全文获取类型
收费全文 | 5029篇 |
免费 | 309篇 |
出版年
2024年 | 7篇 |
2023年 | 35篇 |
2022年 | 97篇 |
2021年 | 171篇 |
2020年 | 132篇 |
2019年 | 134篇 |
2018年 | 210篇 |
2017年 | 155篇 |
2016年 | 241篇 |
2015年 | 324篇 |
2014年 | 350篇 |
2013年 | 384篇 |
2012年 | 433篇 |
2011年 | 416篇 |
2010年 | 306篇 |
2009年 | 222篇 |
2008年 | 256篇 |
2007年 | 246篇 |
2006年 | 227篇 |
2005年 | 174篇 |
2004年 | 145篇 |
2003年 | 114篇 |
2002年 | 113篇 |
2001年 | 62篇 |
2000年 | 41篇 |
1999年 | 61篇 |
1998年 | 30篇 |
1997年 | 20篇 |
1996年 | 11篇 |
1995年 | 16篇 |
1994年 | 13篇 |
1993年 | 8篇 |
1992年 | 16篇 |
1991年 | 19篇 |
1990年 | 11篇 |
1989年 | 13篇 |
1988年 | 10篇 |
1987年 | 8篇 |
1986年 | 8篇 |
1985年 | 5篇 |
1984年 | 8篇 |
1983年 | 8篇 |
1982年 | 8篇 |
1981年 | 5篇 |
1980年 | 7篇 |
1979年 | 7篇 |
1976年 | 5篇 |
1971年 | 5篇 |
1970年 | 4篇 |
1969年 | 5篇 |
排序方式: 共有5338条查询结果,搜索用时 947 毫秒
121.
Julia Blümer Juliana Rey Leif Dehmelt Tomá? Mazel Yao-Wen Wu Philippe Bastiaens Roger S. Goody Aymelt Itzen 《The Journal of cell biology》2013,200(3):287-300
Eukaryotic cells critically depend on the correct regulation of intracellular vesicular trafficking to transport biological material. The Rab subfamily of small guanosine triphosphatases controls these processes by acting as a molecular on/off switch. To fulfill their function, active Rab proteins need to localize to intracellular membranes via posttranslationally attached geranylgeranyl lipids. Each member of the manifold Rab family localizes specifically to a distinct membrane, but it is unclear how this specific membrane recruitment is achieved. Here, we demonstrate that Rab-activating guanosine diphosphate/guanosine triphosphate exchange factors (GEFs) display the minimal targeting machinery for recruiting Rabs from the cytosol to the correct membrane using the Rab-GEF pairs Rab5A–Rabex-5, Rab1A-DrrA, and Rab8-Rabin8 as model systems. Specific mistargeting of Rabex-5/DrrA/Rabin8 to mitochondria led to catalytic recruitment of Rab5A/Rab1A/Rab8A in a time-dependent manner that required the catalytic activity of the GEF. Therefore, RabGEFs are major determinants for specific Rab membrane targeting. 相似文献
122.
123.
Juliana L. França Marcelo R. Pinto Malson N. Lucena Daniela P. Garçon Wagner C. Valenti John C. McNamara Francisco A. Leone 《The Journal of membrane biology》2013,246(7):529-543
The stimulation by Mg2+, Na+, K+, NH4 +, and ATP of (Na+, K+)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na+, K+)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg2+, Na+, and K+ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V M = 115.0 ± 2.3 U mg?1, K 0.5 = 0.10 ± 0.01 mmol L?1. Stimulation by Na+ (V M = 110.0 ± 3.3 U mg?1, K 0.5 = 1.30 ± 0.03 mmol L?1), Mg2+ (V M = 115.0 ± 4.6 U mg?1, K 0.5 = 0.96 ± 0.03 mmol L?1), NH4 + (V M = 141.0 ± 5.6 U mg?1, K 0.5 = 1.90 ± 0.04 mmol L?1), and K+ (V M = 120.0 ± 2.4 U mg?1, K M = 2.74 ± 0.08 mmol L?1) followed single saturation curves and, except for K+, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K I = 12.4 ± 1.3 mol L?1. Complementary inhibition studies suggest the presence of F0F1–, Na+-, or K+-ATPases, but not V(H+)- or Ca2+-ATPases, in the gill microsomal preparation. K+ and NH4 + synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4 +, and K+ of the gill enzyme. 相似文献
124.
