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Culture Fluid (culture + fluid)
Selected AbstractsBiochemical and molecular characterization of a laccase from the edible straw mushroom, Volvariella volvaceaFEBS JOURNAL, Issue 2 2004Shicheng Chen We have isolated a laccase (lac1) from culture fluid of Volvariella volvacea, grown in a defined medium containing 150 µm CuSO4, by ion-exchange and gel filtration chromatography. Lac1 has a molecular mass of 58 kDa as determined by SDS/PAGE and an isoelectric point of 3.7. Degenerate primers based on the N-terminal sequence of purified lac1 and a conserved copper-binding domain were used to generate cDNA fragments encoding a portion of the lac1 protein and RACE was used to obtain full-length cDNA clones. The cDNA of lac1 contained an ORF of 1557 bp encoding 519 amino acids. The amino acid sequence from Ala25 to Asp41 corresponded to the N-terminal sequence of the purified protein. The first 24 amino acids are presumed to be a signal peptide. The expression of lac1 is regulated at the transcription level by copper and various aromatic compounds. RT-PCR analysis of gene transcription in fungal mycelia grown on rice-straw revealed that, apart from during the early stages of substrate colonization, lac1 was expressed at every stage of the mushroom developmental cycle defined in this study, although the levels of transcription varied considerably depending upon the developmental phase. Transcription of lac1 increased sharply during the latter phase of substrate colonization and reached maximum levels during the very early stages (primordium formation, pinhead stage) of fruit body morphogenesis. Gene expression then declined to ,,20,30% of peak levels throughout the subsequent stages of sporophore development. [source] Development of a homologous expression system for rubber oxygenase RoxA from Xanthomonas sp.JOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2010N. Hambsch Abstract Aims:, Natural rubber (poly-[cis -1,4-isoprene]) can be cleaved into 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al by rubber oxygenase A (RoxA) isolated from Xanthomonas sp. RoxA is a novel type of dihaem dioxygenase with unknown cleavage mechanism of the rubber carbon backbone. Analysis of mutant RoxA after mutagenesis could be a way to investigate the function of selected amino acids of RoxA during catalysis. Unfortunately, expression of functional RoxA in recombinant Escherichia coli or in recombinant ,-Proteobacteria such as Pseudomonas putida was not possible in our hands. Therefore, expression of recombinant RoxA in the homologous host, Xanthomonas, was performed. Methods and Results:, A transformation system via electroporation was established, and a conjugation system was optimized for Xanthomonas sp. Inactivation of the chromosomal roxA gene by insertional mutagenesis resulted in inability of Xanthomonas sp. to produce active RoxA and to utilize rubber as a sole source of carbon and energy. When an intact copy of roxA was cloned under control of a rhamnose-inducible promoter in a broad host range vector and was transferred to Xanthomonas sp., high expression levels of functional RoxA in the presence of rhamnose were obtained. Conclusions and Significance and Impact of the Study:, Purification of recombinantly expressed RoxA was simplified because of drastically shortened fermentation times and because separation of RoxA from remaining rubber latex particles was not necessary with rhamnose-induced cultures. About 6 mg purified RoxA were obtained from 1 l of cell-free culture fluid. Purified recombinant RoxA was highly active and revealed comparable spectral properties as RoxA purified from the wild type. The results of our study are the methodical basis for molecular biological manipulation in Xanthomonas sp. and will simplify investigation into the biochemical mechanisms by which rubber can be biodegraded in the environment by this novel extracellular dihaem dioxygenase RoxA. [source] Elicitation of Ethylene by Verticillium albo-atrum Phytotoxins in PotatoJOURNAL OF PHYTOPATHOLOGY, Issue 3 2005B. Mansoori Abstract Petioles from a susceptible cultivar (Désirče) of Solanum tuberosum treated with a low-molecular mass toxin, separated from culture fluid of Verticillium albo-atrum, produced greater quantities of ethylene than did petioles of a tolerant cultivar (Home Guard). Pretreatment of leaflets from cv. Désirče with silver thiosulphite, which inhibits perception of ethylene, prevented the chlorosis and necrosis normally