全文获取类型
收费全文 | 629篇 |
免费 | 60篇 |
出版年
2022年 | 2篇 |
2021年 | 14篇 |
2020年 | 4篇 |
2019年 | 6篇 |
2018年 | 8篇 |
2017年 | 8篇 |
2016年 | 18篇 |
2015年 | 24篇 |
2014年 | 40篇 |
2013年 | 47篇 |
2012年 | 72篇 |
2011年 | 61篇 |
2010年 | 41篇 |
2009年 | 22篇 |
2008年 | 38篇 |
2007年 | 34篇 |
2006年 | 33篇 |
2005年 | 26篇 |
2004年 | 29篇 |
2003年 | 14篇 |
2002年 | 18篇 |
2001年 | 13篇 |
2000年 | 15篇 |
1999年 | 8篇 |
1998年 | 5篇 |
1997年 | 4篇 |
1996年 | 2篇 |
1994年 | 3篇 |
1993年 | 3篇 |
1992年 | 11篇 |
1990年 | 7篇 |
1989年 | 5篇 |
1988年 | 8篇 |
1987年 | 5篇 |
1986年 | 3篇 |
1985年 | 2篇 |
1984年 | 3篇 |
1983年 | 2篇 |
1982年 | 4篇 |
1981年 | 3篇 |
1977年 | 3篇 |
1976年 | 6篇 |
1975年 | 2篇 |
1974年 | 2篇 |
1972年 | 2篇 |
1971年 | 1篇 |
1968年 | 1篇 |
1953年 | 1篇 |
1951年 | 1篇 |
1944年 | 1篇 |
排序方式: 共有689条查询结果,搜索用时 140 毫秒
41.
Jui-Hsin Huang Zhe-Qing Shen Shu-Pei Lien Kuang-Nan Hsiao Chih-Hsiang Leng Chi-Chang Chen Leung-Kei Siu Pele Choi-Sing Chong 《PloS one》2015,10(8)
Clostridium difficile is an emerging pathogen responsible for opportunistic infections in hospitals worldwide and is the main cause of antibiotic-associated pseudo-membranous colitis and diarrhea in humans. Clostridial toxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) on the surface of epithelial cells in the host intestine, disrupting the intestinal barrier and ultimately leading to acute inflammation and diarrhea. The C-terminal receptor-binding domain (RBD) of TcdA, which is responsible for the initial binding of the toxin to host glycoproteins, has been predicted to contain 7 potential oligosaccharide-binding sites. To study the specific roles and functions of these 7 putative lectin-like binding regions, a consensus sequence of TcdA RBD derived from different C. difficile strains deposited in the NCBI protein database and three truncated fragments corresponding to the N-terminal (residues 1–411), middle (residues 296–701), and C-terminal portions (residues 524–911) of the RBD (F1, F2 and F3, respectively) were designed and expressed in Escherichia coli. In this study, the recombinant RBD (rRBD) and its truncated fragments were purified, characterized biologically and found to have the following similar properties: (a) are capable of binding to the cell surface of both Vero and Caco-2 cells; (b) possess Toll-like receptor agonist-like adjuvant activities that can activate dendritic cell maturation and increase the secretion of pro-inflammatory cytokines; and (c) function as potent adjuvants in the intramuscular immunization route to enhance immune responses against weak immunogens. Although F1, F2 and F3 have similar repetitive amino acid sequences and putative oligosaccharide-binding domains, they do not possess the same biological and immunological properties: (i) TcdA rRBD and its fragments bind to the cell surface, but only TcdA rRBD and F3 internalize into Vero cells within 15 min; (ii) the fragments exhibit various levels of hemagglutinin (HA) activity, with the exception of the F1 fragment, which demonstrates no HA activity; and (iii) in the presence of alum, all fragments elicit various levels of anti-toxin A-neutralizing antibody responses, but those neutralizing antibodies elicited by F2 did not protect mice against a TcdA challenge. Because TcdA rRBD, F1 and F3 formulated with alum can elicit immune protective responses against the cytotoxicity of TcdA, they represent potential components of future candidate vaccines against C. difficile-associated diseases. 相似文献
42.
