992 resultados para beta galactosidase
Resumo:
Les infections à Salmonella Typhimurium constituent un problème de taille pour l’industrie porcine et la santé publique car cet animal est un réservoir pour les infections chez l’homme. De plus, on observe, chez des souches appartenant au lysotype (LT) 104, des résistances multiples aux antimicrobiens associées à des septicémies chez les porcs en engraissement, ce qui peut contribuer à la contamination des carcasses. Il faut donc contrôler l’infection au niveau du troupeau. Pour ce faire, il importe donc de mieux caractériser ces souches, comprendre la pathogénie de l’infection et identifier des facteurs de virulence. L’objectif principal de cette étude était de caractériser des isolats de S. Typhimurium provenant de porcs septicémiques et de les comparer avec ceux de porcs sains. Une banque d’isolats provenant de porcs septicémiques (ASC) et de porcs sains à l’abattoir (SSC) était constituée. Le lysotype des isolats a été identifié et ceux-ci ont été caractérisés selon le profil de résistance aux antimicrobiens, le SDS-PAGE et l’immunobuvardage et le PFGE. Chez les isolats ASC, LT 104 représentait 36.4% des isolats et chez les isolats SSC la proportion était de 51.5%. Les isolats pouvaient être résistants jusqu’à douze antimicrobiens, peu importe leur origine. Il n’a toutefois pas été possible d’associer une protéine spécifique au groupe d’isolats ASC. Parmi les souches LT 104, plusieurs groupes génétiques ont été identifiés. Les différentes étapes de la pathogénie de Salmonella ont ensuite été évaluées, dont l’adhésion et l’invasion des isolats des deux banques sur des cellules intestinales humaines. Nos résultats ont démontré que les isolats ASC avaient un pouvoir accru d’invasion comparés aux isolats SSC (P=0.003). Pour un sous-groupe d’isolats sélectionnés selon leur taux d’invasion, des tests de phagocytose, d’apoptose et d’adhésion au mucus intestinal ont été effectués en utilisant la cytométrie en flux. La survie des bactéries après la phagocytose a aussi été évaluée et la méthode MATS a été utilisée pour évaluer l'adhésion aux solvants. Les pourcentages de phagocytose chez les isolats SSC par les monocytes porcins étaient plus élevés que chez les isolats ASC à 15 minutes (P=0.02). Nous n’avons trouvé aucune différence significative pour les autres méthodes utilisées. Nous avons ensuite comparé le génome d’un isolat ASC (#36) à celui d’un isolat SSC (#1) par le SSH pour identifier des facteurs de virulence potentiels. Des clones correspondant à des gènes retrouvés sur le chromosome ainsi que sur des plasmides ont été identifiés. Ces résultats nous ont dirigés vers l’analyse des profils plasmidiques de tous les isolats. Les différents profils étaient retrouvés autant chez les isolats ASC que chez les isolats SSC. Deux profils (PL14 et PL20) étaient observés plus fréquemment chez les isolats LT 104 que chez les isolats d’autres lysotypes (P=0.01 et P=0.01, respectivement). Le séquençage d’un des plasmides de l’isolat ASC, démontrait la présence d’informations génétiques codant pour la réplication et une bêta-galactosidase-α. Il serait intéressant de préciser le rôle exact de ces gènes dans l’infection. Nos travaux suggèrent que les isolats de S. Typhimurium provenant de porcs septicémiques se distinguent par un pouvoir d’invasion accru ainsi que par des taux de phagocytose plus faibles dans les phases initiales de l’infection. Cette étude aura donc permis d’accroître les connaissances sur la pathogénie des infections à S. Typhimurium chez le porc.
