全文获取类型
收费全文 | 6670篇 |
免费 | 688篇 |
国内免费 | 3篇 |
出版年
2022年 | 32篇 |
2021年 | 72篇 |
2020年 | 60篇 |
2019年 | 89篇 |
2018年 | 86篇 |
2017年 | 73篇 |
2016年 | 123篇 |
2015年 | 212篇 |
2014年 | 276篇 |
2013年 | 294篇 |
2012年 | 399篇 |
2011年 | 393篇 |
2010年 | 276篇 |
2009年 | 242篇 |
2008年 | 380篇 |
2007年 | 391篇 |
2006年 | 358篇 |
2005年 | 351篇 |
2004年 | 368篇 |
2003年 | 294篇 |
2002年 | 310篇 |
2001年 | 145篇 |
2000年 | 146篇 |
1999年 | 137篇 |
1998年 | 108篇 |
1997年 | 76篇 |
1996年 | 65篇 |
1995年 | 69篇 |
1994年 | 64篇 |
1993年 | 85篇 |
1992年 | 97篇 |
1991年 | 87篇 |
1990年 | 83篇 |
1989年 | 83篇 |
1988年 | 81篇 |
1987年 | 67篇 |
1986年 | 56篇 |
1985年 | 71篇 |
1984年 | 66篇 |
1983年 | 53篇 |
1982年 | 53篇 |
1981年 | 43篇 |
1980年 | 43篇 |
1979年 | 38篇 |
1978年 | 33篇 |
1977年 | 37篇 |
1976年 | 41篇 |
1975年 | 30篇 |
1973年 | 34篇 |
1972年 | 28篇 |
排序方式: 共有7361条查询结果,搜索用时 31 毫秒
901.
902.
903.
Improved enzyme production by bio-pellets of Aspergillus niger: targeted morphology engineering using titanate microparticles 总被引:1,自引:0,他引:1
Driouch H Hänsch R Wucherpfennig T Krull R Wittmann C 《Biotechnology and bioengineering》2012,109(2):462-471
The present study describes the design of bio-pellet morphologies of the industrial working horse Aspergillus niger strains in submerged culture. The novel approach recruits the intended addition of titanate microparticles (TiSiO(4), 8 μm) to the growth medium. As tested for two recombinant strains producing fructofuranosidase and glucoamylase, the enzyme titer by the titanate-enhanced cultures in shake flasks was increased 3.7-fold to 150 U/mL (for fructofuranosidase) and 9.5-fold to 190 U/mL (for glucoamylase) as compared to the control. This could be successfully utilized for improved enzyme production in stirred tank reactors. Stimulated by the particles, the achieved final glucoamylase activity of 1,080 U/mL (fed-batch) and 320 U/mL (batch) was sevenfold higher as compared to the conventional processes. The major reason for the enhanced production was the close association between the titanate particles and the fungal cells. Already below 2.5 g/L the micromaterial was found inside the pellets, including single particles embedded as 50-150 μm particle aggregates in the center resulting in core shell pellets. With increasing titanate levels the pellet size decreased from 1,700 μm (control) to 300 μm. Fluorescence based resolution of GFP expression revealed that the large pellets of the control were only active in a 200 μm surface layer. This matches with the critical penetration depth for nutrients and oxygen typically observed for fungal pellets. The biomass within the titanate derived fungal pellets, however, was completely active. This was due a reduced thickness of the biomass layer via smaller pellets as well as the core shell structure. Moreover, also the created loose inner pellet structure enabled a higher mass transfer and penetration depths for up to 500 μm. The creation of core-shell pellets has not been achieved previously by the addition of microparticles, for example, made of talc or alumina. Due to this, the present work opens further possibilities to use microparticles for tailor-made morphology design of filamentous fungi, especially for pellet based processes which have a long and strong industrial relevance for industrial production. 相似文献
904.
STK25 Protein Mediates TrkA and CCM2 Protein-dependent Death in Pediatric Tumor Cells of Neural Origin 总被引:1,自引:0,他引:1
B Costa MJ Kean V Ast JD Knight A Mett Z Levy DF Ceccarelli BG Badillo R Eils R König AC Gingras M Fainzilber 《The Journal of biological chemistry》2012,287(35):29285-29289
The TrkA receptor tyrosine kinase induces death in medulloblastoma cells via an interaction with the cerebral cavernous malformation 2 (CCM2) protein. We used affinity proteomics to identify the germinal center kinase class III (GCKIII) kinases STK24 and STK25 as novel CCM2 interactors. Down-modulation of STK25, but not STK24, rescued medulloblastoma cells from NGF-induced TrkA-dependent cell death, suggesting that STK25 is part of the death-signaling pathway initiated by TrkA and CCM2. CCM2 can be phosphorylated by STK25, and the kinase activity of STK25 is required for death signaling. Finally, STK25 expression in tumors is correlated with positive prognosis in neuroblastoma patients. These findings delineate a death-signaling pathway downstream of neurotrophic receptor tyrosine kinases that may provide targets for therapeutic intervention in pediatric tumors of neural origin. 相似文献
905.
906.
Tavares CD O'Brien JP Abramczyk O Devkota AK Shores KS Ferguson SB Kaoud TS Warthaka M Marshall KD Keller KM Zhang Y Brodbelt JS Ozpolat B Dalby KN 《Biochemistry》2012,51(11):2232-2245
Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM. 相似文献
907.
