971 resultados para Extracellular Ca2
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Thse numrise par la Division de la gestion de documents et des archives de l'Universit de Montral
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Glutamatergic dysfunction has been suggested to play an important role in the pathogenesis of hepatic encephalopathy (HE) in acute liver failure (ALF). Increased extracellular brain glutamate concentrations have consistently been described in different experimental animal models of ALF and in patients with increased intracranial pressure due to ALF. High brain ammonia levels remain the leading candidate in the pathogenesis of HE in ALF and studies have demonstrated a correlation between ammonia and increased concentrations of extracellular brain glutamate both clinically and in experimental animal models of ALE Inhibition of glutamate uptake or increased glutamate release from neurons and/or astrocytes could cause an increase in extracellular glutamate. This review analyses the effect of ammonia on glutamate release from (and uptake into) both neurons and astrocytes and how these pathophysiological mechanisms may be involved in the pathogenesis of HE in ALF.
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We previously demonstrated in pigs with acute liver failure (ALF) that albumin dialysis using the molecular adsorbents recirculating system (MARS) attenuated a rise in intracranial pressure (ICP). This was independent of changes in arterial ammonia, cerebral blood flow and inflammation, allowing alternative hypotheses to be tested. The aims of the present study were to determine whether changes in cerebral extracellular ammonia, lactate, glutamine, glutamate, and energy metabolites were associated with the beneficial effects of MARS on ICP. Three randomized groups [sham, ALF (induced by portacaval anastomosis and hepatic artery ligation), and ALF+MARS] were studied over a 6-hour period with a 4-hour MARS treatment given beginning 2 hours after devascularization. Using cerebral microdialysis, the ALF-induced increase in extracellular brain ammonia, lactate, and glutamate was significantly attenuated in the ALF+MARS group as well as the increases in extracellular lactate/pyruvate and lactate/glucose ratios. The percent change in extracellular brain ammonia correlated with the percent change in ICP (r(2) = 0.511). Increases in brain lactate dehydrogenase activity and mitochondrial complex activity for complex IV were found in ALF compared with those in the sham, which was unaffected by MARS treatment. Brain oxygen consumption did not differ among the study groups. Conclusion: The observation that brain oxygen consumption and mitochondrial complex enzyme activity changed in parallel in both ALF- and MARS-treated animals indicates that the attenuation of increased extracellular brain ammonia (and extracellular brain glutamate) in the MARS-treated animals reduces energy demand and increases supply, resulting in attenuation of increased extracellular brain lactate. The mechanism of how MARS reduces extracellular brain ammonia requires further investigation.
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Hyperammonemia is a feature of acute liver failure (ALF), which is associated with increased intracranial pressure (ICP) and brain herniation. We hypothesized that a combination of L-ornithine and phenylacetate (OP) would synergistically reduce toxic levels of ammonia by (1) L-ornithine increasing glutamine production (ammonia removal) through muscle glutamine synthetase and (2) phenylacetate conjugating with the ornithine-derived glutamine to form phenylacetylglutamine, which is excreted into the urine. The aims of this study were to determine the effect of OP on arterial and extracellular brain ammonia concentrations as well as ICP in pigs with ALF (induced by liver devascularization). ALF pigs were treated with OP (L-ornithine 0.07 g/kg/hour intravenously; phenylbutyrate, prodrug for phenylacetate; 0.05 g/kg/hour intraduodenally) for 8 hours following ALF induction. ICP was monitored throughout, and arterial and extracellular brain ammonia were measured along with phenylacetylglutamine in the urine. Compared with ALF + saline pigs, treatment with OP significantly attenuated concentrations of arterial ammonia (589.6 +/- 56.7 versus 365.2 +/- 60.4 mumol/L [mean +/- SEM], P= 0.002) and extracellular brain ammonia (P= 0.01). The ALF-induced increase in ICP was prevented in ALF + OP-treated pigs (18.3 +/- 1.3 mmHg in ALF + saline versus 10.3 +/- 1.1 mmHg in ALF + OP-treated pigs;P= 0.001). The value of ICP significantly correlated with the concentration of extracellular brain ammonia (r(2) = 0.36,P< 0.001). Urine phenylacetylglutamine levels increased to 4.9 +/- 0.6 micromol/L in ALF + OP-treated pigs versus 0.5 +/- 0.04 micromol/L in ALF + saline-treated pigs (P< 0.001).Conclusion:L-Ornithine and phenylacetate act synergistically to successfully attenuate increases in arterial ammonia, which is accompanied by a significant decrease in extracellular brain ammonia and prevention of intracranial hypertension in pigs with ALF.
