全文获取类型
收费全文 | 33937篇 |
免费 | 3055篇 |
国内免费 | 2476篇 |
专业分类
39468篇 |
出版年
2024年 | 89篇 |
2023年 | 418篇 |
2022年 | 813篇 |
2021年 | 1326篇 |
2020年 | 963篇 |
2019年 | 1181篇 |
2018年 | 1158篇 |
2017年 | 797篇 |
2016年 | 1193篇 |
2015年 | 2021篇 |
2014年 | 2247篇 |
2013年 | 2492篇 |
2012年 | 3037篇 |
2011年 | 2831篇 |
2010年 | 1691篇 |
2009年 | 1505篇 |
2008年 | 1832篇 |
2007年 | 1645篇 |
2006年 | 1505篇 |
2005年 | 1238篇 |
2004年 | 1155篇 |
2003年 | 985篇 |
2002年 | 892篇 |
2001年 | 719篇 |
2000年 | 657篇 |
1999年 | 586篇 |
1998年 | 326篇 |
1997年 | 314篇 |
1996年 | 297篇 |
1995年 | 248篇 |
1994年 | 265篇 |
1993年 | 180篇 |
1992年 | 320篇 |
1991年 | 295篇 |
1990年 | 246篇 |
1989年 | 229篇 |
1988年 | 192篇 |
1987年 | 156篇 |
1986年 | 146篇 |
1985年 | 151篇 |
1984年 | 142篇 |
1983年 | 103篇 |
1982年 | 90篇 |
1980年 | 59篇 |
1979年 | 75篇 |
1978年 | 69篇 |
1977年 | 58篇 |
1976年 | 67篇 |
1975年 | 63篇 |
1974年 | 75篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
991.
Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion 下载免费PDF全文
Chia-Li Wei Yunn-Bor Yang Chan-Hui Deng Wen-Chi Liu Jyh-Shing Hsu Yu-Ching Lin Shwu-Huey Liaw Ying-Chieh Tsai 《Applied microbiology》2005,71(12):8873-8880
The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the kcat/Km ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a kcat/Km ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering. 相似文献
992.
A miRNA involved in phosphate-starvation response in Arabidopsis 总被引:27,自引:0,他引:27
Although microRNAs (miRNAs) have been documented to regulate development in plants and animals , the function of miRNAs in physiology is unclear. miR399 has multiple target sites in the 5' untranslated region (UTR) of a gene encoding a putative ubiquitin-conjugating enzyme (UBC) in Arabidopsis thaliana. We report here that miR399 was highly induced, whereas the target UBC mRNA was reduced by low-phosphate (Pi) stress. In transgenic plants with constitutive expression of miR399, UBC mRNA accumulation was suppressed even under high Pi. The expression of transgene UBC mRNA with 5' UTR miR399 target sites, but not the one without 5' UTR, was reduced under low-Pi condition. Furthermore, transgenic Arabidopsis plants with constitutive expression of miR399 accumulated more Pi than the wild-type, and transgenic plants expressing the UBC mRNA without 5' UTR (miRNA-deregulated) showed less inhibition of primary root growth and less induction of a Pi transporter gene by low-Pi stress than those of wild-type plants. We conclude that miR399 downregulates UBC mRNA accumulation by targeting the 5' UTR, and this regulation is important for plant responses to Pi starvation. The results suggest that miRNAs have functional roles for plants to cope with fluctuations in mineral-nutrient availability in the soil. 相似文献
993.
Cheng TL Cheng CM Chen BM Tsao DA Chuang KH Hsiao SW Lin YH Roffler SR 《Bioconjugate chemistry》2005,16(5):1225-1231
Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is a proven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitative analysis of PEG-derivatized molecules is important for both drug development and clinical applications. We previously reported the development of a monoclonal IgM antibody (AGP3) to PEG. We now describe a new IgG1 monoclonal antibody (E11) to PEG and show that it can be used in combination with AGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunits of the PEG backbone and could detect free PEG and PEG-modified proteins by ELISA, immunoblotting, and flow cytometry. Detection sensitivity increased with the length and the number of PEG chains on pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modified enzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detection antibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. In addition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG20,000, PEG2,000-quantum dots, and PEG2,000-liposomes at concentrations as low as 20 ng/mL (1.0 nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show that the sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. These antibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules. 相似文献
994.
