000-fold in the mutant. UV difference spectra measured for the respective enzyme-ligand complexes revealed an approximately 3-fold shift in the equilibrium of the two active site conformers away from that inducing strong pi-electron polarization in the ligand benzoyl ring. Increased substrate binding, decreased ring polarization, and decreased catalytic efficiency indicated that the repositioning of the point charge in the Glu232Asp mutant might affect the orientation of the Asp145 carboxylate with respect to the substrate aromatic ring. The time course for formation and reaction of the arylated enzyme intermediate during a single turnover was measured for wild-type and Glu232Asp mutant dehalogenases. The accumulation of arylated enzyme in the wild-type dehalogenase was not observed in the mutant. This indicates that the reduced turnover rate in the mutant is the result of a slow arylation of Asp145, owing to decreased efficiency in substrate nucleophilic attack by Asp145. To rationalize the experimental observations, a theoretical model is proposed, which computes the potential of mean force for the nucleophilic aromatic substitution step using a hybrid quantum mechanical/molecular mechanical method. To this end, the removal or reorientation of the side chain charge of residue 232, modeled respectively by the Glu232Gln and Glu232Asp mutants, is shown to increase the rate-limiting energy barrier. The calculated 23.1 kcal/mol free energy barrier for formation of the Meisenheimer intermediate in the Glu232Asp mutant represents an increase of 6 kcal/mol relative to that of the wild-type enzyme, consistent with the 5.6 kcal/mol increase calculated from the difference in experimentally determined rate constants. On the basis of the combination of the experimental and theoretical evidence, we hypothesize that the Glu232(B) residue contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile. 相似文献
918.
919.
Identification and Partial Characterization of the Pectin
Methyltransferase “Homogalacturonan-Methyltransferase” from
Membranes of Tobacco Cell Suspensions 下载免费PDF全文
A membrane preparation from tobacco (Nicotiana tabacum L.) cells contains at least one enzyme that is capable of transferring the methyl group from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalacturonan present in the membranes. This enzyme is named homogalacturonan-methyltransferase (HGA-MT) to distinguish it from methyltransferases that catalyze methyletherification of the pectic polysaccharides rhamnogalacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipitation assay was used to measure HGA-MT activity, because published procedures to recover pectic polysaccharides via ethanol or chloroform:methanol precipitation lead to high and variable background radioactivity in the product pellet. Attempts to reduce the incorporation of the 14C-methyl group from SAM into pectin by the addition of the alternative methyl donor 5-methyltetrahydrofolate were unsuccessful, supporting the role of SAM as the authentic methyl donor for HGA-MT. The pH optimum for HGA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 μm, and the maximum initial velocity was 0.81 pkat mg−1 protein. At least 59% of the radiolabeled product was judged to be methylesterified homogalacturonan, based on the release of radioactivity from the product after a mild base treatment and via enzymatic hydrolysis by a purified pectin methylesterase. The released radioactivity eluted with a retention time identical to that of methanol upon fractionation over an organic acid column. Cleavage of the radiolabeled product by endopolygalacturonase into fragments that migrated as small oligomers of HGA during thin-layer chromatography, and the fact that HGA-MT activity in the membranes is stimulated by uridine 5′-diphosphate galacturonic acid, a substrate for HGA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SAM is methylesterified HGA. 相似文献
920.
Barlow SE Bobra SR Elliott MB Brownson RC Haire-Joshu D 《Obesity (Silver Spring, Md.)》2007,15(1):225-232
Objective: To assess, in diverse pediatric practices, the frequency of overweight/obesity (OW/OB) identification during health supervision visits and its association with BMI curve use. Research Methods and Procedures: Pediatricians in public and private practice in St. Louis, MO, participated in a study of the care of chronic conditions during health supervision visits. Requested information from 30 visits per pediatrician of children 6 to 17 years of age included the visit note, the growth chart, and a one‐page questionnaire about patient demographics and visit content. Pediatricians indicated the presence and discussion of common chronic conditions, including OW/OB. Identification was compared with patient BMI category, and associations between identification and patient and visit characteristics, including BMI curve use, were examined. Results: Twenty‐one (40%) of contacted pediatricians returned information from 557 visits. Pediatricians identified OW/OB in 27% of children with a BMI at the 85th to 94th percentile and 86% of children with a BMI at or above the 95th percentile. Identification was higher in adolescents but was not associated with patient sex or race, practice setting, insurance type, or visit length. Only 41% of growth charts were current, and 6.1% had BMI plotted. BMI plotting was associated with OW/OB identification when the BMI was at the 85th to 94th percentile but not when the BMI was at or above the 95th percentile. After controlling for BMI percentile, OW/OB identification was significantly associated with diet counseling (odds ratio, 7.46; 95% confidence interval, 3.42 to 16.24) and exercise counseling (odds ratio, 5.57; 95% confidence interval, 2.61 to 11.90). Discussion: Despite low BMI curve use, pediatricians recognized most overweight/obese children with a BMI at or above the 95th percentile. BMI plotting may increase recognition in mildly overweight children. 相似文献