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101.
Dwight SS Balakrishnan R Christie KR Costanzo MC Dolinski K Engel SR Feierbach B Fisk DG Hirschman J Hong EL Issel-Tarver L Nash RS Sethuraman A Starr B Theesfeld CL Andrada R Binkley G Dong Q Lane C Schroeder M Weng S Botstein D Cherry JM 《Briefings in bioinformatics》2004,5(1):9-22
A scientific database can be a powerful tool for biologists in an era where large-scale genomic analysis, combined with smaller-scale scientific results, provides new insights into the roles of genes and their products in the cell. However, the collection and assimilation of data is, in itself, not enough to make a database useful. The data must be incorporated into the database and presented to the user in an intuitive and biologically significant manner. Most importantly, this presentation must be driven by the user's point of view; that is, from a biological perspective. The success of a scientific database can therefore be measured by the response of its users - statistically, by usage numbers and, in a less quantifiable way, by its relationship with the community it serves and its ability to serve as a model for similar projects. Since its inception ten years ago, the Saccharomyces Genome Database (SGD) has seen a dramatic increase in its usage, has developed and maintained a positive working relationship with the yeast research community, and has served as a template for at least one other database. The success of SGD, as measured by these criteria, is due in large part to philosophies that have guided its mission and organisation since it was established in 1993. This paper aims to detail these philosophies and how they shape the organisation and presentation of the database. 相似文献
102.
Automation aided optimization of cloning,expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies 下载免费PDF全文
Sneha Bairy Lakshmi Narayanan Gopalan Thanuja Gangi Setty Sathya Srinivasachari Lavanyaa Manjunath Jay Prakash Kumar Sai R Guntupalli Sucharita Bose Vinod Nayak Swagatha Ghosh Nitish Sathyanarayanan Rhawnie Caing‐Carlsson Weixiao Yuan Wahlgren Rosmarie Friemann S. Ramaswamy Muniasamy Neerathilingam 《Microbial biotechnology》2018,11(2):420-428
The process of obtaining a well‐expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His‐tagged Gateway vector, followed by their small‐scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large‐scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins. 相似文献
103.
Susanna Esposito Alberto Zampiero Sonia Bianchini Alessandro Mori Alessia Scala Claudia Tagliabue Calogero Sathya Sciarrabba Emilio Fossali Antonio Piralla Nicola Principi 《PloS one》2016,11(4)
To evaluate the predominant human adenovirus (HAdV) species and types associated with pediatric respiratory infections, nasopharyngeal swabs were collected from otherwise healthy children attending an emergency room in Milan, Italy, due to a respiratory tract infection from January 1 to February 28 of two subsequent years, 2013 and 2014. The HAdVs were detected using a respiratory virus panel fast assay (xTAG RVP FAST v2) and with a HAdV-specific real-time polymerase chain reaction; their nucleotides were sequenced, and they were tested for positive selection. Among 307 nasopharyngeal samples, 61 (19.9%) tested positive for HAdV. HAdV was the only virus detected in 31/61 (50.8%) cases, whereas it was found in association with one other virus in 25 (41.0%) cases and with two or more viruses in 5 (8.2%) cases. Human Enterovirus/human rhinovirus and respiratory syncytial virus were the most common co-infecting viral agents and were found in 12 (19.7%) and 7 (11.5%) samples, respectively. Overall, the HAdV strain sequences analyzed were highly conserved. In comparison to HAdV-negative children, those infected with HAdV had a reduced frequency of lower respiratory tract involvement (36.1% vs 55.2%; p = 0.007), wheezing (0.0% vs 12.5%; p = 0.004), and hospitalization (27.9% vs 56.1%; p<0.001). Antibiotic therapy and white blood cell counts were more frequently prescribed (91.9% vs 57.1%; p = 0.04) and higher (17,244 ± 7,737 vs 9,565 ± 3,211 cells/μL; p = 0.04), respectively, in children infected by HAdV-C than among those infected by HAdV-B. On the contrary, those infected by HAdV-B had more frequently lower respiratory tract involvement (57.1% vs 29.7%) but difference did not reach statistical significant (p = 0.21). Children with high viral load were absent from child care attendance for a longer period of time (14.5 ± 7.5 vs 5.5 ± 3.2 days; p = 0.002) and had higher C reactive protein levels (41.3 ± 78.5 vs 5.4 ± 9.6 μg/dL; p = 0.03). This study has shown that HAdV infections are diagnosed more commonly than usually thought and that HAdVs are stable infectious agents that do not frequently cause severe diseases. A trend toward more complex disease in cases due to HAdV species C and in those with higher viral load was demonstrated. However, further studies are needed to clarify factors contributing to disease severity to understand how to develop adequate preventive and therapeutic measures. 相似文献
104.
