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Orotidine-5-monophosphate decarboxylase (OMPdecase) catalyzes the final step in pyrimidine biosynthesis, the conversion of orotidine-5-monophosphate (OMP) to uridine-5-monophosphate. ThepyrF gene, encoding OMPdecase, was isolated from a chromosomal library ofPseudomonas aeruginosa PAO1 by screening for complementation of anEscherichia coli and aP. aeruginosa pyrF mutant. The nucleotide sequence of a 2510-bp chromosomal DNA fragment, complementing both strains, was determined (EMBL accession number X65613). On this a 696-bp open reading frame capable of encoding the 24 kDa OMPdecase was identified. Despite a generally good correspondence to other OMPdecase sequences, theP. aeruginosa gene was unique in that it did not constitute part of an operon. ThepyrF gene was amplified by polymerase chain reaction, overexpressed in the pT7-7/E. coli BL21(DE3) system and purified to near electrophoretic homogeneity by anion exchange chromatography. Characterization of the purified enzyme revealed the following data, aK
m value for OMP of 9.91 M and an isoelectric point of 6.65. No major decrease in enzyme activity was observed in a pH range between 7.8 and 10.2. Gel electrophoresis under nondenaturing conditions suggested that the native form of OMPdecase is the dimer. 相似文献
64.
Miles TJ Barfoot C Brooks G Brown P Chen D Dabbs S Davies DT Downie DL Eyrisch S Giordano I Gwynn MN Hennessy A Hoover J Huang J Jones G Markwell R Rittenhouse S Xiang H Pearson N 《Bioorganic & medicinal chemistry letters》2011,21(24):7483-7488
As part of our wider efforts to exploit novel mode of action antibacterials, we have discovered a series of cyclohexyl-amide compounds that has good Gram positive and Gram negative potency. The mechanism of action is via inhibition of bacterial topoisomerases II and IV. We have investigated various subunits in this series and report advanced studies on compound 7 which demonstrates good PK and in vivo efficacy properties. 相似文献
65.
Jaclyn L. Myers Katherine S. Wetzel Susanne L. Linderman Yang Li Colleen B. Sullivan Scott E. Hensley 《Journal of virology》2013,87(20):11168-11172
Influenza viruses routinely acquire mutations in antigenic sites on the globular head of the hemagglutinin (HA) protein. Since these antigenic sites are near the receptor binding pocket of HA, many antigenic mutations simultaneously alter the receptor binding properties of HA. We previously reported that a K165E mutation in the Sa antigenic site of A/Puerto Rico/8/34 (PR8) HA is associated with secondary neuraminidase (NA) mutations that decrease NA activity. Here, using reverse genetics, we show that the K165E HA mutation dramatically decreases HA binding to sialic acid receptors on cell surfaces. We sequentially passaged reverse-genetics-derived PR8 viruses with the K165E antigenic HA mutation in fertilized chicken eggs, and to our surprise, viruses with secondary NA mutations did not emerge. Instead, viruses with secondary HA mutations emerged in 3 independent passaging experiments, and each of these mutations increased HA binding to sialic acid receptors. Importantly, these compensatory HA mutations were located in the Ca antigenic site and prevented binding of Ca-specific monoclonal antibodies. Taken together, these data indicate that HA antigenic mutations that alter receptor binding avidity can be compensated for by secondary HA or NA mutations. Antigenic diversification of influenza viruses can therefore occur irrespective of direct antibody pressure, since compensatory HA mutations can be located in distinct antibody binding sites. 相似文献
66.
The development of an effective vaccine is urgently needed to fight tuberculosis (TB) which is still the leading cause of death from a single infectious agent worldwide. One of the promising vaccine candidates M72/AS01E consists of two proteins subunits PepA and PPE18 coded by Rv0125 and Rv1196. However, preliminary data indicate a high level of sequence variability among clinical Mycobacterium tuberculosis complex (MTBC) strains that might have an impact on the vaccine efficacy. To further investigate this finding, we determined ppE18 sequence variability in a well-characterized reference collection of 71 MTBC strains from 23 phylogenetic lineages representing the global MTBC diversity. In total, 100 sequence variations consisting of 96 single nucleotide polymorphisms (SNPs), three insertions and one deletion were detected resulting in 141 variable positions distributed over the entire gene. The majority of SNPs detected were non-synonymous (n = 68 vs. n = 28 synonymous). Strains from animal adapted lineages, e.g., M. bovis, showed a significant higher diversity than the human pathogens such as M. tuberculosis Haarlem. SNP patterns specific for different lineages as well as for deeper branches in the phylogeny could be identified. The results of our study demonstrate a high variability of the ppE18 gene even in the N-terminal domains that is normally highly conserved in ppe genes. As the N-terminal region interacts with TLR2 receptor inducing a protective anti-inflammatory immune response, genetic heterogeneity has a potential impact on the vaccine efficiency, however, this has to be investigated in future studies. 相似文献
67.
