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Sushama Chaphalkar Rajdeep Dongre Deepa Joshi Sabita Dey 《Biotechnic & histochemistry》1993,68(3):166-168
A synthetic aromatic polymer has been used for preparing replicas of different microorganisms. This method of preparing highly concentrated (9.6 k) microbiological samples for scanning electron microscopy was compared with a standard method. The micrographs of the replicated samples are satisfactory. This method is rapid, cost effective and produces good results, especially in the case of spore-forming mycelial microorganisms. 相似文献
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Sebastiaan van Heesch Michal Mokry Veronika Boskova Wade Junker Rajdeep Mehon Pim Toonen Ewart de Bruijn James D Shull Timothy J Aitman Edwin Cuppen Victor Guryev 《Genome biology》2013,14(4):R33
Background
The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal.Results
While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing.Conclusion
These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses. 相似文献3.
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Savchenko A Yee A Khachatryan A Skarina T Evdokimova E Pavlova M Semesi A Northey J Beasley S Lan N Das R Gerstein M Arrowmith CH Edwards AM 《Proteins》2003,50(3):392-399
Only about half of non-membrane-bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825-1830; Christendat et al., Prog Biophys Mol Biol 200;73:339-345). This property limits genome-scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X-ray crystallography for 68 pairs of homologous proteins from E. coli and Thermotoga maritima. A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures. Fourteen (eight E. coli and six T. maritima proteins) samples generated NMR spectra of a quality suitable for structure determination and 30 (14 E. coli and 16 T. maritima proteins) samples formed crystals. Only three (one E. coli and two T. maritima proteins) samples both crystallized and had excellent NMR properties. The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies. 相似文献
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The stability of thermophilic proteins: a study based on comprehensive genome comparison 总被引:8,自引:0,他引:8
We address the question of the thermal stability of proteins in thermophiles through comprehensive genome comparison, focussing
on the occurrence of salt bridges. We compared a set of 12 genomes (from four thermophilic archaeons, one eukaryote, six mesophilic
eubacteria, and one thermophilic eubacteria). Our results showed that thermophiles have a greater content of charged residues
than mesophiles, both at the overall genomic level and in alpha helices. Furthermore, we found that in thermophiles the charged
residues in helices tend to be preferentially arranged with a 1–4 helical spacing and oriented so that intra-helical charge
pairs agree with the helix dipole. Collectively, these results imply that intra-helical salt bridges are more prevalent in
thermophiles than mesophiles and thus suggest that they are an important factor stabilizing thermophilic proteins. We also
found that the proteins in thermophiles appear to be somewhat shorter than those in mesophiles. However, this later observation
may have more to do with evolutionary relationships than with physically stabilizing factors. In all our statistics we were
careful to controls for various biases. These could have, for instance, arisen due to repetitive or duplicated sequences.
In particular, we repeated our calculation using a variety of random and directed sampling schemes. One of these involved
making a "stratified sample," a representative cross-section of the genomes derived from a set of 52 orthologous proteins
present roughly once in each genome. For another sample, we focused on the subset of the 52 orthologs that had a known 3D
structure. This allowed us to determine the frequency of tertiary as well as main-chain salt bridges. Our statistical controls
supported our overall conclusion about the prevalence of salt bridges in thermophiles in comparison to mesophiles.
Electronic Publication 相似文献
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Kar P Chakraborti T Roy S Choudhury R Chakraborti S 《Archives of biochemistry and biophysics》2007,466(2):290-299
Using calpastatin antibody we have identified a 145 kDa major band along with two relatively minor bands at 120 kDa and 110 kDa calpastatin molecules in bovine pulmonary artery smooth muscle mitochondria. To the best of our knowledge this is first report regarding the identification of calpastatin in mitochondria. We also demonstrated the presence of micro-calpain in the mitochondria by immunoblot and casein zymogram studies. Immunoblot studies identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of mu-calpain. Additionally 76 kDa, 40 kDa and 18 kDa immunoreactive bands have also been detected. Purification and N-terminal amino acid sequence analysis of the identified proteins confirmed their identity as mu-calpain and calpastatins. Immunoprecipitation study revealed molecular association between mu-calpain and calpastatin in the mitochondria indicating that calpastatin could play an important role in preventing uncontrolled activity of mu-calpain which otherwise may facilitate pulmonary hypertension, smooth muscle proliferation and apoptosis. 相似文献
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Biomarker discovery in biological fluids 总被引:2,自引:0,他引:2
Gao J Garulacan LA Storm SM Opiteck GJ Dubaquie Y Hefta SA Dambach DM Dongre AR 《Methods (San Diego, Calif.)》2005,35(3):291-302
Discovery of novel protein biomarkers is essential for successful drug discovery and development. These novel protein biomarkers may aid accelerated drug efficacy, response, or toxicity decision making based on their enhanced sensitivity and/or specificity. These biomarkers, if necessary, could eventually be converted into novel diagnostic marker assays. Proteomic platforms developed over the past few years have given us the ability to rapidly identify novel protein biomarkers in various biological matrices from cell cultures (lysates, supernatants) to human clinical samples (serum, plasma, and urine). In this article, we delineate an approach to biomarker discovery. This approach is divided into three steps, (i) identification of markers, (ii) prioritization of identified markers, and (iii) preliminary validation (qualification) of prioritized markers. Using drug-induced idiosyncratic hepatotoxicity as a case study, the article elaborates methods and techniques utilized during the three steps of biomarker discovery process. The first step involves identification of markers using multi-dimensional protein identification technology. The second step involves prioritization of a subset of marker candidates based on several criteria such as availability of reagent set for assay development and literature association to disease biology. The last step of biomarker discovery involves development of preliminary assays to confirm the bio-analytical measurements from the first step, as well as qualify the marker(s) in pre-clinical models, to initiate future marker validation and development. 相似文献