The gating of ion channels has widely been modeled by assuming the transition between open and closed states is a memoryless process. Nevertheless, the statistical analysis of an ionic current signal recorded from voltage dependence K(+) single channel is presented. Calculating the sample auto-correlation function of the ionic current based on the digitized signals, rather than the sequence of open and closed states duration time. The results provide evidence for the existence of memory. For different voltages, the ion channel current fluctuation has different correlation attributions. The correlations in data generated by simulation of two Markov models, on one hand, auto-correlation function of the ionic current shows a weaker memory, after a delayed period of time, the attribute of memory does not exist; on the other hand, the correlation depends on the number of states in the Markov model. For V(p)=-60 mV pipette potential, spectral analysis of ion channel current was conducted, the result indicates that the spectrum is not a flat spectrum, the data set from ionic current fluctuations shows considerable variability with a broad 1/f -like spectrum, alpha=1.261+/-0.24. Thus the ion current fluctuations give information about the kinetics of the channel protein, the results suggest the correlation character of ion channel protein nonlinear kinetics regardless of whether the channel is in open or closed state. 相似文献
995.
Cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells through activation of the proapoptotic Bcl-2 family proteins and MAPK pathway 总被引:2,自引:0,他引:2
Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. However, the intracellular death signaling mechanisms by which Cin inhibits tumor cell growth are poorly understood. In this study, we investigated the effect of mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on the stress-responsive MAPK pathway induced by Cin in PLC/PRF/5 cells. Trypan blue staining assay indicated that Cin was cytotoxic to PLC/PRF/5 cells. Cin caused cell cycle perturbation (S-phase arrest) and triggered apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and accumulation of sub-G1 peak. It down-regulated the Bcl-2 and Mcl-1 expression, and up-regulated Bax protein in a time-response manner. Treatment with 1 microM Cin resulted in an activation of caspase-8 and cleavage of Bid to its truncated form in a time-dependent pattern. JNK, ERK and p38 kinases in cells were activated and phosphorylated after Cin treatment. Pre-incubation with SP600125 and SB203580 markedly suppressed the effect of Cin-induced apoptosis, but not PD98059. Both SP600125 and SB203580 significantly prevented the phosphorylation of JNK and p38 proteins, but not ERK. These results conclude that Cin triggers apoptosis in PLC/PRF/5 cells could be through the activation of pro-apoptotic Bcl-2 family (Bax and Bid) proteins and MAPK signaling pathway. 相似文献
996.
997.
998.
Hsu WC Chang HC Chou CY Tsai PJ Lin PI Chang GG 《The Journal of biological chemistry》2005,280(24):22741-22748
The severe acute respiratory syndrome (SARS) coronavirus (CoV) main protease represents an attractive target for the development of novel anti-SARS agents. The tertiary structure of the protease consists of two distinct folds. One is the N-terminal chymotrypsin-like fold that consists of two structural domains and constitutes the catalytic machinery; the other is the C-terminal helical domain, which has an unclear function and is not found in other RNA virus main proteases. To understand the functional roles of the two structural parts of the SARS-CoV main protease, we generated the full-length of this enzyme as well as several terminally truncated forms, different from each other only by the number of amino acid residues at the C- or N-terminal regions. The quaternary structure and K(d) value of the protease were analyzed by analytical ultracentrifugation. The results showed that the N-terminal 1-3 amino acid-truncated protease maintains 76% of enzyme activity and that the major form is a dimer, as in the wild type. However, the amino acids 1-4-truncated protease showed the major form to be a monomer and had little enzyme activity. As a result, the fourth amino acid seemed to have a powerful effect on the quaternary structure and activity of this protease. The last C-terminal helically truncated protease also exhibited a greater tendency to form monomer and showed little activity. We concluded that both the C- and the N-terminal regions influence the dimerization and enzyme activity of the SARS-CoV main protease. 相似文献
999.
本文通过把中国古人类的进化摆到中国这一特定的地理区域、特定的自然地理环境中去作联系的考察,提出如下几点看法:1.中国是世界上很早就为人类所居住的地区之一,受自然地理环境的影响和制约,中国古人类主要生存于自然条件较好的东部季风区域;2.古人类在中国境内主要是由南向北、进而向东北方向扩散和分布;3.中国最早的古人类可能是外来的,可能是从中国南部边境进入中国境内的。 相似文献
1000.
Bach S Knockaert M Reinhardt J Lozach O Schmitt S Baratte B Koken M Coburn SP Tang L Jiang T Liang DC Galons H Dierick JF Pinna LA Meggio F Totzke F Schächtele C Lerman AS Carnero A Wan Y Gray N Meijer L 《The Journal of biological chemistry》2005,280(35):31208-31219
(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs. 相似文献