全文获取类型
收费全文 | 3759篇 |
免费 | 278篇 |
国内免费 | 339篇 |
出版年
2024年 | 9篇 |
2023年 | 52篇 |
2022年 | 125篇 |
2021年 | 203篇 |
2020年 | 126篇 |
2019年 | 153篇 |
2018年 | 153篇 |
2017年 | 100篇 |
2016年 | 150篇 |
2015年 | 243篇 |
2014年 | 246篇 |
2013年 | 307篇 |
2012年 | 350篇 |
2011年 | 283篇 |
2010年 | 162篇 |
2009年 | 190篇 |
2008年 | 231篇 |
2007年 | 202篇 |
2006年 | 170篇 |
2005年 | 132篇 |
2004年 | 121篇 |
2003年 | 109篇 |
2002年 | 94篇 |
2001年 | 68篇 |
2000年 | 65篇 |
1999年 | 67篇 |
1998年 | 31篇 |
1997年 | 41篇 |
1996年 | 19篇 |
1995年 | 26篇 |
1994年 | 17篇 |
1993年 | 18篇 |
1992年 | 19篇 |
1991年 | 10篇 |
1990年 | 18篇 |
1989年 | 11篇 |
1988年 | 16篇 |
1987年 | 7篇 |
1986年 | 8篇 |
1985年 | 7篇 |
1984年 | 5篇 |
1983年 | 3篇 |
1982年 | 2篇 |
1981年 | 2篇 |
1979年 | 1篇 |
1974年 | 1篇 |
1969年 | 1篇 |
1949年 | 1篇 |
1948年 | 1篇 |
排序方式: 共有4376条查询结果,搜索用时 46 毫秒
991.
Selection on phenotypes may cause genetic change. To understand the relationship between phenotype and gene expression from an evolutionary viewpoint, it is important to study the concordance between gene expression and profiles of phenotypes. In this study, we use a novel method of clustering to identify genes whose expression profiles are related to a quantitative phenotype. Cluster analysis of gene expression data aims at classifying genes into several different groups based on the similarity of their expression profiles across multiple conditions. The hope is that genes that are classified into the same clusters may share underlying regulatory elements or may be a part of the same metabolic pathways. Current methods for examining the association between phenotype and gene expression are limited to linear association measured by the correlation between individual gene expression values and phenotype. Genes may be associated with the phenotype in a nonlinear fashion. In addition, groups of genes that share a particular pattern in their relationship to phenotype may be of evolutionary interest. In this study, we develop a method to group genes based on orthogonal polynomials under a multivariate Gaussian mixture model. The effect of each expressed gene on the phenotype is partitioned into a cluster mean and a random deviation from the mean. Genes can also be clustered based on a time series. Parameters are estimated using the expectation-maximization algorithm and implemented in SAS. The method is verified with simulated data and demonstrated with experimental data from 2 studies, one clusters with respect to severity of disease in Alzheimer's patients and another clusters data for a rat fracture healing study over time. We find significant evidence of nonlinear associations in both studies and successfully describe these patterns with our method. We give detailed instructions and provide a working program that allows others to directly implement this method in their own analyses. 相似文献
992.
Liu L Li J Liu J Yuan Z Pierre SV Qu W Zhao X Xie Z 《Free radical biology & medicine》2006,41(10):1548-1556
We have shown that increased production of reactive oxygen species (ROS) was required for ouabain-induced hypertrophy in cultured cardiac myocytes. In the present study we assessed whether long-term exposure of myocytes to nontoxic ROS stress alone is sufficient to induce hypertrophy. A moderate amount of H2O2 was continuously generated in culture media by glucose oxidase. This resulted in a steady increase in intracellular ROS in cultured cardiac myocytes for at least 12 h. Such sustained, but not transient, increase in intracellular ROS at a level comparable to that induced by ouabain was sufficient to stimulate protein synthesis, increase cell size, and change the expression of several hypertrophic marker genes. Like ouabain, glucose oxidase increased intracellular Ca2+ and activated extracellular signal-regulated kinases 1 and 2 (ERK1/2). These effects of glucose oxidase were additive to ouabain-induced cellular changes. Furthermore, glucose oxidase stimulated endocytosis of the plasma membrane Na+/K+-ATPase, resulting in significant inhibition of sodium pump activity. While inhibition of ERK1/2 abolished glucose oxidase-induced increases in protein synthesis, chelating intracellular Ca2+ by BAPTA-AM showed no effect. These results, taken together with our prior observations, suggest that ROS may cross talk with Na+/K+-ATPase, leading to the activation of hypertrophic pathways in cardiac myocytes. 相似文献
993.