Resumo:
Il a été suggéré que l’autophagie pouvait participer au processus fibrotique en favorisant la différenciation du fibroblaste en myofibroblaste. La sénescence cellulaire a aussi été montrée comme impliquée dans la réparation tissulaire et la fibrose. Des liens ont été établis entre autophagie et sénescence. Cette étude a pour but d’investiguer les liens possibles entre autophagie, sénescence et différenciation myofibroblastique afin de mieux comprendre les mécanismes moléculaires régulant la réparation tissulaire et la fibrose. Les fibroblastes carencés en sérum pendant quatre jours montrent des ratios LC3B-II/-I élevés et des niveaux de SQSTM1/p62 diminués. L’augmentation de l’autophagie est accompagnée d’une augmentation de l’expression des marqueurs de différenciation myofibroblastique ACTA2/αSMA et collagènes de type 1 et 3 et de la formation de fibres de stress. Les fibroblastes autophagiques expriment les marqueurs de sénescence CDKN1A (p21) et p16INK4a (p16) et montrent une augmentation de l’activité beta-galactosidase associée à la sénescence. L’inhibition de l’autophagie à l’aide de différents inhibiteurs de phosphoinositide 3-kinase de classe I et de phosphatidylinositol 3-kinase de classe III (PtdIns3K) ou par inhibition génique à l’aide d’ARN interférant ATG7 bloquent l’expression des marqueurs de différenciation et de sénescence. L’expression et la sécrétion de CTGF (connective tissue growth factor) sont augmentées chez les fibroblastes autophagiques. L’inhibition de l’expression du CTGF par interférence génique prévient la différenciation myofibroblastique, démontrant l’importance de ce facteur pro-fibrotique pour la différenciation induite par l’autophagie. La phosphorylation de la kinase RPS6KB1/p70S6K, cible du complexe MTORC1, est abolie dans les fibroblastes autophagiques. La phosphorylation d’AKT à la Ser473, une cible du complexe MTORC2, diminue lors de la carence en sérum des fibroblastes mais est suivie d’une rephosphorylation après 2 jours. Ce résultat suggère la réactivation de MTORC2 lors d’une autophagie prolongée. Ceci a été vérifié par inhibition de l’autophagie dans les fibroblastes carencés en sérum. Les inhibiteurs de PtdIns3K et le siRNA ATG7 bloquent la rephosphorylation d’AKT. L’inhibition de la réactivation de MTORC2, et donc de la rephosphorylation d’AKT, est aussi obtenue par exposition des fibroblastes à la rapamycine, le Torin 1 ou par inhibition génique de RICTOR. Ces traitements inhibent l’augmentation de l’expression du CTGF ainsi que des marqueurs de différenciation et de sénescence, démontrant le rôle central joué par MTORC2 dans ces processus. Le stress oxydant peut induire la sénescence et la carence en sérum est connue pour augmenter la quantité de ROS (reactive oxygen species) dans les cellules. Afin d’investiguer le rôle des ROS dans la différenciation et la sénescence induites par l’autophagie, nous avons incubés les fibroblastes carencés en sérum en présence de N-acetyl-L-cysteine (NAC). Le NAC diminue la production de ROS, diminue les marqueurs d’autophagie, de sénescence et de différenciation myofibroblastique. Le NAC inhibe aussi la phosphorylation d’AKT Ser473. L’ensemble de ces résultats identifient les ROS en association avec une autophagie prolongée comme des nouveaux activateurs du complexe MTORC2. MTORC2 est central pour l’activation subséquente de la sénescence et de la différenciation myofibroblastique.
Resumo:
Anabaena PCC 7120 nifHDK operon is interrupted by an 11 kb DNA element which is excised during the development of heterocysts by Excisase A, encoded by the xisA gene residing on the element. The excision is a site-specific recombination event that occurs at the I I base pair direct repeats flanking the element. Earlier work showed the excision of the I I kb element in Escherichia coli at a frequency 0.3%. We report here the excision of this element at 1.1% and 1.98% in E. coli DH5 alpha, and 1.9% and 10.9% in E. coli JM 101 when grown on Luria broth and minimal media, respectively. Excision of nifD element in isogenic recA(-) (RK1) and recA(+) (RK2) E. coli JM101 P1 transductants, showed similar results to that of E. coli JM101 and DH5 alpha, respectively. A plasmid pMX32, carrying a xisA defective 11 kb element, showed no excision in E. coli RK2 strain. In contrast to Anabaena PCC 7120, excision of nifD element did not increase in E. call DH5 alpha grown in iron-deficient conditions. A PxisA::lacZ transcriptional fusion, used to detect the expression of elusive xisA gene, showed maximal beta-galactosidase activity in the stationary phase. The results suggest that the excision event in E. coli may involve additional factors, such as RecA and that the physiological status can influence the excision of nifD element. (C) 2007 Elsevier Ltd. All rights reserved.
