全文获取类型
收费全文 | 3556篇 |
免费 | 242篇 |
国内免费 | 378篇 |
出版年
2024年 | 10篇 |
2023年 | 95篇 |
2022年 | 89篇 |
2021年 | 135篇 |
2020年 | 169篇 |
2019年 | 218篇 |
2018年 | 172篇 |
2017年 | 200篇 |
2016年 | 159篇 |
2015年 | 154篇 |
2014年 | 251篇 |
2013年 | 344篇 |
2012年 | 143篇 |
2011年 | 220篇 |
2010年 | 158篇 |
2009年 | 190篇 |
2008年 | 183篇 |
2007年 | 201篇 |
2006年 | 155篇 |
2005年 | 155篇 |
2004年 | 133篇 |
2003年 | 108篇 |
2002年 | 95篇 |
2001年 | 49篇 |
2000年 | 41篇 |
1999年 | 35篇 |
1998年 | 33篇 |
1997年 | 47篇 |
1996年 | 32篇 |
1995年 | 23篇 |
1994年 | 20篇 |
1993年 | 21篇 |
1992年 | 26篇 |
1991年 | 13篇 |
1990年 | 17篇 |
1989年 | 9篇 |
1988年 | 6篇 |
1987年 | 7篇 |
1986年 | 6篇 |
1985年 | 12篇 |
1984年 | 11篇 |
1983年 | 10篇 |
1982年 | 2篇 |
1981年 | 7篇 |
1979年 | 2篇 |
1978年 | 2篇 |
1977年 | 3篇 |
1976年 | 2篇 |
1974年 | 1篇 |
1971年 | 1篇 |
排序方式: 共有4176条查询结果,搜索用时 31 毫秒
991.
992.
Pasqualina D’Ursi Federica Chiappori Ivan Merelli Ermanna Rovida 《Biochemical and biophysical research communications》2009,383(4):445-449
The H5N1 virus neuraminidase structure was solved in two different conformations depending on the inhibitor concentration. In the absence of oseltamivir or at a low concentration, the neuraminidase structure assumes an open form that closes at a high oseltamivir concentration due to the shift of the so-called 150-loop near the active site. Although the close conformation is similar to all the other structurally known neuraminidase types, it doesn’t appear to be the most likely physiological condition for N1.To investigate the specific ligand binding properties of the open form, we screened by docking simulation, a large dataset of ligands and compared the results with closed form. The virtual screening procedure was implemented in a docking pipeline that also performs a step-by-step, target specific, filtering approach for data reduction. The selected ligands display binding ability involving multiple sites of interaction including the active site and an adjacent cavity made available by the 150-loop shift. Two ligands are especially interesting and are proposed as substituents to design oseltamivir derivatives specifically suited for the open conformation. 相似文献
993.
Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[32P]pyrophosphate (PPi) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (Pi) concentrations after degradation by inorganic pyrophosphatase of the PPi released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A4, one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[32P]PPi exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover. 相似文献
994.
The prevalence of drug-resistant strains of Mycobacterium tuberculosis (M. tb) emphasizes the need for new antitubercular drugs. An essential component of the drug discovery process is the development of tools to rapidly screen potential drug libraries against important biological targets. Similarly to well-documented M. tb targets, the antigen 85 (Ag85) enzymes are involved in the maintenance of the mycobacterial cell wall. The products synthesized by these mycolyltransferases are the cell wall components most responsible for the reduced permeability of drugs into the bacterial cell, thereby linking Ag85 activity directly with drug resistance. This article presents the development of a high-throughput colorimetric assay suitable for direct monitoring of the enzymatic activity. The assay uses a synthetic substrate containing three chemical moieties: an octanoyl fatty acid, β-d-glucose, and p-nitrophenyl. In the context of the assay, Ag85 catalyzes the removal of the fatty acid and releases p-nitrophenyl-β-d-glucoside. The glucoside is hydrolyzed by β-glucosidase to release the p-nitrophenolate chromophore. With this assay, the KM and kcat values of Ag85C were determined to be 0.047 ± 0.008 mM and 0.062 s−1, respectively. In addition, the assay exhibits a Z′ value of 0.81 ± 0.06, indicating its suitability for high-throughput screening applications and drug development. 相似文献
995.
This study describes generation of an anti-PBAN receptor (PBAN-R) antiserum and its employment for the characterization of the PK/PBAN-R(s). The antiserum recognized, in a specific and dose-dependent manner, the presence of PBAN-R in pheromone gland membrane preparations of three female moths: Heliothis peltigera, Helicoverpa armigera and Spodoptera littoralis. It also reacted specifically with the S. littoralis larval receptor in vivo, most likely by competing with the ligand on the binding site and consequently inhibiting cuticular melanization. Despite its ability to react with the receptor of H. peltigera in dot blot experiments, the antiserum did not react with the receptor in vivo and failed to inhibit sex pheromone biosynthesis. The antiserum was also used to develop two microplate binding assays. The Ab described in this study is the first raised against an insect neuropeptide (Np) receptor to be used in vivo, and its employment for characterization of the PK/PBAN-R(s) may thus provide important information on the mode of action of this Np family. The present study adds important information on the difference between the receptors in the two moth species, hints at the possible existence of receptor subtypes, and provides a platform for the development of a high-throughput assay (HTA) for screening of PK/PBAN agonists and antagonists. 相似文献
996.