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Le remodelage cardiaque est le processus par lequel la structure ou la fonction cardiaque change en rponse un dsquilibre pathophysiologique tel qu'une maladie cardiaque, un contexte d'arythmie prolonge ou une modification de l'quilibre hormonal. Le systme rnine-angiotensine (SRA) est un systme hormonal largement tudi et il est impliqu dans de nombreuses activits associes au remodelage cardiovasculaire. Lexistence d'un systme circulatoire coupl un systme de tissus locaux est une reprsentation classique, cependant de nouvelles donnes suggrent un SRA indpendant et fonctionnellement actif l'chelle cellulaire. La comprhension de l'activit intracellulaire du SRA pourrait mener de nouvelles pistes thrapeutiques qui pourraient prvenir un remodelage cardiovasculaire dfavorable. L'objectif de cette thse tait d'lucider le rle du SRA intracellulaire dans les cellules cardiaques. Rcemment, les rcepteurs coupls aux protines G (RCPG), les protines G et leurs effecteurs ont t dtects sur des membranes intracellulaires, y compris sur la membrane nuclaire, et les concepts de RCPG intracellulaires fonctionnels sont en voie d'tre accepts comme une ralit. Nous avons ds lors fait l'hypothse que la signalisation du SRA dlimitant le noyau tait implique dans le contrle de l'expression des gnes cardiaques. Nous avons dmontr la prsence de rcepteurs d'angiotensine de type-1 (AT1R) et de type-2 (AT2R) nuclaires dans les cardiomyocytes ventriculaires adultes et dans une fraction nuclaire purifie de tissu cardiaque. Des quantits d'Ang II ont t dtectes dans du lysat de cardiomyocytes et des microinjections d'Ang-II-FITC ont donn lieu des liaisons prfrentielles aux sites nuclaires. L'analyse transcriptionnelle prouve que la synthse d'ARN de novo dans des noyaux isols stimuls l'Ang-II, et l'expression des ARNm de NF-B taient beaucoup plus importants lorsque les noyaux taient exposs de l'Ang II par rapport aux cardiomyocytes intacts. La stimulation des AT1R nuclaires a engendr une mobilisation de Ca2+ via les rcepteurs de l'inositol trisphosphate (IP3R), et le blocage des IP3R a diminu la rponse transcriptionnelle. Les mthodes disponibles actuellement pour l'tude de la signalisation intracrine sont limites aux mthodes indirectes. L'un des objectifs de cette thse tait de synthtiser et caractriser des analogues d'Ang-II cellule-permants afin dtudier spcifiquement dans les cellules intactes l'activit intracellulaire du SRA. Nous avons synthtis et caractris pharmacologiquement des analogues photosensibles Ang-II encapsule en incorporant un groupement 4,5-dimthoxy-2-nitrobenzyl (DMNB) photoclivable sur les sites actifs identifis du peptide. Chacun des trois analogues d'Ang II encapsule synthtiss et purifis: [Tyr(DMNB)4]Ang-II, Ang-II-ODMNB et [Tyr(DMNB)4]Ang-II-ODMNB a montr une rduction par un facteur deux ou trois de l'affinit de liaison envers AT1R et AT2R dans les dosages par liaison comptitive et une activit rduite dans la contraction de l'aorte thoracique. La photostimulation de [Tyr(DMNB)4]Ang-II dans des cellules HEK a augment la phosphorylation d'ERK1/2 (via AT1R) et la production de cGMP (via AT2R) alors que dans les cardiomyocytes isols elle gnrait une augmentation de Ca2+ nucloplasmique et initiait la synthse d'ARNr 18S et d'ARNm du NF-B. Les fibroblastes sont les principaux gnrateurs de remodelage cardiaque structurel, et les fibroblastes auriculaires sont plus ractifs aux stimuli profibrotiques que les fibroblastes ventriculaires. Nous avons mis l'hypothse que lAng-II intracellulaire et l'activation des AT1R et AT2R nuclaires associs contrlaient les profils d'expression des gnes des fibroblastes via des systmes de signalisation distincts et de ce fait jouaient un rle majeur dans le dveloppement de la fibrose cardiaque. Nous avons remarqu que les fibroblastes auriculaires expriment lAT1R et lAT2R nuclaire et l'Ang-II au niveau intracellulaire. Lexpression d'AT1R nuclaire a t rguls positivement dans les cas dinsuffisance cardiaque (IC), tandis que l'AT2R nuclaire a t glycosyl post-traductionnellement. La machinerie protique des protines G, y compris Gq/11, Gi/3, et G, a t observe dans des noyaux isols de fibroblastes. AT1R et AT2R rgulent l'initiation de la transcription du fibroblaste via les voies de transduction de signal d'IP3R et du NO. La photostimulation de [Tyr(DMNB)4]Ang-II dans une culture de fibroblastes auriculaire dclenche la libration de Ca2+ nucloplasmique, la prolifration, et la synthse et scrtion de collagne qui ne sont pas inhibes par les bloqueurs d'AT1R et/ou AT2R extracellulaires.
