首页 | 本学科首页   官方微博 | 高级检索  
文章检索
  按 检索   检索词:      
出版年份:   被引次数:   他引次数: 提示:输入*表示无穷大
  收费全文   2062篇
  免费   100篇
  国内免费   1篇
  2023年   3篇
  2021年   19篇
  2020年   11篇
  2019年   12篇
  2018年   23篇
  2017年   28篇
  2016年   29篇
  2015年   52篇
  2014年   94篇
  2013年   162篇
  2012年   113篇
  2011年   130篇
  2010年   91篇
  2009年   69篇
  2008年   106篇
  2007年   152篇
  2006年   138篇
  2005年   122篇
  2004年   171篇
  2003年   131篇
  2002年   150篇
  2001年   20篇
  2000年   24篇
  1999年   27篇
  1998年   33篇
  1997年   27篇
  1996年   17篇
  1995年   22篇
  1994年   26篇
  1993年   25篇
  1992年   15篇
  1991年   8篇
  1990年   16篇
  1989年   7篇
  1988年   6篇
  1987年   5篇
  1986年   3篇
  1985年   4篇
  1984年   4篇
  1983年   9篇
  1982年   8篇
  1981年   8篇
  1980年   7篇
  1979年   7篇
  1978年   6篇
  1977年   3篇
  1975年   5篇
  1974年   3篇
  1969年   2篇
  1965年   2篇
排序方式: 共有2163条查询结果,搜索用时 156 毫秒
81.
82.
It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105–115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC50. This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.  相似文献   
83.
Nitrogen transformations were studied in flooded and non-flooded vertical flow columns with and without a rice plant. Influent (average concentration: NH4+-N: 40 mg L?1; NO3?-N: 0.15 mg L?1; and NO2?-N: 4.0 mg L?1) was supplied at 1.25 cm d?1 during stage 1 (20 May–5 August) and at 2.50 cm d?1 at stage 2 (6 August–26 October), which resulted in an average nitrogen loading of 156 g m?2 during the entire experimental period. Total nitrogen (T-N) removal efficiencies exceeded 90% in vertical flow systems with rice plants. Nitrogen assimilated by the rice plants in the flooded column accounted for 60% of the total input nitrogen, while that in the non-flooded column accounted for 36% of the total input. The remaining nitrogen appeared to be removed through biogeochemical pathways. Although some nitrogen flowed out, most input nitrogen was also removed even in the flooded and non-flooded unplanted columns.A high-resolution vertical distribution investigation showed the changes of nitrogen forms in soil water. In the flooded condition, there were high ammonium and high nitrite concentrations in the upper layers. The concentrations of ammonium and nitrite simultaneously decreased with depth increasing, suggesting that anaerobic ammonia oxidation (anammox) may occur in these anaerobic conditions. In contrast, the distributions of nitrogen in the non-flooded columns with elevated water level suggested that nitrification–denitrification route was the major removal mechanism, whether or not rice plants were present.  相似文献   
84.
Autochthonous bioaugmentation and its possible application to oil spills   总被引:1,自引:0,他引:1  
Bioaugmentation for oil spills is a much more promising technique than is biostimulation. However, the effectiveness of bioaugmentation is variable, because the survival and the xenobiotic-degrading ability of introduced microorganisms are highly dependent on environmental conditions. As an alternative, autochthonous bioaugmentation (ABA) is proposed to overcome these difficulties. The ABA method is like a ready-made bioaugmentation technology. In ABA, microorganisms indigenous to the contaminated site or predicted contamination site that are well-characterized and potentially capable of degrading oils are used, and these microorganisms should be enriched under conditions where bioaugmentation will be conducted. It is possible to obtain information in advance on the chemical and physical characteristics of potential oil spill sites and of oils that might be spilled. The application of ABA in the coastal areas of Hokkaido Prefecture, Japan, is considered here, because Hokkaido is located south of Sakhalin Island, Russia, where development of oil fields is in progress. If oil spills in this region were well characterized in advance, ABA could be a feasible technology in the near future.  相似文献   
85.
High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14 h, including 8 h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).  相似文献   
86.
Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3β-hydroxysterol Δ24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.  相似文献   
87.
Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10°C. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10°C. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4°C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 108 CFU/g at 10°C after 7 to 12 days.  相似文献   
88.
We have recently identified a novel protein named hornerin, the structural features of which are most similar to those of profilaggrin, an essential protein for keratinization of epidermal tissues. In this study we examined the expression of hornerin compared with that of profilaggrin in various mouse tissues. Hornerin was expressed in the upper epidermis of newborn mouse skin, as was profilaggrin. In addition, both hornerin and profilaggrin were expressed in the tongue, esophagus, and forestomach. In all four tissues, immunostaining for hornerin and profilaggrin showed a granular pattern, and most of the signals for the two proteins were co-localized on keratohyalin granules. This was confirmed by double immunoelectron microscopy. Within keratohyalin granules, hornerin was detected more frequently in the periphery, whereas profilaggrin was equally distributed. A quantitative RT-PCR revealed that both genes were expressed at highest levels in the forestomach and at the next highest levels in skin. Profilaggrin mRNA was most abundant in the forestomach. In skin, the amount of hornerin mRNA was more than fourfold greater than the amount of profilaggrin mRNA. These results form the basis for a better understanding of possible overlapping and/or differential functions of hornerin and profilaggrin.  相似文献   
89.
We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (DeltaPsi) and pH difference (DeltapH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K(+) diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K(+) permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with DeltaPsi formed by valinomycin-K(+) did not significantly alter 9-AA fluorescence, but unexpectedly dissipated DeltaPsi. The DeltaPsi preformed by valinomycin-K(+) decreased gradually following the addition of increasing concentrations of anacardic acid. The DeltaPsi dissipation rate was dependent on the pre-existing magnitude of DeltaPsi, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of DeltapH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by DeltaPsi, and a 'proton carrier' that dissipates the transmembrane proton gradient formed.  相似文献   
90.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号