全文获取类型
收费全文 | 108525篇 |
免费 | 1504篇 |
国内免费 | 1961篇 |
专业分类
111990篇 |
出版年
2024年 | 42篇 |
2023年 | 238篇 |
2022年 | 574篇 |
2021年 | 948篇 |
2020年 | 570篇 |
2019年 | 761篇 |
2018年 | 12395篇 |
2017年 | 11060篇 |
2016年 | 8098篇 |
2015年 | 1609篇 |
2014年 | 1558篇 |
2013年 | 1692篇 |
2012年 | 5634篇 |
2011年 | 14020篇 |
2010年 | 12704篇 |
2009年 | 8862篇 |
2008年 | 10540篇 |
2007年 | 11976篇 |
2006年 | 805篇 |
2005年 | 998篇 |
2004年 | 1367篇 |
2003年 | 1371篇 |
2002年 | 1039篇 |
2001年 | 501篇 |
2000年 | 379篇 |
1999年 | 254篇 |
1998年 | 165篇 |
1997年 | 156篇 |
1996年 | 130篇 |
1995年 | 110篇 |
1994年 | 109篇 |
1993年 | 116篇 |
1992年 | 122篇 |
1991年 | 137篇 |
1990年 | 60篇 |
1989年 | 62篇 |
1988年 | 58篇 |
1987年 | 45篇 |
1986年 | 22篇 |
1985年 | 27篇 |
1984年 | 30篇 |
1983年 | 32篇 |
1982年 | 9篇 |
1972年 | 246篇 |
1971年 | 274篇 |
1965年 | 13篇 |
1962年 | 24篇 |
1956年 | 5篇 |
1944年 | 12篇 |
1940年 | 10篇 |
排序方式: 共有10000条查询结果,搜索用时 15 毫秒
981.
Hisashi Mizutani Hideaki Sugawara Ashley M. Buckle Takeshi Sangawa Ken-ichi Miyazono Jun Ohtsuka Koji Nagata Tomoki Shojima Shohei Nosaki Yuqun Xu Delong Wang Xiao Hu Masaru Tanokura Kei Yura 《BMC structural biology》2017,17(1):4
Background
More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.Results
We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.Conclusion
REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.982.
L-精氨酸是一种半必需氨基酸,广泛应用于食品、制药、饲料等行业。【目的】当前对L-精氨酸生产菌株的研究,极少涉及离子转运领域。在本研究中,发现在发酵时适量添加外源K~+有利于促进钝齿棒杆菌(Corynebacterium crenatum) SYPA5-5合成L-精氨酸。【方法】在C. crenatum SYPA5-5发酵培养基外源添加0.5 g/L和2.5 g/L的K_3PO_4,取对数期发酵样品进行转录组数据分析,挖掘出K~+转运相关的阳离子转运ATP酶CTAP1以及单价阳离子/H~+逆转运蛋白Mrp1A,研究其在C. crenatum SYPA5-5快速合成L-精氨酸阶段,对菌株生长及L-精氨酸合成的影响。【结果】对基因ctap1和mrp1分别进行敲除和过表达,深入研究突变株对L-精氨酸合成的影响。研究发现同时过表达离子转运蛋白CTAP1和Mrp1A更有利于胞内离子、pH稳态和渗透压调节,最终提高L-精氨酸的产量。在补料分批发酵中分别过表达Mrp1A、CTAP1以及同时过表达Mrp1A和CTAP1的菌株L-精氨酸产量分别达到61.4 g/L、63.9 g/L和65.3 g/L,产率分别为0.383 g/g、0.392 g/g和0.395 g/g,比C. crenatum SYPA5-5分别提高了34.9%、38.0%和39.1%。【结论】CTAP1是特异性的K~+转运ATP酶,可以将培养基中的K~+运输到胞内。同时Mrp1A可将胞内K~+和Na~+等单价阳离子运输到胞外,将胞外H~+运输至胞内,中和胞内L-精氨酸所导致的碱性环境,从而维持胞内pH稳定。CTAP1和Mrp1A的研究为解析离子转运机制和L-精氨酸合成之间的联系奠定了基础。 相似文献
983.
Hélène Pereira Jean-François Martin Charlotte Joly Jean-Louis Sébédio Estelle Pujos-Guillot 《Metabolomics : Official journal of the Metabolomic Society》2010,6(2):207-218
In order to study the effect of a diet on metabolites found in body fluids such as plasma, we have developed and validated
a UPLC/MS method. While methods using NMR have been well established to analyse different biological tissues, recent studies
have described robust untargeted UPLC-MS methods for plasma analysis. One major concern when profiling plasma is the presence
of an important quantity of proteins which have to be precipitated without any loss of metabolites prior to LC/MS analysis.
The utilization of untargeted approaches in nutritional metabolomics still suffers from the lack of identification of specific
biomarkers. We therefore suggest an alternative method still using a global approach but focusing at the same time on metabolites
previously described in human plasma in order to detect biomarkers of metabolic dysregulations. Thus, to fulfil our objectives,
analytical parameters were tested (i) the anticoagulant type for sample collection, (ii) the protein precipitation method
and (iii) UPLC/MS analytical conditions. Three protein precipitation methods and two anticoagulants were tested and compared.