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Department of Biotechnology, Cochin University of Science and Technology
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The beta-glucosidase enzyme purified from the marine fungus, Aspergillus sydowii BTMFS 55 showed a good yield of enzyme production under solid state fermentation. The statistical optimization of the media components revealed that moisture content, concentration of peptone and inoculum are the major parameters which supported the maximal enzyme production. The purified enzyme showed low pH activity and stability, glucose tolerance and activation by ethanol. It could produce ethanol from wheat bran and rice straw by simultaneous saccharification and fermentation with yeast.The glucosidase purified from Aspergillus sydowii BTMFS 55 shows great potential for several biotechnological applications such as the production of bio-ethanol from agricultural biomass and improvement in the aromatic character of wines and fruit juices through the hydrolysis of flavour glucosidic precursors. There is immense scope for the application of this marine fungus in the biofuel production besides in other industries provided further studies are pursued in exploiting this enzyme and the organism particularly scale up studies with respect to application. There is also ample scope for cloning of the gene encoding beta-glucosidase in domesticated hosts such as Pichia pastoris or S. cerevisiae that can produce ethanol directly from cellulosic biomass.
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Commercially, Pleurotus spp. of mushroom are cultivated in bags. After mushroom cultivation, spent substrate remains as residual material. Proper recycling of spent substrate is beneficial for our economy. Spent substrate can be utilized for various other value added purposes through the proper knowledge of its components. Composition of various components depends on the activity of extracellular enzymes in the spent substrate. The present study was conducted to know the enzyme profile of some major extracellular enzymes - cellulase, hemicellulase (xylanase), pectinase and ligninase (lignin peroxidase and laccase) and to estimate cellulose, hemicellulose, pectin and lignin in the substrate. The use of spent substrate as a source of fibre and ethanol, and in the biodegradation of phenol by Pleurotus spp. was also investigated
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During last decades there has been a continuous growth of aquaculture industries all over the world and taking into consideration the spurt in freshwater ornamental fish aquaculture and trade in Kerala, the present study was aimed to assess the prevalence of various motile Aeromonas spp. in fresh water ornamental fishes and associated carriage water. The extracellular virulence factors and the antibiogram of the isolates were also elucidated. Various species of motile aeromonads such as Aeromonas caviae, A. hydrophila, A. jandaei, A. schubertii, A. sobria, A. trota and A. veronii were detected. Aeromonas sobria predominated both fish and water samples. Extracellular enzymes and toxins produced by motile aeromonds are important elements of bacterial virulence. The production of extracellular virulence factors - proteases, lipase, DNase and haemolysin by the isolates were studied. All the isolates from both fish and water samples produced gelatinase and nuclease but the ability to produce lipase, caseinase and haemolysins was found to vary among isolates from different sources. Among the 15 antibiotics to which the isolates were tested, all the isolates were found to be sensitive to chloramphenicol, ciprofloxacin and gentamicin and resistant to amoxycillin. Local aquarists maintain the fish in crowded stressful conditions, which could trigger infections by the obligate/ opportunistic pathogenic members among motile aeromonads
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An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.