Raquel Fonseca-Maldonado Davi Serradella Vieira Juliana Sanchez Alponti Eric Bonneil Pierre Thibault Richard John Ward 《The Journal of biological chemistry》2013,288(35):25522-25534
Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex8–16GlcNAc2 modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex9GlcNAc2 at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions. 相似文献
125.
Gabriela Turk Yanina Ghiglione Juliana Falivene María Eugenia Socias Natalia Laufer Romina Soledad Coloccini Ana María Rodriguez María Julia Ruiz María ángeles Pando Luis David Giavedoni Pedro Cahn Omar Sued Horacio Salomon María Magdalena Gherardi 《Journal of virology》2013,87(13):7445-7462
The important role of the CD8+ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8+ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8+ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4+ T-cell set points were observed in PHI subjects with higher HIV-specific CD8+ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8+ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection. 相似文献
126.
127.
Jaqueline Raymondi Silva Larissa Campos de Medeiros Vinícius Pinho dos Reis Juliana Helena Chávez Thiago Demarchi Munhoz Gustavo Puia Borges Otavio Augusto Brioschi Soares Carlos Henrique Coelho de Campos Rosangela Zacarias Machado Cristiane Divan Baldani Maria Luana Cristiny Rodrigues Silva Joice Lara Maia Faria Edson Elias da Silva Luiz Tadeu Moraes Figueiredo 《Memórias do Instituto Oswaldo Cruz》2013,108(7):921-923
Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country. 相似文献
128.
129.
João Vidigal Mafalda M. Dias Fabiana Fernandes Marco Patrone Cláudia Bispo Cláudia Andrade Rui Gardner Manuel J.T. Carrondo Paula M. Alves Ana P. Teixeira 《Journal of biotechnology》2013
Insect cell lines such as Sf9 and High Five™ have been widely used to produce recombinant proteins mostly by the lytic baculovirus vector system. We have recently established an expression platform in Sf9 cells using a fluorescence-based recombinase mediated cassette exchange (RMCE) strategy which has similar development timelines but avoids baculovirus infection. To expedite cell engineering efforts, a robust fluorescence-activated cell sorting (FACS) protocol optimized for insect cells was developed here. The standard sorting conditions used for mammalian cells proved to be unsuitable, resulting in post-sorting viabilities below 10% for both cell lines. We found that the extreme sensitivity to the shear stress displayed by Sf9 and High Five™ cells was the limiting factor, and using Pluronic F-68 in the cell suspension could increase post-sorting viabilities in a dose dependent manner. The newly developed protocol was then used to sort stable populations of both cell lines tagged with a DsRed-expressing cassette. Before sorting, the average fluorescence intensity of the Sf9 cell population was 3-fold higher than that of the High Five™ cell population. By enriching with the 10% strongest DsRed-fluorescent cells, the productivity of both cell populations could be successfully improved. The established sorting protocol potentiates the use of RMCE technology for recombinant protein production in insect cells. 相似文献
130.
E. Rodriguez R. Azevedo C. Remédios T. Almeida P. Fernandes C. Santos 《Journal of plant physiology》2013
Chromium (Cr), as a mutagenic agent in plants, has received less attention than other metal pollutants. To understand if Cr induces microsatellite instability (MSI), Pisum sativum seedlings were exposed for 28 days to different concentrations of Cr(VI) up to 2000 mg L−1, and the genetic instability of ten microsatellites (SSRs) was analyzed. In plants exposed to Cr(VI) up to 1000 mg L−1, MSI was never observed. However, roots exposed to 2000 mg L−1 displayed MSI in two of the loci analyzed, corresponding to a mutation rate of 8.3%. SSR2 (inserted in the locus for plastid photosystem I 24 kDa light harvesting protein) and SSR6 (inserted in the locus for P. sativum glutamine synthetase) from Cr(VI)-treated roots presented alleles with, respectively, less 6 bp and more 3 bp than the corresponding controls. This report demonstrates that: (a) SSRs technique is sensitive to detect Cr-induced mutagenicity in plants, being Cr-induced-MSI dose and organ dependent (roots are more sensitive); (b) two Cr-sensitive loci are related with thylakoid photophosphorylation and with glutamine synthetase, respectively; (c) despite MSI is induced by Cr(VI), it only occurs in plants exposed to concentrations higher than 1000 mg L−1 (values rarely found in real scenarios). Considering these data, we also discuss the known functional changes induced by Cr(VI) in photosynthesis and in glutamine synthetase activity. 相似文献