Neuronal tau, through its proline-rich domain and the microtubule binding domain, binds to RNA non-sequence-specifically via electrostatic interaction. This binding inhibits the activity of tau. Tau and RNA were also found to co-localize in SH-SY5Y cells suggesting that RNA has opportunities to interact with tau in cells. 相似文献
994.
Provins L Christophe B Danhaive P Dulieu J Durieu V Gillard M Lebon F Lengelé S Quéré L van Keulen B 《Bioorganic & medicinal chemistry letters》2006,16(7):1834-1839
SAR around 4,6-diaminopyrimidine derivatives allowed the discovery of the first potent dual M(3) antagonists and PDE4 inhibitors. Various chemical modulations around that scaffold led to the discovery of ucb-101333-3 which is characterized by the most interesting profile on both targets. 相似文献
995.
研究利用intein的蛋白质反式剪接功能在大肠杆菌中对凝血VIII因子(FVIII)重链和轻链的连接作用,将B结构域大部分缺失型FVIII(BDD-FVIII)于满足剪接所需的保守性氨基酸Ser1657前断裂为重链和轻链,分别与split mini Ssp DnaB intein的106个氨基酸的N端(Int-N)和48个氨基酸的C端(Int-C)融合,构建到原核表达载体pBV220。诱导表达后SDS-PAGE分析可见预期大小的BDD-FVIII蛋白条带,Western blotting用FVIII特异性抗体证明其为剪接所产生的BDD-FVIII蛋白,表明intein可有效连接BDD-FVIII的重链和轻链。为进一步甲型血友病基因治疗研究应用intein以双腺相关病毒载体(AAV)携带FVIII基因,克服单个AAV载体的容量限制提供了依据。 相似文献
996.
997.
Julien Vaubourgeix Fabienne Bardou Fanny Boissier Sylviane Julien Patricia Constant Olivier Ploux Mamadou Daffé Anna?k Quémard Lionel Mourey 《The Journal of biological chemistry》2009,284(29):19321-19330
S-Adenosylmethionine-dependent methyltransferases (AdoMet-MTs) constitute a large family of enzymes specifically transferring a methyl group to a range of biologically active molecules. Mycobacterium tuberculosis produces a set of paralogous AdoMet-MTs responsible for introducing key chemical modifications at defined positions of mycolic acids, which are essential and specific components of the mycobacterial cell envelope. We investigated the inhibition of these mycolic acid methyltransferases (MA-MTs) by structural analogs of the AdoMet cofactor. We found that S-adenosyl-N-decyl-aminoethyl, a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain, inhibited MA-MTs from Mycobacterium smegmatis and M. tuberculosis strains, both in vitro and in vivo, with IC50 values in the submicromolar range. By contrast, S-adenosylhomocysteine, the demethylated reaction product, and sinefungin, a general AdoMet-MT inhibitor, did not inhibit MA-MTs. The interaction between Hma (MmaA4), which is strictly required for the biosynthesis of oxygenated mycolic acids in M. tuberculosis, and the three cofactor analogs was investigated by x-ray crystallography. The high resolution crystal structures obtained illustrate the bisubstrate nature of S-adenosyl-N-decyl-aminoethyl and provide insight into its mode of action in the inhibition of MA-MTs. This study has potential implications for the design of new drugs effective against multidrug-resistant and persistent tubercle bacilli.One-third of the world population is infected with the tubercle bacillus, Mycobacterium tuberculosis, and tuberculosis kills one person every 20 s. The inhaled pathogenic bacilli are taken up by phagocytosis by pulmonary macrophages, which, together with lymphocytes and dendritic cells, form granulomas. The bacilli persist in the granuloma until their reactivation, dissemination into the lungs, and the triggering of disease. The natural resistance of persistent tubercle bacilli to drugs and the emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains are two main concerns in the treatment of the disease. A survey carried out by the Centers for Disease Control and Prevention and the World Health Organization between 2000 and 2004 reported that 20% of 17,690 M. tuberculosis isolates from 49 countries were multidrug-resistant, and 2% were extensively