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Beauveria sp. BTMF S10 isolated from marine sediment produced extracellular L-glutaminase. Maximal L- glutaminase yield (46.9 U/ml) was obtained in a medium supplemented with 1% (w/v) yeast extract and sorbitol, 9% (w/v) sodium chloride and 0.2% (w/v) methionine, initial pH 9.0 and at 27 C after 108 h. This enzyme was inducible and growth-associated.
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Four species of bacteria which included Pseudomonas fluorescens, Vibrio cho!erae and Vibrio costicola were observed to produce glutaminase both as extracellular and intracellular fractions. Comparatively both the fractions were higher in mineral media supplemented with 1% glutamine than in nutrient broth added with or without glutamine. Extracellular glutaminase production was about 2.6-6.8 times greater than the intracellular production by all the tested strains
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A potential fungal strain producing extracellular -glucosidase enzyme was isolated from sea water and identified as ^J = BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of ^= in the GenBank. A sequential optimization strategy was used to enhance the production of -glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence -glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for -glucosidase production. The enzyme was purified by (NH4)2SO4 precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ~95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50C. It showed high affinity towards NPG and enzyme has a h and s~ of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a h of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of p~~=~ in presence of cellulase and the purified -glucosidase of ^= BTMFS 55.
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An alkaline protease gene (Eap) was isolated for the first time from a marine fungus, Engyodontium album. Eap consists of an open reading frame of 1,161 bp encoding a prepropeptide consisting of 387 amino acids with a calculated molecular mass of 40.923 kDa. Homology comparison of the deduced amino acid sequence of Eap with other known proteins indicated that Eap encode an extracellular protease that belongs to the subtilase family of serine protease (Family S8). A comparative homology model of the Engyodontium album protease (EAP) was developed using the crystal structure of proteinase K. The model revealed that EAP has broad substrate specificity similar to Proteinase K with preference for bulky hydrophobic residues at P1 and P4. Also, EAP is suggested to have two disulfide bonds and more than two Ca2? binding sites in its 3D structure; both of which are assumed to contribute to the thermostable nature of the protein.
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L-Glutamine amidohydrolase (L-glutaminase, EC 3.5.1.2) is a therapeutically and industrially important enzyme. Because it is a potent antileukemic agent and a flavor-enhancing agent used in the food industry, many researchers have focused their attention on L-glutaminase. In this article, we report the continuous production of extracellular L-glutaminase by the marine fungus Beauveria bassiana BTMF S-10 in a packed-bed reactor. Parameters influencing bead production and performance under batch mode were optimized in the order-support (Na-alginate) concentration, concentration of CaCl2 for bead preparation, curing time of beads, spore inoculum concentration, activation time, initial pH of enzyme production medium, temperature of incubation, and retention time. Parameters optimized under batch mode for L-glutaminase production were incorporated into the continuous production studies. Beads with 12 108 spores/g of beads were activated in a solution of 1% glutamine in seawater for 15 h, and the activated beads were packed into a packed-bed reactor. Enzyme production medium (pH 9.0) was pumped through the bed, and the effluent was collected from the top of the column. The effect of flow rate of the medium, substrate concentration, aeration, and bed height on continuous production of L-glutaminase was studied. Production was monitored for 5 h in each case, and the volumetric productivity was calculated. Under the optimized conditions for continuous production, the reactor gave a volumetric productivity of 4.048 U/(mLh), which indicates that continuous production of the enzyme by Ca-alginate-immobilizedspores is well suited for B. bassiana and results in a higher yield of enzyme within a shorter time. The results indicate the scope of utilizing immobilized B. bassiana for continuous commercial production of L-glutaminase