Quantitative trait loci (QTL) hotspots (genomic locations affecting many traits) are a common feature in genetical genomics studies and are biologically interesting since they may harbor critical regulators. Therefore, statistical procedures to assess the significance of hotspots are of key importance. One approach, randomly allocating observed QTL across the genomic locations separately by trait, implicitly assumes all traits are uncorrelated. Recently, an empirical test for QTL hotspots was proposed on the basis of the number of traits that exceed a predetermined LOD value, such as the standard permutation LOD threshold. The permutation null distribution of the maximum number of traits across all genomic locations preserves the correlation structure among the phenotypes, avoiding the detection of spurious hotspots due to nongenetic correlation induced by uncontrolled environmental factors and unmeasured variables. However, by considering only the number of traits above a threshold, without accounting for the magnitude of the LOD scores, relevant information is lost. In particular, biologically interesting hotspots composed of a moderate to small number of traits with strong LOD scores may be neglected as nonsignificant. In this article we propose a quantile-based permutation approach that simultaneously accounts for the number and the LOD scores of traits within the hotspots. By considering a sliding scale of mapping thresholds, our method can assess the statistical significance of both small and large hotspots. Although the proposed approach can be applied to any type of heritable high-volume "omic" data set, we restrict our attention to expression (e)QTL analysis. We assess and compare the performances of these three methods in simulations and we illustrate how our approach can effectively assess the significance of moderate and small hotspots with strong LOD scores in a yeast expression data set. 相似文献
908.
Paine A Kirchner H Immenschuh S Oelke M Blasczyk R Eiz-Vesper B 《Journal of immunology (Baltimore, Md. : 1950)》2012,188(4):1620-1629
The glycoprotein CD86 is an important costimulatory molecule that has been shown to be predominantly expressed on APCs, such as dendritic cells, macrophages, and B cells. More recently, CD86 was also detected on T cells in specific pathological conditions. The mechanisms of how CD86 might be induced and its functional role in T cells are not well understood. In the present study, we showed that treatment with IL-2 markedly upregulated CD86, but not CD80, in human CD4(+) and CD8(+) T cells. This upregulation occurred in the absence of bystander cells, and isolated naive CD4(+) or CD8(+) T cells exhibited different time-dependent CD86-expression patterns in response to IL-2. Upregulation of CD86 on activated T cells was reduced by Abs that block IL-2 and IL-2Rα (CD25), indicating a receptor-mediated mechanism. IL-2-dependent CD86 upregulation was blocked by pharmacological inhibitors of the NFAT and mammalian target of rapamycin pathways and was largely reduced by simultaneous exposure to IFN-α. Importantly, a marked increase in CD86 on T cells was also observed in vivo in IL-2-treated patients. In conclusion, IL-2 upregulates CD86 expression on human CD4(+) and CD8(+) T cells via a receptor-dependent mechanism that involves the NFAT and mammalian target of rapamycin pathways. 相似文献
909.
Keller MA Watschinger K Lange K Golderer G Werner-Felmayer G Hermetter A Wanders RJ Werner ER 《Journal of lipid research》2012,53(7):1410-1416
The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome
(SLS) patient cells not only impairs the conversion of fatty aldehydes into their
corresponding fatty acid but also has an effect on connected pathways. Alteration of
the lipid profile in these cells is thought to be responsible for severe symptoms
such as ichtyosis, mental retardation, and spasticity. Here we present a novel
approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring
pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the
culture medium of living cells using HPLC separation and fluorescence detection. Our
results show that in fibroblasts from SLS patients, fatty aldehyde is not
accumulating but is converted readily into fatty alcohol. In control cells, in
contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did
not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower
concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed
to induce toxicity in SLS patient and in control cells. 相似文献
910.
Kim GY Ligons DL Hong C Luckey MA Keller HR Tai X Lucas PJ Gress RE Park JH 《Journal of immunology (Baltimore, Md. : 1950)》2012,188(12):5859-5866
All T cells are dependent on IL-7 for their development and for homeostasis. Foxp3(+) regulatory T cells (Tregs) are unique among T cells in that they are dependent on IL-2. Whether such IL-2 dependency is distinct from or in addition to an IL-7 requirement has been a confounding issue, particularly because of the absence of an adequate experimental system to address this question. In this study, we present a novel in vivo mouse model where IL-2 expression is intact but IL-7 expression was geographically limited to the thymus. Consequently, IL-7 is not available in peripheral tissues. Such mice were generated by introducing a thymocyte-specific IL-7 transgene onto an IL-7 null background. In these mice, T cell development in the thymus, including Foxp3(+) Treg numbers, was completely restored, which correlates with the thymus-specific expression of transgenic IL-7. In peripheral cells, however, IL-7 expression was terminated, which resulted in a general paucity of T cells and a dramatic reduction of Foxp3(+) Treg numbers. Loss of Tregs was further accompanied by a significant reduction in Foxp3(+) expression levels. These data suggest that peripheral IL-7 is not only necessary for Treg survival but also for upregulating Foxp3 expression. Collectively, we assessed the effect of a selective peripheral IL-7 deficiency in the presence of a fully functional thymus, and we document a critical requirement for in vivo IL-7 in T cell maintenance and specifically in Foxp3(+) cell homeostasis. 相似文献