associated with exposure to the toxin. Similarly, application of aminoethoxyvinylglycine (AVG) an inhibitor of aminocyclopropane-1-carboxylic acid (ACC) synthase, to petioles of cv. Désirče reduced toxin-induced ethylene synthesis and symptom development. The data indicate that, in part, Verticillium -toxin acts through induction of ethylene biosynthesis in the host tissues, and different responses of susceptible and tolerant potato cultivars to V. albo-atrum are the result of differential production of ethylene. [source] Development of fluoroapatite chromatography for the purification of monoclonal antibodyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17-18 2010Paul K. Ng Abstract Monoclonal antibody was purified from tissue culture fluid using ceramic fluoroapatite chromatography. Efficiency of the capture step was shown to be sensitive to pH and phosphate concentration. More than 90% of host cell proteins were removed by ceramic fluoroapatite chromatography. Studies regarding pH control and metal adsorption are presented. [source] Mechanism of antibody reduction in cell culture production processesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Yung-Hsiang Kao Abstract We recently observed a significant disulfide reduction problem during the scale-up of a manufacturing process for a therapeutic antibody using a CHO expression system. Under certain conditions, extensive reduction of inter-chain disulfide bonds of an antibody produced by CHO cell culture may occur during the harvest operations and/or the protein A chromatography step, resulting in the observation of antibody fragments (light chain, heavy chain, and various combination of both) in the protein A pools. Although all conditions leading to disulfide reduction have not been completely identified, an excessive amount of mechanical cell lysis generated at the harvest step appears to be an important requirement for antibody reduction (Trexler-Schmidt et al., 2010). We have been able to determine the mechanism by which the antibody is reduced despite the fact that not all requirements for antibody reduction were identified. Here we present data strongly suggesting that the antibody reduction was caused by a thioredoxin system or other reducing enzymes with thioredoxin-like activity. The intracellular reducing enzymes and their substrates/cofactors apparently were released into the harvest cell culture fluid (HCCF) when cells were exposed to mechanical cell shear during harvest operations. Surprisingly, the reducing activity in the HCCF can last for a long period of time, causing the reduction of inter-chain disulfide bonds in an antibody. Our findings provide a basis for designing methods to prevent the antibody reduction during the manufacturing process. Biotechnol. Bioeng. 2010;107:622,632. © 2010 Wiley Periodicals, Inc. [source] Development of a high throughput protein a well-plate purification method for monoclonal antibodiesBIOTECHNOLOGY PROGRESS, Issue 5 2009Jennifer Hopp Abstract We have developed a new high throughput method for the purification of monoclonal antibodies from harvested cell culture fluid for analytical characterization. This method uses Protein A resin in a 96 well-plate format with protein loading sufficient to perform multiple analyses per well. Resin and buffer conditions were optimized to obtain aggregate and charge variant comparability with three preparative Protein A purified monoclonal antibodies. We are able to successfully demonstrate comparability for aggregate within 0.25% based upon size-exclusion chromatography. Acidic species were found to be within 2% from the preparative purified control based upon cation-exchange chromatography, 5% based upon capillary zone electrophoresis, and 3% based upon imaged capillary isoelectric focusing. Glycan distribution was analyzed and was within 1% of the preparative purified controls. A tryptic digest was performed and all peaks in the preparative purified control were found in the first elution from the well-plate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] "Reverse degradomics", monitoring of proteolytic trimming by multi-CE and confocal detection of fluorescent substrates and reaction productsELECTROPHORESIS, Issue 13 2009Helene Piccard Abstract A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96-channel capillary DNA sequencer with CE-LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi-channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi-quantified in a