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Extracellular Proteases (extracellular + protease)
Selected AbstractsTissue-type plasminogen activator-plasmin-BDNF modulate glutamate-induced phase-shifts of the mouse suprachiasmatic circadian clock in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2009Xiang Mou Abstract The mammalian circadian clock in the suprachiasmatic nucleus (SCN) maintains environmental synchrony through light signals transmitted by glutamate released from retinal ganglion terminals. Brain-derived neurotrophic factor (BDNF) is required for light/glutamate to reset the clock. In the hippocampus, BDNF is activated by the extracellular protease, plasmin, which is produced from plasminogen by tissue-type plasminogen activator (tPA). We provide data showing expression of proteins from the plasminogen activation cascade in the SCN and their involvement in circadian clock phase-resetting. Early night glutamate application to SCN-containing brain slices resets the circadian clock. Plasminogen activator inhibitor-1 (PAI-1) blocked these shifts in slices from wild-type mice but not mice lacking its stabilizing protein, vitronectin (VN). Plasmin, but not plasminogen, prevented inhibition by PAI-1. Both plasmin and active BDNF reversed ,2 -antiplasmin inhibition of glutamate-induced shifts. ,2 -Antiplasmin decreased the conversion of inactive to active BDNF in the SCN. Finally, both tPA and BDNF allowed daytime glutamate-induced phase-resetting. Together, these data are the first to demonstrate expression of these proteases in the SCN, their involvement in modulating photic phase-shifts, and their activation of BDNF in the SCN, a potential ,gating' mechanism for photic phase-resetting. These data also demonstrate a functional interaction between PAI-1 and VN in adult brain. Given the usual association of these proteins with the extracellular matrix, these data suggest new lines of investigation into the locations and processes modulating mammalian circadian clock phase-resetting. [source] Sequencing and characterization of a novel serine metalloprotease from Burkholderia pseudomalleiFEMS MICROBIOLOGY LETTERS, Issue 1 2000May-Ann Lee Abstract Burkholderia pseudomallei, a Gram-negative bacterium is found in the soil and water, mainly in Southeast Asia and Northern Australia. It is responsible for melioidosis in human and animals. The bacteria produce several potential virulent factors such as extracellular protease, hemolysin, lipase and lecithinase. The isolation of virulence genes and the study of their functions will contribute to our understanding of bacterial pathogenesis. Previous studies have implicated protease as a contributing virulence factor in the pathogenesis of some bacteria. Three out of 5000 clones screened from a genomic DNA library of B. pseudomallei were found to express protease activity. The clones were found to have the same sequence. The nucleotide sequence revealed an open reading frame (designated as metalloprotease A, mprA) encoding a 500-amino acid protein, MprA, with an estimated molecular mass of 50,241 Da. The predicted amino acid sequence shares homology with the subtilisin family of serine proteases. [source] Characterization of a broad pH range protease of Candida caseinolyticaJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2001M. Poza Aims:,The study of a protease secreted by Candida caseinolytica for use in future industrial applications. Methods and Results:,Growth of Candida caseinolytica on a medium containing milk induced a rapid production of an extracellular enzyme able to hydrolyse casein. The crude extract was applied to both Sephacryl S-200 and DEAE-Biogel A columns, obtaining one peak of activity showing a molecular mass of , 30 kDa and three active peaks, respectively. These four peaks showed the same biochemical parameters. In all cases, an extremely broad pH range of action was determined. Conclusions:,Candida caseinolytica secretes high levels of an extracellular protease when grown either in rotary shakers or in batch-fermenters. Significance and Impact of the Study:,The biochemical properties of this enzyme suggest its possible industrial application in the brewing industry, in the formulation of certain type of detergents and in the fur and leather industries, among others. [source] Statistical optimization of medium components for extracellular protease production by an extreme haloarchaeon, Halobacterium sp.LETTERS IN APPLIED MICROBIOLOGY, Issue 1 2009SP1(1) Abstract Aims:, Optimization