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  • Biochemistry and Biotechnology  (10)
  • Spacecraft Design, Testing and Performance  (6)
  • 1995-1999  (16)
  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 332-338 
    ISSN: 0006-3592
    Keywords: α-hydroxynitrile lyase ; cassava ; cyanogenesis ; cyanohydrin ; Escherichia coli expression vector ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The coding sequence of the cyanogenic α-hydroxynitrile lyase gene of Manihot esculenta Crantz (cassava) was cloned in the plasmid vector pMal-c2 and expressed in Escherichia coli strain JM105. DNA sequencing showed that the recombinant plasmid contained the same sequence as the cDNA clone pHNL10. Peptide sequencing of the recombinant protein showed that the N-terminus was heterogeneous, with either four or six additional amino acid residues compared with the native protein. Circular dichroism spectra indicated similar secondary structure contents for both proteins. Enzyme assays showed that specific activity of native and recombinant proteins were 0.24 and 0.26 mmol CN-/mg/min, respectively; that both proteins had optimal activity at 40°C and pH 5.5; and that both proteins were inhibited by the serine protease inhibitor phenyl-methane sulfonyl flouride (PMSF). Isoelectric focusing of native and recombinant protein revealed multiple isoforms for both proteins; the recombinant protein had a more basic mean isoelectric point (pl) (5.1) than the native protein (4.5). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 332-338, 1997.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 112-119 
    ISSN: 0006-3592
    Keywords: biodegradation ; self-cycling fermentation ; phenol ; Pseudomonas putida ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Self-cycling fermentation (SCF) in a stirred tank reactor was applied to the biodegradation of phenol by Pseudomonas putida. The technique resulted in stable and repeatable performance. Complete substrate consumption was achieved under all operating conditions investigated. SCF resulted in substrate utilization rates as high as 14.5 kg of phenol per cubic meter of fermentor volume per day of fermentation, higher than those that have been reported for batch, CSTR, and packed column fermentors. A mathematical model of the self-cycling fermentation process was expanded to include inhibitory substrate-microorganism combinations, and was shown to provide a good fit to both end-of-cycle and intracycle experimental data. © 1996 John Wiley & Sons, Inc.
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  • 3
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Escherichia coli ; SWISS-2DPAGE database ; Immobilized pH gradient ; Sequence tag ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The combination of two-dimensional polyacrylamide gel electrophoresis (2-DPAGE), computer image analysis and several protein identification techniques allowed the Escherichia coli SWISS-2DPAGE database to be established. This is part of the ExPASy molecular biology server accessible through the WWW at the URL address http://www.expasy.ch/ch2d/ch2d-top.html. Here we report recent progress in the development of the E. coli SWISS-2DPAGE database. Proteins were separated with immobilized pH gradients in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. To increase the resolution of the separation and thus the number of identified proteins, a variety of wide and narrow range immobilized pH gradients were used in the first dimension. Micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and spots were visualized by amido black staining. Protein identification techniques such as amino acid composition analysis, gel comparison and microsequencing were used, as well as a recently described Edman “sequence tag” approach. Some of the above identification techniques were coupled with database searching tools. Currently 231 polypeptides are identified on the E. coli SWISS-2DPAGE map: 64 have been identified by N-terminal microsequencing, 39 by amino acid composition, and 82 by sequence tag. Of 153 proteins putatively identified by gel comparison, 65 have been confirmed. Many proteins have been identified using more than one technique. Faster progress in the E. coli proteome project will now be possible with advances in biochemical methodology and with the completion of the entire E. coli genome.
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  • 4
    ISSN: 0173-0835
    Keywords: Two-dimensional polyacrylamide gel electrophoresis ; Human blood platelets ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional (2-D) maps of cytosol and enriched-membrane platelet proteins has allowed the identification of more than 25 spots by three different methods: matching of the platelet gels with other 2-D reference maps, immunoblotting with chemiluminescence detection, and N-terminal sequencing. Different G protein (guanosine triphosphate-binding protein) subunits, cytoskeletal proteins, and proteins common to the human liver, red blood cells and plasma were identified. The two platelet protein maps presented here contribute to the project of identification of human cell and body fluid proteins. They may serve as working tools since platelets are popular models for the study of central nervous system neurotransmitter systems and stimulus-response coupling mechanisms.