drug-resistant (1). The development of new drugs effective against persistent and drug-resistant bacilli has therefore become a priority.The thick lipid-rich envelope of the Mycobacterium genus is characterized by the presence of mycolic acids (MAs),4 very long chain (C60–C90) α-alkylated β-hydroxylated fatty acids (2). MAs are the major components of the mycomembrane (3, 4) lipid bilayer, which plays a key role in both the architecture and permeability of the mycobacterial envelope. The MA biosynthetic pathway is essential for mycobacterial survival. MAs are generated by Claisen condensation between two fatty acyl chains as follows: the very long meromycoloyl chain (C40–C60) and a shorter saturated chain (C22–C26) (2). The different types of MAs are defined by the presence of decorations introduced at proximal and distal positions of the meromycolic chain (Fig. 1A) by a family of paralogous S-adenosylmethionine-dependent methyltransferases (AdoMet-MTs), the mycolic acid methyltransferases (MA-MTs). These chemical modifications are known to be important for the pathogenicity, virulence, and persistence of M. tuberculosis. For example, the cis-cyclopropane introduced at the proximal position of α-MAs by PcaA has an impact on the persistence of the tubercle bacillus within infected organisms (5). Furthermore, the keto and methoxy groups, with a vicinal methyl ramification at the distal position of oxygenated MAs, play a role in M. tuberculosis virulence in the mouse model of infection (6) and have recently been reported to be involved in host-pathogen interplay. Indeed, oxygenated MAs have been shown to modulate IL-12p40 production by macrophages (7) and to trigger the in vitro differentiation of monocyte-derived macrophages into foamy macrophages, which house the bacillus in a dormant state, within granulomas (8). Oxygenated MA biosynthesis requires the Hma (MmaA4) methyltransferase (Fig. 1B), as demonstrated by the absence of the oxygenated form in an M. tuberculosis hma knock-out mutant (6, 9). These results suggest that the enzymes responsible for adding the decorations to MAs, including oxygenated groups in particular, may be relevant pharmacological targets for the development of new antituberculous drugs (10).Open in a separate windowFIGURE 1.A, structures of MAs from M. tuberculosis and M. smegmatis. D, distal position; P, proximal position. Enzymes involved in the introduction of decorations on the meromycolic chain are indicated. B, proposed reaction scheme for the introduction of oxygenated groups. m = 17, 19; n, unknown; X, unknown carrier.Based on the essential role played by MA-MTs in the physiopathology of tuberculosis, several studies have investigated the possible inhibition of this family of enzymes. A recent study revealed that the antituberculous drug thiacetazone and its chemical analogs inhibited MA cyclopropanation at concentrations in the micromolar range (11). Another study, based on mixtures of crude extracts of heat-inactivated mycobacteria and recombinant Escherichia coli overproducing MA-MTs, suggested that the incorporation of [3H]AdoMet into growing meromycolic chains is inhibited by a high concentration (1 mg/ml, i.e. 2.6 mm) of S-adenosyl-l-homocysteine (AdoHcy) or sinefungin (12), the demethylated reaction product and a natural structural analog of AdoMet, respectively (Fig. 2). By contrast, AdoHcy and sinefungin are strong inhibitors of other AdoMet-MTs in vitro, including the cyclopropane fatty-acid synthase (CFAS) from E. coli (Ki of 30 and 0.22 μm, respectively) (13, 14). However, they are active only against the isolated enzyme, whereas S-adenosyl-N-decyl-aminoethyl (SADAE), a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain (Fig. 2), is active against CFAS both in vitro (Ki,app = 6 μm) and in vivo (complete inhibition at 150 μm) (15). The broad screening of possible inhibitors of MA-MTs with an in vitro mini-assay poses a major challenge, as these enzymes most likely use very long meromycolic chains as substrates. In this context, the similarity between CFAS and Hma in terms of their sequences (31% sequence identity) and substrates may be useful, as it suggests that SADAE may inhibit MA-MTs (15).Open in a separate windowFIGURE 2.Structure of AdoMet and of the AdoHcy, sinefungin, and SADAE analogs.We report here our investigations of the interactions between Hma and SADAE, as compared with those between Hma and AdoHcy or sinefungin, and the potential impact of these interactions on the activities of Hma and other MA-MTs and mycobacterial growth. Our high resolution crystallographic characterization of the Hma-SADAE interaction illustrates the bisubstrate nature of the ligand, which is strongly correlated with its strong inhibitory properties. 相似文献
998.
Manish B. Shah Cheryl Ingram‐Smith Leroy L. Cooper Jun Qu Yu Meng Kerry S. Smith Andrew M. Gulick 《Proteins》2009,77(3):685-698
The acyl‐AMP forming family of adenylating enzymes catalyze two‐step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X‐ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl‐CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl‐CoA synthetase and 4‐chlorobenzoate:CoA ligase. Most interestingly, the acyl‐binding pocket of the new structure is compared with other acyl‐ and aryl‐CoA synthetases. A comparison of the acyl‐binding pocket of the acyl‐CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl‐binding pocket. Proteins 2009. © 2009 Wiley‐Liss, Inc. 相似文献
999.
A simple and rapid method was devised for determination of tryptophan, based on the Belousov-Zhabotinskii (B-Z) oscillating
chemical system. Changes in oscillating period and amplitude were linearly proportional to the negative logarithm of l-tryptophan concentration over the range of 6.44 × 10−7–2.55 × 10−4 M, with the regression coefficients of near unity and a lower detection limit of 6.5 × 10−8 M. d-tryptophan was also examined although it is rarely found in most biological fluids, and perhaps not at all in natural proteins.
The change of period against to negative logarithm of d-tryptophan concentration over the range of 4.9 × 10−5–8.24 × 10−4 M is linear. Because the optimum conditions for determination of l- and d-tryptophan are not the same, a little amount of d-tryptophan does not affect the determination of l-tryptophan. Various influences were studied and a possible mechanism of perturbation to the B-Z oscillator by tryptophan
was also discussed. Spectrophotometry and fluorescence spectrophotofluorimetry were used for comparision and confirmation
of the results. 相似文献
1000.
A strain that was capable of transforming the phytosterol in corn flour and soybean flour was isolated from soil and identified as Fusarium moniliforme Sheld. The main transformation product was purified by high performance liquid chromatography (HPLC), and was characterized by nuclear magnetic resonance (NMR), mass spectrum (MS), and infrared spectrum (IR). The results indicated that the product was 4-androstene-3,17-dione (AD). The production of AD was increased with the increase of initial concentration of corn flour while the yield of AD was decreased. The yield of AD was lower in the media with only soybean flour. Sulfate–phosphate–ferric method (SPF) was first used for determination of the total phytosterol content in corn flour or soybean flour. The measured value by SPF method matched reasonably well with that by HPLC, which indicated the validity of SPF method. 相似文献