Ling Ma Jann-Tay Wang Tsu-Lan Wu L. Kristopher Siu Yin-Ching Chuang Jung-Chung Lin Min-Chi Lu Po-Liang Lu 《PloS one》2015,10(9)
The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four bla
OXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured bla
CTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the bla
OXA-48 gene in all four isolates was identical to pKPOXA-48N1, a bla
OXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify bla
OXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of bla
OXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern. 相似文献
43.
44.
Xinghuo Wu Shek Man Chim Vincent Kuek Bay Sie Lim Siu To Chow Jinmin Zhao Shuhua Yang Vicki Rosen Jennifer Tickner Jiake Xu 《FEBS letters》2014
Bone remodeling is regulated by secreted factors in the bone microenvironment. However, data regarding osteoclast-derived factors that influence osteoblast differentiation are lacking. Here, we show that HtrA1 is produced as a secreted protein during osteoclastogenesis, and negatively regulates osteoblast differentiation. Exogenous addition of recombinant HtrA1 attenuates osteoblast differentiation and BMP2-induced Smad1/5/8, ERK1/2 and p38 phosphorylation in pre-osteoblasts. Our studies imply a unique mode of crosstalk in which HtrA1 is produced by both osteoclasts and osteoblasts and negatively regulates osteoblast differentiation, suggesting that HtrA1 may mediate the fine tuning of paracrine and autocrine regulations during bone remodeling processes. 相似文献
45.
Kim-Tat Teoh Yu-Lam Siu Wing-Lim Chan Marc A. Schl��ter Chia-Jen Liu J. S. Malik Peiris Roberto Bruzzone Benjamin Margolis B��atrice Nal 《Molecular biology of the cell》2010,21(22):3838-3852
Intercellular tight junctions define epithelial apicobasal polarity and form a physical fence which protects underlying tissues from pathogen invasions. PALS1, a tight junction-associated protein, is a member of the CRUMBS3-PALS1-PATJ polarity complex, which is crucial for the establishment and maintenance of epithelial polarity in mammals. Here we report that the carboxy-terminal domain of the SARS-CoV E small envelope protein (E) binds to human PALS1. Using coimmunoprecipitation and pull-down assays, we show that E interacts with PALS1 in mammalian cells and further demonstrate that the last four carboxy-terminal amino acids of E form a novel PDZ-binding motif that binds to PALS1 PDZ domain. PALS1 redistributes to the ERGIC/Golgi region, where E accumulates, in SARS-CoV–infected Vero E6 cells. Ectopic expression of E in MDCKII epithelial cells significantly alters cyst morphogenesis and, furthermore, delays formation of tight junctions, affects polarity, and modifies the subcellular distribution of PALS1, in a PDZ-binding motif-dependent manner. We speculate that hijacking of PALS1 by SARS-CoV E plays a determinant role in the disruption of the lung epithelium in SARS patients. 相似文献
46.
Leroi V. DeSouza Olga Krakovska Marlene M. Darfler David B. Krizman Alexander D. Romaschin Terence J. Colgan K. W. Michael Siu 《Proteomics》2010,10(17):3108-3116
Formalin‐fixed paraffin‐embedded (FFPE) tissues are the primary and preferred medium for archiving patients' samples. Here we demonstrate relative quantifications of protein biomarkers in extracts of laser microdissected epithelial cells from FFPE endometrial carcinoma tissues versus those from normal proliferative endometria by means of targeted proteomic analyses using LC–multiple reaction monitoring (MRM) MS with MRM Tags for Relative and Absolute Quantitation (mTRAQ) labeling. Comparable results of differential expressions for pyruvate kinase isoform M2 (PK‐M2) and polymeric Ig receptor were observed between analyses on laser microdissected epithelial cells from FFPE tissues and corresponding homogenates from frozen tissues of the same individuals that had previously been analyzed and reported. We also identified PK‐M2 in the normal proliferative phase of the endometrium. Other biomarkers in addition to PK‐M2 and polymeric Ig receptor were also observed but not consistently and/or were at levels below the threshold for quantification. 相似文献
47.