Marla Tuffin Dominique Anderson Cal Heath Don A. Cowan Professor 《Biotechnology journal》2009,4(12):1671-1683
Metagenomics emerged in the late 1990s as a tool for accessing and studying the collective microbial genetic material in the environment. The advent of the technology generated great excitement, as it has provided new opportunities and technologies for studying the wealth of microbial genetic diversity in the environment. Metagenomics has been widely predicted to access new dimensions of protein sequence space. A decade on, we review how far we have actually moved into new sequence space (and other aspects of protein space) using metagenomic tools. While several novel enzyme activities and protein structures have been identified through metagenomic strategies, the greatest advancement has been made in the isolation of novel protein sequences, some of which have no close relatives, form deeply branched lineages and even represent novel families. This is particularly true for glycosyl hydrolases and lipase/esterases, despite the fact that these activities are frequently screened for in metagenomic studies. However, there is much room for improvement in the methods employed and they will need to be addressed so that access to novel biocatalytic activities can be widened. 相似文献
997.
Rojas JL Martín J Tormo JR Vicente F Brunati M Ciciliato I Losi D Van Trappen S Mergaert J Swings J Marinelli F Genilloud O 《Marine Genomics》2009,2(1):33-41
During the MICROMAT project, the bacterial diversity of microbial mats growing in the benthic environment of Antarctic lakes was accessed for the discovery of novel antibiotics. In all, 723 Antarctic heterotrophic bacteria belonging to novel and/or endemic taxa in the α-, β- and γ-subclasses of the Proteobacteria, the Bacteroidetes branch, and of the high and low percentage G+C Gram-positives, were isolated, cultivated in different media and at different temperatures, and then screened for the production of antimicrobial activities. A total of 6348 extracts were prepared by solid phase extraction of the culture broths or by biomass solvent extraction. 122 bacteria showed antibacterial activity against the Gram-positives Staphylococcus aureus and to a lower extent Enterococcus faecium, and versus the Gram-negative Escherichia coli. Few of these strains showed also some antifungal activity against Cryptococcus neoformans, Aspergillus fumigatus and to a lower extent Candida albicans. LC–MS fractionation of extracts from a subset of strains (hits) that exhibited relatively potent antibacterial activities evidenced a chemical novelty that was further investigated. Two strains of Arthrobacter agilis produced potent antibacterial compounds with activity against Gram-positives and possibly related to novel cyclic thiazolyl peptides. To our knowledge, this is the first report of new antibiotics produced by bacteria from benthic microbial mats from Antarctic lakes. With no doubts these microbial assemblages represent an extremely rich source for the isolation of new strains producing novel bioactive metabolites with the potential to be developed as antibiotic compounds. 相似文献
998.
Phosphatase of regenerating liver-1 (PRL-1) is a novel target for potentially treating cancer metastases. Although its specific
biochemical role in these processes has yet to be delineated, considerable evidence suggests the phosphatase activity of PRL-1
is required for promoting cancer and metastasis. PRL-1 belongs to the protein tyrosine phosphatase (PTPase) family and functions
using the CX5R consensus active site motif. Like other PTPases, PRL-1 is inhibited by oxidation at its active site Cys, however, disulfide
bond formation occurs unusually readily in wild-type PRL-1. Chemical shift assignments are available for oxidized wild type,
but numerous, substantial changes are observed in the spectra upon reduction. Because the reduced form is active, we sought
to identify a stable mutant that would resist oxidation and be useful for facilitating drug screening and development using
NMR-based assays. We present here NMR assignments for a full-length, reduced and active form of PRL-1, PRL-1-C170S-C171S,
that is well suited for this purpose. 相似文献
999.
Takayuki Y. Nara Hideaki Togashi Chisato Sekikawa Kengo Sakaguchi Fujio Mizukami Tatsuo Tsunoda 《Biotechnology progress》2009,25(4):1071-1077
We established a 96‐well‐plate‐based refolding screening system using zeolite. In this system, protein denatured and solubilized with 6 M guanidine hydrochloride is adsorbed onto zeolite placed in a 96‐well plate. The refolding conditions can be tested by incubating the samples with refolding buffers under various conditions of pH, salts, and additives. In this study, we chose green fluorescent protein as the model protein. Green fluorescent protein was expressed as inclusion bodies, and we tested the effects of four pH conditions and six additives on its refolding. The results demonstrate that green fluorescent protein was more efficiently refolded with zeolite than with the conventional dilution method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
1000.