of medium components for extracellular protease production by Halobacterium sp. SP1(1) using statistical approach. Methods and Results:, The significant factors influencing the protease production as screened by Plackett,Burman method were identified as soybean flour and FeCl3. Response surface methodology such as central composite design was applied for further optimization studies. The concentrations of medium components for higher protease production as optimized using this approach were (g l,1): NaCl, 250; KCl, 2; MgSO4, 10; tri-Na-citrate, 1·5; soybean flour, 10 and FeCl3, 0·16. This statistical optimization approach led to production of 69·44 ± 0·811 U ml,1 of protease. Conclusions:, Soybean flour and FeCl3 were identified as important factors controlling the production of extracellular protease by Halobacterium sp. SP1(1). The statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 3·9-fold increase in extracellular protease production. Significance and Impact of the Study:, The present study is the first report on statistical optimization of medium components for production of haloarchaeal protease. The study also explored the possibility of using extracellular protease produced by Halobacterium sp. SP1(1) for various applications like antifouling coatings and fish sauce preparation using cheaper raw material. [source] Pleomorphism of the marine bacterium Teredinobacter turniraeLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2001G.M. Ferreira Aims:,A morphology transition for the marine bacterium, Teredinobacter turnirae is reported. Methods and Results:,When grown in the rod-shaped morphology, the cells require high concentrations of NaCl (0·3 mol l,1) and secrete extracellular protease and endoglucanase activity. When this bacterium is grown in a medium containing casein as a sole carbon and nitrogen source, a major change in morphology to a stable aggregated form is obtained. Conclusions:,In the aggregated morphology, much higher protease production rates (170 Units ml,1 d,1 for aggregates vs. 15 Units ml,1 d,1 for rods, for the same initial biomass) and negligible endoglucanase titres are obtained. In addition, the aggregated morphology does not require sodium chloride for growth. Significance and Impact of the Study:,The phenomenon reported here describes a novel relationship between the cell morphology and the biochemical characteristics of the bacterium. [source] Protease-Activated Receptors: A Means of Converting Extracellular Proteolysis into Intracellular SignalsIUBMB LIFE, Issue 6 2002E. J. Mackie Abstract Protease-activated receptors (PARs) mediate cellular responses to a variety of extracellular proteases. The four known PARs constitute a subgroup of the family of seven-transmembrane domain G protein-coupled receptors and activate intracellular signalling pathways typical for this family of receptors. Activation of PARs involves proteolytic cleavage of the extracellular domain, resulting in formation of a new N terminus, which acts as a tethered ligand. PAR-1, -3, and -4 are relatively selective for activation by thrombin whereas PAR-2 is activated by a variety of proteases, including trypsin and tryptase. Recent studies in mice genetically incapable of expressing specific PARs have defined roles for PAR-1 in vascular development, and for PAR-3 and -4 in platelet activation, which plays a fundamental role in blood coagulation. PAR-1 has also been implicated in a variety of other biological processes including inflammation, and brain and muscle development. Responses mediated by PAR-2 include contraction of intestinal smooth muscle, epithelium-dependent smooth muscle relaxation in the airways and vasculature, and potentiation of inflammatory responses. The area of PAR research is rapidly expanding our understanding of how cells communicate and control biological functions, in turn increasing our knowledge of disease processes and providing potential targets for therapeutic intervention. [source] Tat dependent export of E. coli phytase AppA by using the PhoD-specific transport system of Bacillus subtilisJOURNAL OF BASIC MICROBIOLOGY, Issue 5 2004Roman Gerlach It has been shown recently that the twin-arginine signal peptide of Bacillus subtilis phosphodiesterase PhoD (SPPhoD) can mediate Tat dependent transport of proteins via its specific Tat-transport components. In order to test the use of Tat dependent transport signals for heterologous product synthesis, Escherichia coli phytase AppA was expressed under control of PhoD-specific export signals in B. subtilis. Induction of Tat components TatAd/TatCd was mediated by using a functionally altered PhoR/PhoP signal transduction system which regulates the expression of these components. AppA was highly susceptible to host specific extracellular proteases. Expression of appA in B. subtilis wprA strain resulted in the stable production of AppA. A fusion protein consisting of SPPhoD and mature AppA remained unprocessed, while introduction of the AppA signal peptidase cleavage site resulted in efficient processing of the fusion protein. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1LETTERS IN APPLIED MICROBIOLOGY, Issue 3 2006T. Miyaji Abstract Aims:, The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. Methods and Results:, Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11·0. In BPP-A, zein was better substrate than casein at pH 13·0, whereas casein was better one than zein at pH 11·0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. Conclusion:, A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. Significance and Impact of the Study:, This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates. [source] The asgE locus is required for cell,cell signalling during Myxococcus xanthus developmentMOLECULAR MICROBIOLOGY, Issue 4 2000Anthony G. Garza In response to starvation, Myxococcus xanthus undergoes a multicellular developmental process that produces a dome-shaped fruiting body structure filled with differentiated cells called myxospores. Two insertion mutants that block the final stages of fruiting body morphogenesis and reduce sporulation efficiency were isolated and characterized. DNA sequence analysis revealed that the chromosomal insertions are located in open reading frames ORF2 and asgE, which are separated by 68 bp. The sporulation defect of cells carrying the asgE insertion can be rescued phenotypically when co-developed with wild-type cells, whereas the sporulation efficiency of cells carrying the ORF2 insertion was not improved when mixed with wild-type cells. Thus, the asgE insertion mutant appears to belong to a class of developmental mutants that are unable to produce cell,cell signals required for M. xanthus development, but they retain the ability to respond to them when they are provided by wild-type cells. Several lines of evidence indicate that asgE cells fail to produce normal levels of A-factor, a cell density signal. A-factor consists of a mixture of heat-stable amino acids and peptides, and at least two heat-labile extracellular proteases. The asgE mutant yielded about 10-fold less heat-labile A-factor and about twofold less heat-stable A-factor than wild-type cells, suggesting that the primary defect of asgE cells is in the production or release of heat-labile A-factor. [source] Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2008Lidia Westers Abstract Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases. [source] Activity, molecular mass and hydrolysis on baker's yeast protein of extracellular proteases from the putative probiotic bacteria Microbacterium sp. strain 8L and Exiguobacterium mexicanum strain 8NAQUACULTURE RESEARCH, Issue 1 2009César Orozco-Medina Abstract The bacteria Microbacterium sp. 8L and Exiguobacterium mexicanum 8N are known to improve the culture of Artemia franciscana using baker's yeast as food. Using spectrophotometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), substrate-SDS-PAGE and pH-stat in vitro -digestibility assays, the activity, molecular mass and hydrolysis on baker's yeast protein of proteases from extracellular polymeric substances (EPS) of the strains 8L and 8N along with the pathogenic strains Microbacterium sp. 8R and Vibrio parahaemolyticus 588 CECT (Vp) were studied. The EPSs of 8L and 8R showed one activity band, on which the serine inhibitor phenylmethylsulphonyl fluoride (PMSF) had no effect. The EPSs of 8N showed four bands; two were unaffected by PMSF, whereas one was affected, and the other was partially affected. The EPSs of Vp showed two bands, one partially inhibited by PMSF. No inhibitory effects from 1-chloro-3-tosylamido-7-amino-2-heptanone (trypsin inhibitor) were observed in the protease bands of the studied bacteria. The EPSs of 8L and 8N showed a similar degree of hydrolysis (pH-stat). The EPSs of 8L had the lowest Dice index of similarity of yeast protein profiles at 1 h of reaction. We conclude that the strain 8L could benefit A. franciscana by providing bacterial proteases for digestion of baker's yeast. [source] | ||||||||||