The method utilizing blood collection on heparin and methanol precipitation was chosen for giving the most reproducible results
while keeping the complexity of the sample. Finally, a validation was proposed to evaluate the stability of this analytical
method applied to a large batch of samples for nutritional metabolomic studies. 相似文献
984.
杉木连栽土壤对其幼树生长的影响 总被引:6,自引:5,他引:6
在会同林区采用3种不同连栽杉木次数的土壤,进行杉木幼树盆栽试验.结果表明,杉木连栽不利其幼树生长.连裁使幼树高生长下降37—40%,基径下降19—28%,冠幅下降21—29%,总生物量减少45—50%.杉木连栽还造成土壤养分递减,即腐殖质减少7—28%,速效养分下降23—28%,土壤微生物数量和活性也大大降低.因此,杉木纯林连栽经营方式应尽快改变. 相似文献
985.
Yan Liu Zheng-lian Xue Shao-peng Chen Zhou Wang Yong Zhang Wei-liang Gong Zhi-ming Zheng 《Journal of industrial microbiology & biotechnology》2016,43(6):751-760
To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K2, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl2 before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains. 相似文献
986.
The Effect of Sterilization Methods on the Physical Properties of Silk Sericin Scaffolds 总被引:1,自引:0,他引:1
Protein-based biomaterials respond differently to sterilization methods. Since protein is a complex structure, heat, or irradiation
may result in the loss of its physical or biological properties. Recent investigations have shown that sericin, a degumming
silk protein, can be successfully formed into a 3-D scaffolds after mixing with other polymers which can be applied in skin
tissue engineering. The objective of this study was to investigate the effectiveness of ethanol, ethylene oxide (EtO) and
gamma irradiation on the sterilization of sericin scaffolds. The influence of these sterilization methods on the physical
properties such as pore size, scaffold dimensions, swelling and mechanical properties, as well as the amount of sericin released
from sericin/polyvinyl alcohol/glycerin scaffolds, were also investigated. Ethanol treatment was ineffective for sericin scaffold
sterilization whereas gamma irradiation was the most effective technique for scaffold sterilization. Moreover, ethanol also
caused significant changes in pore size resulting from shrinkage of the scaffold. Gamma-irradiated samples exhibited the highest
swelling property, but they also lost the greatest amount of weight after immersion for 24 h compared with scaffolds obtained
from other sterilization methods. The results of the maximum stress test and Young’s modulus showed that gamma-irradiated
and ethanol-treated scaffolds are more flexible than the EtO-treated and untreated scaffolds. The amount of sericin released,
which was related to its collagen promoting effect, was highest from the gamma-irradiated scaffold. The results of this study
indicate that gamma irradiation should have the greatest potential for sterilizing sericin scaffolds for skin tissue engineering. 相似文献
987.
988.
Eftimie R Dushoff J Bridle BW Bramson JL Earn DJ 《Bulletin of mathematical biology》2011,73(12):2932-2961
Recent advances in virology, gene therapy, and molecular and cell biology have provided insight into the mechanisms through
which viruses can boost the anti-tumor immune response, or can infect and directly kill tumor cells. A recent experimental
report (Bridle et al. in Molec. Ther. 18(8):1430–1439, 2010) showed that a sequential treatment approach that involves two viruses that carry the same tumor antigen leads to an improved
anti-tumor response compared to the effect of each virus alone. In this article, we derive a mathematical model to investigate
the anti-tumor effect of two viruses, and their interactions with the immune cells. We discuss the conditions necessary for
permanent tumor elimination and, in this context, we stress the importance of investigating the long-term effect of non-linear
interactions. In particular, we discuss multi-stability and multi-instability, two complex phenomena that can cause abrupt
transitions between different states in biological and physical systems. In the context of cancer immunotherapies, the transitions
between a tumor-free and a tumor-present state have so far been associated with the multi-stability phenomenon. Here, we show
that multi-instability can also cause the system to switch from one state to the other. In addition, we show that the multi-stability
is driven by the immune response, while the multi-instability is driven by the presence of the virus. 相似文献
989.
Tong Lin Jing Li Jun-jun Shao Guo-zheng Cong Jun-zheng Du Shan-dian Gao Hui-yun Chang 《Virologica Sinica》2011,(4)
In order to develop an anti-FMDV A Type monoclonal antibody (mAb),BABL/c mice were immunized with FMDV A type.Monoclonal antibodies (mAbs) 7B11 and 8H4 against Foot-and-mouth disease virus (FMDV) serotype A were produced by fusing SP2/0 myeloma cells with splenocyte from the mouse immunized with A/AV88.The microneutralization titer of the mAbs 7B11 and 8H4 were 1024 and 512,respectively.Both mAbs contain kappa light chains,the mAbs were IgG1.In order to define the mAbs binding epitopes,the reactivity of the... 相似文献
990.
Hernández-Hernández A Rincón-Arano H Recillas-Targa F Ortiz R Valdes-Quezada C Echeverría OM Benavente R Vázquez-Nin GH 《Chromosoma》2008,117(1):77-87
The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during
meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that
are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of
the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic
sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major
structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic
acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats,
satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization
of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs
and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences
play a key role in anchoring the chromosome to the protein scaffold of the SC.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献