全文获取类型
收费全文 | 6266篇 |
免费 | 58篇 |
国内免费 | 67篇 |
出版年
2023年 | 8篇 |
2022年 | 27篇 |
2021年 | 39篇 |
2020年 | 51篇 |
2019年 | 74篇 |
2018年 | 49篇 |
2017年 | 31篇 |
2016年 | 38篇 |
2015年 | 129篇 |
2014年 | 392篇 |
2013年 | 410篇 |
2012年 | 417篇 |
2011年 | 601篇 |
2010年 | 500篇 |
2009年 | 272篇 |
2008年 | 216篇 |
2007年 | 274篇 |
2006年 | 187篇 |
2005年 | 171篇 |
2004年 | 177篇 |
2003年 | 180篇 |
2002年 | 122篇 |
2001年 | 45篇 |
2000年 | 74篇 |
1999年 | 87篇 |
1998年 | 69篇 |
1997年 | 67篇 |
1996年 | 84篇 |
1995年 | 83篇 |
1994年 | 78篇 |
1993年 | 77篇 |
1992年 | 50篇 |
1991年 | 54篇 |
1990年 | 50篇 |
1989年 | 64篇 |
1988年 | 47篇 |
1987年 | 42篇 |
1986年 | 45篇 |
1985年 | 67篇 |
1984年 | 187篇 |
1983年 | 168篇 |
1982年 | 173篇 |
1981年 | 113篇 |
1980年 | 111篇 |
1979年 | 110篇 |
1978年 | 25篇 |
1977年 | 21篇 |
1976年 | 16篇 |
1975年 | 5篇 |
1974年 | 5篇 |
排序方式: 共有6391条查询结果,搜索用时 343 毫秒
921.
The formation of pores by membrane-inserted diphtheria toxin is closely linked to the translocation of its catalytic chain
across membranes. In this report a number of aromatic polyanionic molecules were identified that inhibit toxin-induced leakage
of molecules from model membrane vesicles. One inhibitor, Cibacron blue, totally blocked pore formation. Aniline blue and
Fast Green decreased the size of the molecule released by a given concentration of toxin. Amaranth appeared to reduce the
maximal amount of leakage, without greatly affecting the size of the molecule released at a given toxin concentration. Finally,
Ponceau S and Cibacron brilliant red appeared to exhibit a mixture of these various types of inhibition. The inhibitors neither
prevented the conformational transition of the toxin to form a hydrophobic state at low pH, nor (with the exception of Cibacron
Brilliant Red) appeared to strongly inhibit toxin binding to model membranes. Additional experiments showed release of trapped
materials from model membranes by isolated T domain of the toxin was similar to that by whole toxin. The effects of inhibitors
on T domain induced release was also similar to that they have on whole toxin. Therefore, it is likely that the inhibition
of pore formation by whole toxin involves inhibitor interaction with the T domain. The inhibitors identified in this study
may be helpful for development of agents that interfere with toxin action in vivo.
Received: 10 March 1999/Revised: 22 June 1999 相似文献
922.
细胞色素P450超家族蛋白质基于结构知识的序列联配 总被引:4,自引:0,他引:4
应用计算机程序对细胞色素P450蛋白20个家族的代表序列进行初步多序列联配后,利用结构知识对匹配结果加以调整,调整的依据是P450蛋白二级结构特征和某些超二级结构特征的保守性,三维结构空间配置的保守性,在结构与功能重要残基的保守性,亲水和疏水片段的保守性。大量定点突变实验、选择性化学修饰实验、抗多肽抗体结合实验等能很好地确证该联配结果的正确性。联配结果对真核P450蛋白膜结合方式、活性位点残基分布、与氧化还原偶联蛋白有相互作用的残基的分布等作出了合理的推断。细胞色素P450超家族蛋白基于结构知识的序列联配的尝试对P450蛋白结构与功能关系研究提供了参考 相似文献
923.
924.
Detection of cell viability in cultures of hyperthermophiles 总被引:2,自引:0,他引:2
925.