Sethuraman M Clavreul N Huang H McComb ME Costello CE Cohen RA 《Free radical biology & medicine》2007,42(6):823-829
p21ras GTPase is the protein product of the most commonly mutated human oncogene and has been identified as a target for reactive oxygen and nitrogen species. Posttranslational modification of reactive thiols, by reversible S-glutathiolation and S-nitrosation, and potentially also by irreversible oxidation, may have significant effects on p21ras activity. Here we used an isotope-coded affinity tag (ICAT) and mass spectrometry to quantitate the reversible and irreversible oxidative posttranslational thiol modifications of p21ras caused by peroxynitrite (ONOO(-)) or glutathione disulfide (GSSG). The activity of p21ras was significantly increased after exposure to GSSG, but not to ONOO(-). The results of LC-MS/MS analysis of tryptic peptides of p21ras treated with ONOO(-) showed that ICAT labeling of Cys(118) was decreased by 47%, whereas Cys(80) was not significantly affected and was thereby shown to be less reactive. The extent of S-glutathiolation of Cys(118) by GSSG was 53%, and that of the terminal cysteines was 85%, as estimated by the decrease in ICAT labeling. The changes in ICAT labeling caused by GSSG were reversible by chemical reduction, but those caused by peroxynitrite were irreversible. The quantitative changes in thiol modification caused by GSSG associated with increased activity demonstrate the potential importance of redox modulation of p21ras. 相似文献
105.
Sethuraman N Fraser MJ Eggleston P O'Brochta DA 《Insect biochemistry and molecular biology》2007,37(9):941-951
The post-integration activity of piggyBac transposable element gene vectors in Aedes aegypti mosquitoes was tested under a variety of conditions. The embryos from five independent transgenic lines of Ae. aegypti, each with a single integrated non-autonomous piggyBac transposable element gene vector, were injected with plasmids containing the piggyBac transposase open-reading frame under the regulatory control of the Drosophila melanogaster hsp70 promoter. No evidence for somatic remobilization was detected in the subsequent adults whereas somatic remobilization was readily detected when similar lines of transgenic D. melanogaster were injected with the same piggyBac transposase-expressing plasmid. Ae. aegypti heterozygotes of piggyBac reporter-containing transgenes and piggyBac transposase-expressing transgenes showed no evidence of somatic and germ-line remobilization based on phenotypic and molecular detection methods. The post-integration mobility properties of piggyBac in Ae. aegypti enhance the utility of this gene vector for certain applications, particularly those where any level of vector remobilization is unacceptable. 相似文献
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107.
M D Driscoll G Sathya L F Saidi M S DeMott R Hilf R A Bambara 《Molecular endocrinology (Baltimore, Md.)》1999,13(6):958-968
Estrogen-inducible genes contain an enhancer called the estrogen response element (ERE), a double-stranded inverted repeat. The estrogen receptor (ER) is generally thought to bind to the double-stranded ERE. However, some reports provide evidence that an ER homodimer can bind a single strand of the ERE and suggest that single-stranded ERE binding is the preferred binding mode for ER. Since these two models describe quite different mechanisms of receptor action, we have attempted to reconcile the observations. Analyzing DNA structure by nuclease sensitivity, we found that two identical molecules of a single strand of DNA containing the ERE sequence can partially anneal in an antiparallel manner. Bimolecular annealing produces double-stranded inverted repeats, with adjacent unannealed tails. The amount of annealing correlates exactly with the ability of ER to bind bimolecular EREs. Either strand of an ERE could anneal to itself in a way that would bind ER. We conclude that ER binds only the annealed double-stranded ERE both in vitro and in vivo. 相似文献
108.
Gavin C. Barnard Angela R. Kull Nathan S. Sharkey Seemab S. Shaikh Alissa M. Rittenhour Irina Burnina Youwei Jiang Fang Li Heather Lynaugh Teresa Mitchell Juergen H. Nett Adam Nylen Thomas I. Potgieter Bianka Prinz Sandra E. Rios Dongxing Zha Natarajan Sethuraman Terrance A. Stadheim Piotr Bobrowicz 《Journal of industrial microbiology & biotechnology》2010,37(9):961-971
The methylotrophic yeast Pichia pastoris has recently been engineered to express therapeutic glycoproteins with uniform human N-glycans at high titers. In contrast to the current art where producing therapeutic proteins in mammalian cell lines yields a final product with heterogeneous N-glycans, proteins expressed in glycoengineered P. pastoris can be designed to carry a specific, preselected glycoform. However, significant variability exists in fermentation performance between genotypically similar clones with respect to cell fitness, secreted protein titer, and glycan homogeneity. Here, we describe a novel, multidimensional screening process that combines high and medium throughput tools to identify cell lines producing monoclonal antibodies (mAbs). These cell lines must satisfy multiple selection criteria (high titer, uniform N-glycans and cell robustness) and be compatible with our large-scale production platform process. Using this selection process, we were able to isolate a mAb-expressing strain yielding a titer (after protein A purification) in excess of 1 g/l in 0.5-l bioreactors. 相似文献
109.
R. Sathya B. V. Pradeep J. Angayarkanni M. Palaniswamy 《Biotechnology and Bioprocess Engineering》2009,14(6):788-794
Agro-industrial residues, a cheap source of energy have high potential in the area of fermentation for the production of enzymes.
Twenty agro-industrial residues were evaluated to check the possibility of potential utilization of substrates in SSF for
milk clotting enzyme protease production by Mucor circinelloides. In this study, dhal husk holds the greatest promise for cost effective production of the milk clotting enzyme. The dhal
husk supported maximum milk clotting protease production, and yield was improved with the supplementation of sucrose and yeast
extract as carbon and nitrogen source, respectively. Among all the physico-chemical parameters tested, the best results were
obtained in a medium having moisture content of 20% at pH 7.0, when inoculated with 30% of spore suspension and incubated
at 30°C for 5 days. The activity was increased further on addition of Ca2+, Cu2+, and Mg2+ ions. The purified milk-clotting protease obtained from M. circinelloides was successfully applied and compared with commercial rennet in the manufacture of a cheddar cheese. 相似文献
110.