A bacterial field isolate recovered from infected tomato plants in a green-house at Sidi Rehal, a region near Casablanca city (Morocco), was identified as the gammaproteobacterium Pseudomonas syringae pv. tomato DC300...A bacterial field isolate recovered from infected tomato plants in a green-house at Sidi Rehal, a region near Casablanca city (Morocco), was identified as the gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 strain, the causal agent of bacterial speck. The bacterial isolate was characterized by morphological, biochemical and molecular biological tests, its growth curves carried out in various culture media, and its phytopathogenicity verified by infection tests. A screening was performed to evaluate the antibacterial activity of methanolic extracts of 12 selected Moroccan plants against the P. syringae pv. tomato DC3000 isolate, and Agar-well diffusion and Broth microdilution methods were used to determine minimum inhibitory and minimum bactericidal concentrations. Among the methanolic extracts tested, only those of Nigella sativa, Geranuim robertianum, Aizoon canariense and Rubia peregrine showed clear inhibitory and bactericidal activities, although the highest values were achieved with N. sativa, a plant used in Morocco as a spice, condiment and medicinal treatment.展开更多
Staphylococcus aureus is one of the main pathogenic agents responsible for nosocomial and community-acquired bacterial infections. The pathogenicity of this Gram-positive bacterium is ensured by its different adhesion...Staphylococcus aureus is one of the main pathogenic agents responsible for nosocomial and community-acquired bacterial infections. The pathogenicity of this Gram-positive bacterium is ensured by its different adhesion factors. Collagen and the extracellular glycoprotein adhesin are among the Staphylococcus most important virulence factors. It has been shown that most of the S. aureus strains carry the ica operon, responsible for biofilm production. However, the coexpression of the icaA and the icaD genes is necessary for complete biofilm synthesis. The aim of our study was to study a collection of 15 clinical strains of S. aureus from different sources for the presence of can and icaD genes coding intercellular adhesion proteins. We also intended to estimate the strains’ ability to form biofilms by the red Cong method and to test the adhesion ability of S. aureus to the ciliated protist Tetrahymena pyriformis, which we used as a novel cellular adhesion model. Finally, we checked the adhesion’s inhibition capacity of some plants extracts. The molecular detection of adhesion genes revealed that 80% of strains are cna positive, and 73% are icaD positive. Qualitative biofilm production of S. aureus revealed that 66.6% of strains were slime producers. The adhesion test revealed that 20% of strains are strongly adhering to T. pyriformis and that the Clematis cirrhosa extract has an anti-adhering effect of S. aureus to the ciliate T. pyriformis.展开更多
According to molecular biology, genomic and proteomic data, the phytopathogenic gamma-proteobacterium Pseudomonas syringae pv. tomato DC3000 produces a number of proteins that may promote infection and draw nutrients ...According to molecular biology, genomic and proteomic data, the phytopathogenic gamma-proteobacterium Pseudomonas syringae pv. tomato DC3000 produces a number of proteins that may promote infection and draw nutrients from plants. Remarkably, P. syringae DC3000 strain possesses three paralogous gap genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes with different predicted molecular sizes and metabolic functions. As GAPDH was shown to be a virulence factor in other microbial pathogens, in the current study, we analyzed the expression levels of each paralogous gap gene by realtime PCR to understand the actual impact of their protein products on P. syringae virulence. We found that all of them were strongly induced during the infection process. Nevertheless, proteomic analysis of culture supernatants revealed that only Class I GAPDH1 encoded by the gap1 gene was identified as an extracellular protein in infective cells. These results strongly suggest that this GAPDH should play a role in the infective process, including its well-know enzymatic function in the glycolytic metabolic pathway.展开更多