Improved methods for in situ enzymatic amplification and detection of low copy number genes in bacteria 总被引:2,自引:0,他引:2
Daniel Jacobsa Mark L Anglesa Amanda E Goodmana Brett A Neilana 《FEMS microbiology letters》1997,152(1):65-73
We present alternative and improved protocols for in situ analysis of single copy genes in prokaryotes. Primed in situ amplification (PRINS) and cycle PRINS were used to detect, via the incorporation of a fluorescein labelled nucleotide, the presence of specific genes carried on both high and low copy number plasmids in individual cells of Escherichia coli and a marine bacterium, SW5. The optimised protocols described enabled a significant reduction in non-specific signals whilst maintaining high fluorescent activity via labelled nucleotide incorporation. In addition, nucleic acids were amplified linearly and were retained within the permeabilised microbial cells. These methods provide considerable advances in sensitivity, specificity and reliability compared to current protocols for bacterial in situ nucleic acid amplification. 相似文献
926.
Mario A. Cepeda Jacob JH. Pelling Caitlin L. Evered Hon S. Leong Sashko Damjanovski 《Experimental cell research》2017,350(1):169-183
Membrane-type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease that regulates ECM degradation, proMMP-2 activation, and varied cellular processes including migration and viability. MT1-MMP is believed to be a central mediator of tumourigenesis whose role is dictated by its functionally distinct protein domains. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain, exemplifying diverse regulatory functions. To further our understanding of the multifunctional contributions of MT1-MMP to cellular processes, we overexpressed cytoplasmic domain altered constructs in MCF-7 breast cancer cells and analyzed migration and viability in 2D culture conditions, morphology in 3D Matrigel culture, and tumorigenic ability in vivo. We found that the cytoplasmic domain was not needed for MT1-MMP mediated migration promotion, but was necessary to maintain viability during serum depravation in 2D culture. Similarly, during 3D Matrigel culture the cytoplasmic domain of MT1-MMP was not needed to initiate a protrusive phenotype, but was necessary to prevent colony blebbing when cells were serum deprived. We also tested in vivo tumorigenic potential to show that cells expressing cytoplasmic domain altered constructs demonstrated a reduced ability to vascularize tumours. These results suggest that the cytoplasmic domain regulates MT1-MMP function in a manner required for cell survival, but is dispensable for cell migration. 相似文献
927.
José E. Guzmán-Flores Adrián F. Alvarez Sebastián Poggio Marina Gavilanes-Ruiz Dimitris Georgellis 《Analytical biochemistry》2017
Lipid rafts or membrane microdomains have been proposed to compartmentalize cellular processes by spatially organizing diverse molecules/proteins in eukaryotic cells. Such membrane microdomains were recently reported to also exist in a few bacterial species. In this work, we report the development of a procedure for membrane microdomain isolation from Escherichia coli plasma membranes as well as a method to purify the latter. The method here reported could easily be adapted to other gram-negative bacteria, wherein the isolation of this kind of sub-membrane preparation imposes special difficulties. The analysis of isolated membrane microdomains might provide important information on the nature and function of these bacterial structures and permit their comparison with the ones of eukaryotic cells. 相似文献
928.
Shiro Okumura Tetsuyuki Akao Satoko Yamashita Tokio Ichimatsu Kuniyo Inouye 《Cytotechnology》2005,47(1-3):59-67
Proteins of plasma membrane could be an index of purification of the plasma membrane of animal cells. A convenient method
is proposed for determining the plasma membrane proteins by a surface plasmon resonance (SPR) biosensor. Biotinylated proteins
were observed only in the peripheral areas of MOLT-4 cells which were treated by 5-[5-(N-succinimidyloxycarbonyl) pentylamido] hexyl-d-biotinamide. The proteins on HeLa cells were also biotinylated. And then the membrane samples of the HeLa cells were injected
onto the avidin-immobilized SPR-surface, and components bound non-specifically on the surface were removed by a washout solution.
The amount of biotinylated protein (BP) was determined directly from the absolute resonance unit (RU) after injection of the
washout solution. In the method a reference surface was not needed. The amount of BP bound to the surface was gradually attenuated
with the repeated injection, and a method for calibrating the RU value was introduced by considering the ratio of attenuation
by every injection. The correlation between the BP titer calculated by the calibration and the theoretically-estimated one
was greatly improved. Three cycles of the BP determination on a sensor surface was performed successfully. During the purification
process of membrane fractions, the degree of purification as judged by the BP titer was in good agreement with the degree
of increase in aminopeptidase N activity in the membrane fraction. Thus, the BP titer could be used as an index for purification
of plasma membrane. 相似文献
929.
930.
This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of Escherichia coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A600 8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the 13CO chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy. 相似文献