The Influence of Different Land Uses on the Structure of Archaeal Communities in Amazonian Anthrosols Based on 16S rRNA and amoA Genes 总被引:2,自引:0,他引:2
Soil from the Amazonian region is usually regarded as unsuitable for agriculture because of its low organic matter content and low pH; however, this region also contains extremely rich soil, the Terra Preta Anthrosol. A diverse archaeal community usually inhabits acidic soils, such as those found in the Amazon. Therefore, we hypothesized that this community should be sensitive to changes in the environment. Here, the archaeal community composition of Terra Preta and adjacent soil was examined in four different sites in the Brazilian Amazon under different anthropic activities. The canonical correspondence analysis of terminal restriction fragment length polymorphisms has shown that the archaeal community structure was mostly influenced by soil attributes that differentiate the Terra Preta from the adjacent soil (i.e., pH, sulfur, and organic matter). Archaeal 16S rRNA gene clone libraries indicated that the two most abundant genera in both soils were Candidatus nitrosphaera and Canditatus nitrosocaldus. An ammonia monoxygenase gene (amoA) clone library analysis indicated that, within each site, there was no significant difference between the clone libraries of Terra Preta and adjacent soils. However, these clone libraries indicated there were significant differences between sites. Quantitative PCR has shown that Terra Preta soils subjected to agriculture displayed a higher number of amoA gene copy numbers than in adjacent soils. On the other hand, soils that were not subjected to agriculture did not display significant differences on amoA gene copy numbers between Terra Preta and adjacent soils. Taken together, our findings indicate that the overall archaeal community structure in these Amazonian soils is determined by the soil type and the current land use. 相似文献
48.
Diversity and identification of methanogenic archaea and sulphate-reducing bacteria in sediments from a pristine tropical mangrove 总被引:1,自引:0,他引:1
Rodrigo Gouvêa Taketani Caio Augusto Yoshiura Armando Cavalcante Franco Dias Fernando Dini Andreote Siu Mui Tsai 《Antonie van Leeuwenhoek》2010,97(4):401-411
Mangrove sediments are anaerobic ecosystems rich in organic matter. This environment is optimal for anaerobic microorganisms,
such as sulphate-reducing bacteria and methanogenic archaea, which are responsible for nutrient cycling. In this study, the
diversity of these two functional guilds was evaluated in a pristine mangrove forest using denaturing gradient gel electrophoresis
(DGGE) and clone library sequencing in a 50 cm vertical profile sampled every 5.0 cm. DGGE profiles indicated that both groups
presented higher richness in shallow samples (0–30 cm) with a steep decrease in richness beyond that depth. According to redundancy
analysis, this alteration significantly correlated with a decrease in the amount of organic matter. Clone library sequencing
indicated that depth had a strong effect on the selection of dissimilatory sulphate reductase (dsrB) operational taxonomic units (OTUs), as indicated by the small number of shared OTUs found in shallow (0.0 cm) and deep
(40.0 cm) libraries. On the other hand, methyl coenzyme-M reductase (mcrA) libraries indicated that most of the OTUs found in the shallow library were present in the deep library. These results
show that these two guilds co-exist in these mangrove sediments and indicate important roles for these organisms in nutrient
cycling within this ecosystem. 相似文献
49.
Tucholska M Scozzaro S Williams D Ackloo S Lock C Siu KW Evans KR Marshall JG 《Analytical biochemistry》2007,370(2):228-245
Blood peptides can be concentrated, extracted, and analyzed with strong signal-to-noise ratios by precipitation in organic solvents followed by extraction in water. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) hybrid quadrupole time-of-flight (Qq-TOF) were used to analyze the precipitated and extracted endogenous peptides from fetal calf serum. C18 solid-phase extraction with or without prior precipitation in ammonium sulfate, size exclusion chromatography, dealbuminization, dye affinity chromatography, ultrafiltration, and differential precipitation in organic solvents were compared. Hundreds of different ions could be observed by MALDI in the various fractions. It appeared that some peptides were freely dissolved and that not all peptides in blood were obliged to remain bound to albumin or other high-molecular-mass proteins. Mass spectra with high signal-to-noise ratios were obtained from polypeptides precipitated with organic solvents followed by extraction of the peptides from the pellet with water. The peptides extracted from organic precipitates were analyzed by nano liquid chromatography (LC)-ESI-Qq-TOF. In addition to many commonly abundant serum proteins, apparent low-abundance peptides associated with cancer biology from proteins such as insulin-like growth factor II, thymosin beta4 and beta9, plasminogen, coagulation factors, and extracellular matrix protein 1 were observed. 相似文献
50.
We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells. 相似文献