high-throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein-2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest. [source] Response to alkaline stress by root canal bacteria in biofilmsINTERNATIONAL ENDODONTIC JOURNAL, Issue 5 2007L. E. Chávez de Paz Abstract Aim, To determine whether bacteria isolated from infected root canals survive alkaline shifts better in biofilms than in planktonic cultures. Methodology, Clinical isolates of Enterococcus faecalis, Lactobacillus paracasei, Olsenella uli, Streptococcus anginosus, S. gordonii, S. oralis and Fusobacterium nucleatum in biofilm and planktonic cultures were stressed at pH 10.5 for 4 h, and cell viability determined using the fluorescent staining LIVE/DEAD BacLight bacterial viability kit. In addition, proteins released into extracellular culture fluids were identified by Western blotting. Results,Enterococcus faecalis, L. paracasei, O. uli and S. gordonii survived in high numbers in both planktonic cultures and in biofilms after alkaline challenge. S. anginosus, S. oralis and F. nucleatum showed increased viability in biofilms compared with planktonic cultures. Alkaline exposure caused all planktonic cultures to aggregate into clusters and resulted in a greater extrusion of cellular proteins compared with cells in biofilms. Increased levels of DnaK, HPr and fructose-1,6-bisphosphate aldolase were observed in culture fluids, especially amongst streptococci. Conclusions, In general, bacteria isolated from infected roots canals resisted alkaline stress better in biofilms than in planktonic cultures, however, planktonic cells appeared to use aggregation and the extracellular transport of specific proteins as survival mechanisms. [source] Anti-influenza virus activity of crude extract of Ribes nigrum L.PHYTOTHERAPY RESEARCH, Issue 2 2003Yoko M. Knox Abstract This experiment was designed to detect the antiviral activities of crude fruit extracts of wild Ribes nigrum L. (Kurokarin extract) against influenza virus types A and B. Kurokarin extract was prepared as follows: fruits of Ribes nigrum L. were heated at 50,°C in a heating tank, and then ground under anaerobic conditions. The extracts were centrifuged, and the supernatant fluid was filtered and sterilized by infrared rays. The crude extract was diluted with Eagle's minimum essential medium (MEM) and the solution was adjusted to a pH 7.2 with 0.1 N or 1 N NaOH. Proven anti-influenza virus effects of the extracts were shown. The concentration of extract required to inhibit the plaque formation of both IVA and IVB by 50% (IC50) was 3.2,,g/mL. Both IVA and IVB were directly inactivated up to 99% by 10,,g/mL of the extract at pH 2.8, and 95% to 98% by this dose at pH 7.2. The growth of IVA in cells treated with 10 and 100,,g/mL of the extract for 6,h after infection was completely suppressed. Virus titres in culture fluids of the cells treated with 100,,g/mL of Kurokarin extract for 1,h at 8 to 9,h after infection, were completely suppressed, indicating that the extract inhibited the virus release from the infected cells. Copyright © 2003 John Wiley & Sons, Ltd. [source] Effects of secretory leucocyte protease inhibitor on the production of the anti-inflammatory cytokines, IL-10 and transforming growth factor-beta (TGF- ,), by lipopolysaccharide-stimulated macrophagesCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2000C. Sano We studied the effects of secretory leucocyte protease inhibitor (SLPI) on the production of the anti-inflammatory cytokines, IL-10 and TGF- ,, by lipopolysaccharide (LPS)-stimulated macrophages, using half-sized SLPI (1/2 SLPI) containing the C-terminal domain (Arg58 -Ala107). ELISA testing of macrophage culture fluids showed a temporary production of IL-10 by the macrophages in the early phase (24 h) after LPS stimulation at low (1 ng/ml) or high (10 ,g/ml) concentrations. On the other hand, TGF- , production was initiated after day 3 and progressively increased. 1/2 SLPI significantly increased IL-10 and TGF- , production by macrophages in response to a low dose as well as a high dose of LPS. Reverse transcription-polymerase chain reaction analysis showed that 1/2 SLPI caused a significant increase in the expression of both IL-10 and TGF- , mRNAs by LPS-stimulated macrophages. Thus, although the profile of macrophage TGF- , production by LPS-stimulated macrophages is markedly different from that of their IL-10 production, SLPI causes an up-regulation of the production of these anti-inflammatory cytokines by LPS-stimulated macrophages. [source] | ||||||||||||||||