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  • 5
    ISSN: 0173-0835
    Keywords: Amniotic fluid ; Two-dimensional polyacrylamide gel electrophoresis ; Immobilized pH gradient ; Protein map ; Gel matching ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Using updated technical procedures (immobilized pH gradients for isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: IPG/SDS-PAGE) we provide a two-dimensional (2-D) map of amniotic fluid (AF) proteins. This map comprises over 800 silver-stained spots. Over 150 spots have been identified by matching on the net with human plasma and cerebrospinal fluid maps available from SWISS 2DPAGE database; several additional spots were assigned by immunoblotting and/or microanalytical techniques. This report details our investigation on AF proteins focusing on the 17th week of gestation, when AF is most commonly used for clinical evaluation of fetal disorders. As a whole, the map displays a number of potential markers for fetal development and for gestation abnormalities. The 2-D electrophoretic technique allows the monitoring of all these proteins at the same time along with additional spots that may prove of diagnostic significance.
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  • 6
    ISSN: 0173-0835
    Keywords: Ductal breast carcinoma ; Breast biopsy ; Two-dimensional polyacrylamide gel electrophoresis ; Reference map ; Immobilized pH gradient ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Reference two-dimensional (2-D) gels are presented for human breast ductal carcinoma and histologically normal tissue. Whole biopsy fragments were analyzed, including epithelial and nonepithelial components. Thirty-five spots have been assigned by gel matching to the human liver SWISS-2DPAGE reference map and/or to the human primary keratinocyte IPG map from the Danish Center for Human Genome. N-terminal microsequencing was applied to confirm randomly chosen matching assignments and to identify six new spots. Protein expression profiles in ductal carcinoma and in normal breast tissue appeared to be similar, except for a pattern consisting of 32 spots, which were highly expressed in all carcinoma specimens, and less intense and occasionally undetectable in normal tissue. This difference was statistically significant. Assignment has been obtained for several spots, namely GRP94, GRP78, GRP75, mitochondrial HSP60, calreticulin, protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase, collagen-binding protein 2, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, thioredoxin, cytochrome c oxidase VA subunit, tubulin β isoform and macrophage migration inhibitory factor (MIF). The cancer- and tissue-specificity of the described pattern was assessed by matching to the Swiss-2DPAGE human liver, hepatoma, lymphoma, erythroleukemia reference maps. The pattern of 32 spots was found to be indicative of epithelial neoplasia.
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  • 7
    ISSN: 0173-0835
    Keywords: Colonic neoplasms ; Rectal neoplasms ; Two-dimensional polyacrylamide gel electrophoresis ; Cell separation ; Antibodies' monoclonal diagnostic use ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: New diagnostic and prognostic markers are needed in colorectal cancer. They can be found by differential analysis at DNA, RNA or protein level. The accuracy of phenotypic comparisons of tumor and normal tissues depends on the purity of the samples. We present an effective method to identify and isolate proteins that are differentially expressed under altered conditions, and a two-dimensional reference protein map of the normal human colonic epithelium Normal colonic mucosa, primary tumors and liver metastases were prepared in the operating room. After washing in an ice-cold medium containing protease inhibitors, crypts were isolated by mechanical preparation without using metalloproteinases. Epithelial cells were then selected using Ber-EP4 Dynabeads. The samples were denaturated before processing for immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis according to SWISS-2DPAGE standards. The samples contained more than 95% epithelial cells as confirmed by fluorescence-activated cell sorting using pan-anticytokeratin antibodies. Cell surfaces were not damaged, as assessed by scanning electronic microscope. A protein reference map of the normal colonic epithelium was defined. Using gel matching, N-terminal sequencing and/or immunoblotting techniques, 60 polypeptides - including proteins specifically expressed in colorectal epithelium - have now been identified. This reproducible method of sample preparation permits the comparison of protein patterns found in various pathological states with the present reference map (http://www.expasy.ch). Some of these patterns might provide diagnostic or prognostic markers, or even molecular targets for therapy in the future.