W Tumor proliferation of cancer cells requires a high intake of oxygen by angiogenesis. Deep cancer cells suffer from asphyxia and meet their energy needs through the enzymes of glycolysis. The anti-angio- genesis app...W Tumor proliferation of cancer cells requires a high intake of oxygen by angiogenesis. Deep cancer cells suffer from asphyxia and meet their energy needs through the enzymes of glycolysis. The anti-angio- genesis approach has been recognized for therapeutic purposes, but the deep cancers, difficult to reach by this therapy, could be targeted by inhibiting an enzyme of the glycolytic cycle. Our work focused on the study of the expression of GAPDH, a key enzyme of glycolysis, in cervix, breast and prostate tumors, for two approaches: Fundamental and targeted therapeutics. 60 samples, taken at the Anatomopathology laboratory of the Pasteur Institute of Morocco, were examined histologically and immunohistochemically, demonstrating the expression and cellular localization of GAPDH. The three organs have shown an overex-pression of GAPDH in tumor tissues. At the cellular level, the localization of GAPDH in cancer tissue is diffuse but mostly nuclear whereas it remains focused at the membrane and/or the cytoplasm in benign tumor tissues. From these results we could assume that GAPDH is involved in the cancer process and draws attention to a possible new nuclear role that could be either specific to one form or different isoforms of GAPDH enzyme.展开更多
Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterizati...Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.展开更多
Phosphorus is one of the bioelements most needed as a compound cell by living organisms. Phosphorus is involved in several pathologies: in human with bone and kidney diseases, in mammals with metabolism disorder (gluc...Phosphorus is one of the bioelements most needed as a compound cell by living organisms. Phosphorus is involved in several pathologies: in human with bone and kidney diseases, in mammals with metabolism disorder (glucose, insulin···), in microorganisms whose phosphorus is involved in cell growth. Phosphorus has various forms including pyrophosphate, a by-product of multiple pathways of biosynthesis. Enzymes that hydrolyze pyrophosphate are called inorganic pyrophosphatases (PPases). Two major types of inorganic pyrophosphatases are distinguished: the soluble pyrophosphatases (sPPases) and the membrane pyrophosphatases (mPPases or H+/Na+-PPases). They play a key role in the control of intracellular inorganic pyrophosphate level and produce an important ions gradient (H+ or Na+) to the cells. In this work, we primarily focused on the physiological study in a phosphate-poor medium of two models Tetrahymena thermophile and Tetrahymena pyriformis, following the mobility, the growth and the morphology of cells. Secondly, we evaluated the enzymatic activity of soluble and membrane pyrophosphatases in both species grown in the same complex medium. A decrease of cell growth is correlated with unusual morphologies and different mobility in the stress medium. The measurement of soluble and membrane inorganic pyrophosphatases activities also shows a decrease which illustrates the lack of phosphate found in the stress medium. Deficiency of phosphate is a limiting factor for protozoan growth. These results indicate that Tetrahymena can be used as a model of cellular stress and consists of a target to study inorganic pyrophosphatases for a better understanding of phosphate cycle in higher organisms.展开更多
文摘A bacterial field isolate recovered from infected tomato plants in a green-house at Sidi Rehal, a region near Casablanca city (Morocco), was identified as the gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 strain, the causal agent of bacterial speck. The bacterial isolate was characterized by morphological, biochemical and molecular biological tests, its growth curves carried out in various culture media, and its phytopathogenicity verified by infection tests. A screening was performed to evaluate the antibacterial activity of methanolic extracts of 12 selected Moroccan plants against the P. syringae pv. tomato DC3000 isolate, and Agar-well diffusion and Broth microdilution methods were used to determine minimum inhibitory and minimum bactericidal concentrations. Among the methanolic extracts tested, only those of Nigella sativa, Geranuim robertianum, Aizoon canariense and Rubia peregrine showed clear inhibitory and bactericidal activities, although the highest values were achieved with N. sativa, a plant used in Morocco as a spice, condiment and medicinal treatment.