Resumo:
TGF-beta1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets alphavbeta3 and alpha5beta1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-beta1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or beta-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-beta1 for 24 h, but not with PDGF-BB, interferon-gamma, TNF-alpha, nor PMA, increased alphavbeta3 and alpha5beta1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus beta-galactosidase activity increased from 35 +/- 5% (controls) to 75 +/- 5% after TGF-beta1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered beta-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-beta1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases.
Resumo:
A novel strain of Bifidobacterium bifidum NCIMB 41171, isolated from a faecal sample from a healthy human volunteer and able to express beta-galactosidase activity, was used in synthesis reactions for the production of galactooligosaccharide from lactose. The beta-galactosidase activity of whole bifidobacterial cells showed an optimum activity at pH 6.8-7.0 and 40 degrees C. The transgalactosylation activity of the B. bifidum cells from 50% (w/w) lactose resulted in a galactooligosaccharide mixture (20% w/w) comprising (w/w): 25% disaccharides, 35% trisaccharides, 25% tetrasaccharides and 15% pentasaccharides. Using different initial lactose concentrations, the conversion rate to galactooligosaccharides was maximum (35%) when 55% (w/w) lactose was used. In fermentation experiments, B. bifidum showed an increased preference towards the produced galactooligosaccharide mixture, displaying higher growth rate and short-chain fatty acid production when compared with commercially available oligosaccharides.
Resumo:
Type III secretion systems of enteric bacteria enable translocation of effector proteins into host cells. Secreted proteins of verotoxigenic Escherichia coli O157 strains include components of a translocation apparatus, EspA, -B, and -D, as well as "effectors" such as the translocated intimin receptor (Tir) and the mitochondrion-associated protein (Map). This research has investigated the regulation of LEE4 translocon proteins, in particular EspA. EspA filaments could not be detected on the bacterial cell surface when E. coli O157:H7 was cultured in M9 minimal medium but were expressed from only a proportion of the bacterial population when cultured in minimal essential medium modified with 25 mM HEPES. The highest proportions of EspA-filamented bacteria were detected in late exponential phase, after which filaments were lost rapidly from the bacterial cell surface. Our previous research had shown that human and bovine E. coli O157:H7 strains exhibit marked differences in EspD secretion levels. Here it is demonstrated that the proportion of the bacterial population expressing EspA filaments was associated with the level of EspD secretion. The ability of individual bacteria to express EspA filaments was not controlled at the level of LEE1-4 operon transcription, as demonstrated by using both beta-galactosidase and green fluorescent protein (GFP) promoter fusions. All bacteria, whether expressing EspA filaments or not, showed equivalent levels of GFP expression when LEEI-4 translational fusions were used. Despite this, the LEE4-espADB mRNA was more abundant from populations with a high proportion of nonsecreting bacteria (low secretors) than from populations with a high proportion of secreting and therefore filamented bacteria (high secretors). This research demonstrates that while specific environmental conditions are required to induce LEEI-4 expression, a further checkpoint exists before EspA filaments are produced on the bacterial surface and secretion of effector proteins occurs. This checkpoint in E. coli O157:H7 translocon expression is controlled by a posttranscriptional mechanism acting on LEE4-espADB mRNA. The heterogeneity in EspA filamentation could arise from phase-variable expression of regulators that control this posttranscriptional mechanism.