Conservation of plasmids among plant-pathogenic Pseudomonas syringae isolates of diverse origins 总被引:3,自引:0,他引:3
Thirty isolates of Pseudomonas syringae pv. tabaci, pv. angulata (pathogens on tobacco), pv. coronafaciens, and pv. striafaciens (pathogens on oats) were examined for plasmid DNAs. The strains were obtained from plants throughout the world, some over 50 years ago. Of the 22 tobacco pathogens, 16 contain predominantly one type of plasmid, the pJP27.00 type. The remaining six tobacco-specific strains do not harbor detectable plasmids. The oat pathogens contain one, two, or three plasmids. DNA homology studies indicate that the plasmid DNAs are highly conserved. More importantly, the plasmids harbored by strains isolated from one host plant are conserved most stringently; e.g., the plasmids from the tobacco pathogens are, with one exception, indistinguishable by restriction endonuclease digestion and Southern hybridization. There is also extensive homology among plasmids indigenous to the oat-specific P. syringae pv. coronafaciens and pv. striafaciens strains. 相似文献
68.
Fargol Mazaheri Nicolas Snaidero Gernot Kleinberger Charlotte Madore Anna Daria Georg Werner Susanne Krasemann Anja Capell Dietrich Trümbach Wolfgang Wurst Bettina Brunner Sebastian Bultmann Sabina Tahirovic Martin Kerschensteiner Thomas Misgeld Oleg Butovsky Christian Haass 《EMBO reports》2017,18(7):1186-1198
69.
Isolation and characterization of circulating tumor cells using a novel workflow combining the CellSearch® system and the CellCelector™ 下载免费PDF全文
Martin Horst Dieter Neumann Helen Schneck Yvonne Decker Susanne Schömer André Franken Volker Endris Nicole Pfarr Wilko Weichert Dieter Niederacher Tanja Fehm Hans Neubauer 《Biotechnology progress》2017,33(1):125-132
Circulating tumor cells (CTC) are rare cells which have left the primary tumor to enter the blood stream. Although only a small CTC subgroup is capable of extravasating, the presence of CTCs is associated with an increased risk of metastasis and a shorter overall survival. Understanding the heterogeneous CTC biology will optimize treatment decisions and will thereby improve patient outcome. For this, robust workflows for detection and isolation of CTCs are urgently required. Here, we present a workflow to characterize CTCs by combining the advantages of both the CellSearch® and the CellCelector? micromanipulation system. CTCs were isolated from CellSearch® cartridges using the CellCelector? system and were deposited into PCR tubes for subsequent molecular analysis (whole genome amplification (WGA) and massive parallel multigene sequencing). By a CellCelector? screen we reidentified 97% of CellSearch® SKBR‐3 cells. Furthermore, we isolated 97% of CellSearch®‐proven patient CTCs using the CellCelector? system. Therein, we found an almost perfect correlation of R2 = 0.98 (Spearman's rho correlation, n = 20, p < 0.00001) between the CellSearch® CTC count (n = 271) and the CellCelector? detected CTCs (n = 252). Isolated CTCs were analyzed by WGA and massive parallel multigene sequencing. In total, single nucleotide polymorphisms (SNPs) could be detected in 50 genes in seven CTCs, 12 MCF‐7, and 3 T47D cells, respectively. Taken together, CTC quantification via the CellCelector? system ensures a comprehensive detection of CTCs preidentified by the CellSearch® system. Moreover, the isolation of CTCs after CellSearch® using the CellCelector? system guarantees for CTC enrichment without any contaminants enabling subsequent high throughput genomic analyses on single cell level. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:125–132, 2017 相似文献
70.
Anton Pauw Sunshine A. Van Bael Halton A. Peters Steven D. Allison José L. C. Camargo Miguel Cifuentes-Jara Aurlstela Conserva Teresa Garcia Restom Tamara Heartsill-Scalley Scott A. Mangan Gabriela Nunez-lturri Elsie Rivera-Ocasio Mark Rountree Susanne Vetter Carolina Volkmer de Castllho 《Biotropica》2004,36(3):410-413