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  • 8
    ISSN: 0173-0835
    Keywords: Kidney ; Two-dimensional polyacrylamide gel electrophoresis ; Renal cell carcinoma ; Database ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Renal cell carcinoma (RCC), a human kidney cancer from the proximal tubular epithelium, accounts for about 3% of adult malignancies. Molecular and cytogenetic analysis have highlighted deletions, translocations, or loss of heterozygosity in the 3p21-p26, a putative RCC locus, as well as in 6q, 8p, 9pq, and 14pq. Studies on phenotypic expression of human kidney tissue and on post-translational modifications in RCC have not yet provided a marker for early renal cell carcinoma diagnosis. Current dignostic methods do not help to detect the tumor before advanced stages. We therefore used two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to study normal and tumor kidney tissues in ten patients suffering from RCC. A human kidney protein map in the SWISS-2DPAGE database accessible through the ExPASy WWW Molecular Biology Server was established. Of 2789 separated polypeptides, 43 were identified by gel comparison, amino acid analysis, N-terminal sequencing, and/or immunodetection. The comparison between normal and tumor kidney tissues showed four polypeptides to be absent in RCC. One of them was identified as ubiquinol cytochrome c reductase (UQCR), whose locus has elsewhere been tentatively assigned to chromosome 19p12 or chromosome 22. A second polypeptide was identified as mitochondrial NADH-ubiquinone oxido reductase complex I whose locus is located on chromosome 18p 11.2 and chromosome 19q 13.3. These result suggest that the lack of UQCR and of mitochondrial NADH-ubiquinone oxidoreductase complex I expression in RCC may be caused by unknown deletions, or by changes in gene transcription or translation. It might indicate that mitochondrial disfunction plays a major role in RCC genesis or evolution.
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  • 9
    ISSN: 0173-0835
    Keywords: Yeast ; SWISS-2DPAGE ; Two-dimensional polyacrylamide gel electrophoresis ; Protein database ; Protein mapping ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The systematic sequencing of the yeast genome will soon be completed. A new challenge has been launched by the EUROFAN (European Functional Analysis) project whose goal is to elucidate the physiological and biochemical function of newly discovered open reading frames (ORF) from yeast. One of the approaches is to use protein-based technologies such as two-dimensional gel eletrophoresis and protein identification in order to establish a yeast reference map. Modified protein patterns can be compared to the reference map which hopefully will help identify changes related, for example, to growth processes or developmental events. This paper describes the yeast SWISS-2DPAGE database in which charge separation was obtained using immobilized pH gradient (IPG). Proteins identified by gel comparison, amino acid composition analysis and/or microsequencing are recorded and described in an accessible uniform format. We have identified more than one hundred polypeptides, several of which were newly mapped. In addition, the yeast SWISS-2DPAGE database can be freely accessed through the World Wide Web (WWW) network on the ExPASy molecular biology server.
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  • 10
    ISSN: 0173-0835
    Keywords: Escherichia coli ; SWISS-2DPAGE database ; Two-dimensional polyacrylamide gel electrophoresis ; Immobilized pH gradients ; Protein pattern comparison ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Numerous Escherichia coli proteins have already been characterized by two-dimensional gel electrophoresis (2-D PAGE), using carrier ampholytes in the first dimension (VanBogelen, R. A., Sankar, P., Clark, R. L., Bogan, J. A. and Neidhardt, F. C., Electrophoresis 1992, 13, 1014-1054). We present here a reference protein map of E. coli obtained with immobilized pH gradients (IPG) and available in a SWISS-2DPAGE format. Out of the protein spots identified in the E. coli gene protein database by Neidhardt's group, 153 have been identified on the E.coli SWISS-2DPAGE database map by gel comparison and most of them were confirmed either by the analysis of amino acid composition (AAC) and/or N-terminal microsequencing. Additionally, five as yet unsequenced proteins were found. The E. coli SWISS-2DPAGE database is part of the ExPASy molecular biology server accessible through the Word Wide Web network.
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