Resumo:
During seedling establishment, cotyledons of the rain forest tree Hymenaea courbaril mobilize storage cell wall xyloglucan to sustain growth. The polysaccharide is degraded and its products are transported to growing sink tissues. Auxin from the shoot controls the level of xyloglucan hydrolytic enzymes. It is not yet known how important the expression of these genes is for the control of storage xyloglucan degradation. In this work, partial cDNAs of the genes xyloglucan transglycosylase hydrolase (HcXTH1) and beta-galactosidase (HcBGAL1), both related to xyloglucan degradation, and two other genes related to sucrose metabolism [alkaline invertase (HcAlkIN1) and sucrose synthase (HcSUS1)], were isolated. The partial sequences were characterized by comparison with sequences available in the literature, and phylogenetic trees were assembled. Gene expression was evaluated at intervals of 6 h during 24 h in cotyledons, hypocotyl, roots, and leaves, using 45-d-old plantlets. HcXTH1 and HcBGAL1 were correlated to xyloglucan degradation and responded to auxin and light, being down-regulated when transport of auxin was prevented by N-1-naphthylphthalamic acid (NPA) and stimulated by constant light. Genes related to sucrose metabolism, HcAlkIN1 and HcSUS1, responded to inhibition of auxin transport in consonance with storage mobilization in the cotyledons. A model is proposed suggesting that auxin and light are involved in the control of the expression of genes related to storage xyloglucan mobilization in seedlings of H. courbaril. It is concluded that gene expression plays a role in the control of the intercommunication system of the source-sink relationship during seeding growth, favouring its establishment in the shaded environment of the rain forest understorey.
Resumo:
Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.
Resumo:
The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated beta-galactosidase (SA-beta Gal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP`s deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187. (C) 2011 Elsevier B.V. All rights reserved.
Resumo:
Ingestion of transgalactosylated oligosaccharides (TOS) and other non-digestible oligosaccharides (NDOs) induces a significant increase in Bifidobacterium, Lactobacillus and some desirable species of Streptococcus populations in the gut of human and other animals (prebiotic effect). This change in the intestinal flora is responsible for several beneficial physiological effects such as a decrease of putrefactive products in the feces, lower blood cholesterol content, higher Ca2+ absorption, a smaller loss of bone tissue in ovariotomized female rats and a lower incidence of colon cancer. beta-Galactosidase from Penicillium simplicissimum, a strain isolated from soil, showed high galactosyltransferase activity when incubated with a highly concentrated lactose solution. Optimum pH and temperature ranges for hydrolytic activity were 4.0-4.6 and 55-60 degrees C, respectively, for a lactose concentration of 5.0% (w/v). Maximal galactosyltransferase activity was obtained at pH 6.5 and 50 degrees C and TOS synthesis was positively associated with lactose concentration in the reaction medium. Thus, when 50 ml of a 60% (w/v) lactose solution was incubated with 26.6 U of beta-galactosidase under the best pH and temperature conditions for transferase activity, a final product with 30.5% TOS (183 mg ml(-1)), 27.5% residual lactose and 42.0% monosaccharides was obtained. (C) 1999 Elsevier B.V. Ltd. All rights reserved.
Resumo:
The study was carried out with the objective to evaluate the effects of the inclusion of the wheat bran (WB) with or without supplementation of an enzymatic complex (EC) on the performance of semi-heavy hens in the egg-production phase. A total of 288 Lohmann Brown pullets were used, distributed to a completely randomized design in 4 x 2 factorial arrangement, composed by four WB levels (0, 3, 6 and 9%) in the ration and enzymatic complex supplementation (0 or 100g/100 kg diet), with eight treatments and six replicates of six birds. The enzymatic complex contained the enzymes beta-galactosidase, galactomananase, xilanase and alpha-glucanase. Feed intake, final body weight, egg production, egg weight, egg mass, egg mass feed conversion or egg dozen feed conversion was not affected by WB inclusion in the diets. Egg shell specific gravity deteriorated as WB levels increase in the diets. None of the characteristics was affected by the enzymatic complex supplementation, except for egg weight, that improved from 62.74 to 64.28 g. Then, the use up to 9.0% of wheat bran in the ration is recommended for semi-heavily chickens in the production phase. The supplementation of alpha-galactosidase, galactomannanase, xylanase and alpha-glucanase improve egg weight.
Resumo:
Problems related to the systemic administration of drugs, such as biodistribution, difficulty of targeting, necessity of high doses to achieve adequate levels of the drug in specific sites, toxicity, and undesirable side effects have lead to the development of systems able to direct the drug to specific sites in the body. Among the possible organs to the targeting of drugs, the colon can be used for local and systemic therapies. By developing such systems some models have been tested, using pH dependent release, release controlled by enzymatic degradation, time controlled release systems and pressure controlled release systems. This review presents an overview of the colonic release of drugs and the strategies used to achieve such targeting.
Resumo:
Pós-graduação em Química - IBILCE
Resumo:
Background: Solobacterium moorei is a volatile sulfide compound (VSC)-producing Gram-positive anaerobic bacterium that has been associated with halitosis. The aim of this study was to investigate the effects of green tea extract and its major constituent epigallocatechin-3-gallate (EGCG) on growth and severalhalitosis-related properties of S. moorei.Methods: A microplate dilution assay was used to determine the antibacterial activity of green tea extract and EGCG against S. moorei. Their effects on bacterial cell membrane integrity were investigated by transmission electron microscopy and a fluorescence-based permeability assay. Biofilm formation was quantified by crystal violet staining. Adhesion of FITC-labeled S. moorei to oral epithelial cells was monitored by fluorometry. The modulation of beta-galactosidase gene expression in S. moorei was evaluated by quantitative RT-PCR.Results: The green tea extract as well as EGCG inhibited the growth of S. moorei, with MIC values of 500 and 250 mu g/ml, respectively. Transmission electron microscopy analysis and a permeabilization assay brought evidence that the bacterial cell membrane was the target of green tea polyphenols. Regarding the effects of green tea polyphenols on the S. moorei colonization properties, it was found that biofilm formation on EGCG-treated surfaces was significantly affected, and that green tea extract and EGCG can cause the eradication of pre-formed S. moorei biofilms. Moreover, both the green tea extract and EGCG were found to reduce the adherence of S. moorei to oral epithelial cells. The beta-galactosidase activity of S. moorei, which plays a key role in VSC production, was dose-dependently inhibited by green tea polyphenols. In addition, EGCG at 1/2 MIC significantly decreased the beta-galactosidase gene expression.Conclusion: Our study brought evidence to support that green tea polyphenols possess a number of properties that may contribute to reduce S. moorei-related halitosis. Therefore, these natural compounds may be of interest to be used to supplement oral healthcare products.
Resumo:
(Diurnal changes in storage carbohydrate metabolism in cotyledons of the tropical tree Hymenaea courbaril L. (Leguminosae)). The cotyledons of Hymenaea courbaril store large amounts of xyloglucan, a cell wall polysaccharide that is believed to serve as storage for the period of seedling establishment. During storage mobilisation, xyloglucan seems to be degraded by a continuous process that starts right after radicle protrusion and follows up to the establishment of photosynthesis. Here we show evidence that events related to the hydrolases activities and production (alpha-xylosidase, beta-galactosidase, beta-glucosidase and xyloglucan endo-beta-transglucosilase) as well as auxin, showed changes that follow the diurnal cycle. The period of higher hydrolases activities was between 6pm and 6am, which is out of phase with photosynthesis. Among the enzymes, alpha-xilosidase seems to be more important than beta-glucosidase and beta-galactosidase in the xyloglucan disassembling mechanism. Likewise, the sugars related with sucrose metabolism followed the rhythm of the hydrolases, but starch levels were shown to be practically constant. A high level of auxin was observed during the night, what is compatible with the hypothesis that this hormone would be one of the regulators of the whole process. The probable biological meaning of the existence of such a complex control mechanism during storage mobilisation is likely to be related to a remarkably high level of efficiency of carbon usage by the growing seedling of Hymenaea courbaril, allowing the establishment of very vigorous seedlings in the tropical forest.