文摘Staphylococcus aureus is one of the main pathogenic agents responsible for nosocomial and community-acquired bacterial infections. The pathogenicity of this Gram-positive bacterium is ensured by its different adhesion factors. Collagen and the extracellular glycoprotein adhesin are among the Staphylococcus most important virulence factors. It has been shown that most of the S. aureus strains carry the ica operon, responsible for biofilm production. However, the coexpression of the icaA and the icaD genes is necessary for complete biofilm synthesis. The aim of our study was to study a collection of 15 clinical strains of S. aureus from different sources for the presence of can and icaD genes coding intercellular adhesion proteins. We also intended to estimate the strains’ ability to form biofilms by the red Cong method and to test the adhesion ability of S. aureus to the ciliated protist Tetrahymena pyriformis, which we used as a novel cellular adhesion model. Finally, we checked the adhesion’s inhibition capacity of some plants extracts. The molecular detection of adhesion genes revealed that 80% of strains are cna positive, and 73% are icaD positive. Qualitative biofilm production of S. aureus revealed that 66.6% of strains were slime producers. The adhesion test revealed that 20% of strains are strongly adhering to T. pyriformis and that the Clematis cirrhosa extract has an anti-adhering effect of S. aureus to the ciliate T. pyriformis.
文摘According to molecular biology, genomic and proteomic data, the phytopathogenic gamma-proteobacterium Pseudomonas syringae pv. tomato DC3000 produces a number of proteins that may promote infection and draw nutrients from plants. Remarkably, P. syringae DC3000 strain possesses three paralogous gap genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes with different predicted molecular sizes and metabolic functions. As GAPDH was shown to be a virulence factor in other microbial pathogens, in the current study, we analyzed the expression levels of each paralogous gap gene by realtime PCR to understand the actual impact of their protein products on P. syringae virulence. We found that all of them were strongly induced during the infection process. Nevertheless, proteomic analysis of culture supernatants revealed that only Class I GAPDH1 encoded by the gap1 gene was identified as an extracellular protein in infective cells. These results strongly suggest that this GAPDH should play a role in the infective process, including its well-know enzymatic function in the glycolytic metabolic pathway.
文摘W Tumor proliferation of cancer cells requires a high intake of oxygen by angiogenesis. Deep cancer cells suffer from asphyxia and meet their energy needs through the enzymes of glycolysis. The anti-angio- genesis approach has been recognized for therapeutic purposes, but the deep cancers, difficult to reach by this therapy, could be targeted by inhibiting an enzyme of the glycolytic cycle. Our work focused on the study of the expression of GAPDH, a key enzyme of glycolysis, in cervix, breast and prostate tumors, for two approaches: Fundamental and targeted therapeutics. 60 samples, taken at the Anatomopathology laboratory of the Pasteur Institute of Morocco, were examined histologically and immunohistochemically, demonstrating the expression and cellular localization of GAPDH. The three organs have shown an overex-pression of GAPDH in tumor tissues. At the cellular level, the localization of GAPDH in cancer tissue is diffuse but mostly nuclear whereas it remains focused at the membrane and/or the cytoplasm in benign tumor tissues. From these results we could assume that GAPDH is involved in the cancer process and draws attention to a possible new nuclear role that could be either specific to one form or different isoforms of GAPDH enzyme.
文摘Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.
文摘Phosphorus is one of the bioelements most needed as a compound cell by living organisms. Phosphorus is involved in several pathologies: in human with bone and kidney diseases, in mammals with metabolism disorder (glucose, insulin···), in microorganisms whose phosphorus is involved in cell growth. Phosphorus has various forms including pyrophosphate, a by-product of multiple pathways of biosynthesis. Enzymes that hydrolyze pyrophosphate are called inorganic pyrophosphatases (PPases). Two major types of inorganic pyrophosphatases are distinguished: the soluble pyrophosphatases (sPPases) and the membrane pyrophosphatases (mPPases or H+/Na+-PPases). They play a key role in the control of intracellular inorganic pyrophosphate level and produce an important ions gradient (H+ or Na+) to the cells. In this work, we primarily focused on the physiological study in a phosphate-poor medium of two models Tetrahymena thermophile and Tetrahymena pyriformis, following the mobility, the growth and the morphology of cells. Secondly, we evaluated the enzymatic activity of soluble and membrane pyrophosphatases in both species grown in the same complex medium. A decrease of cell growth is correlated with unusual morphologies and different mobility in the stress medium. The measurement of soluble and membrane inorganic pyrophosphatases activities also shows a decrease which illustrates the lack of phosphate found in the stress medium. Deficiency of phosphate is a limiting factor for protozoan growth. These results indicate that Tetrahymena can be used as a model of cellular stress and consists of a target to study inorganic pyrophosphatases for a better understanding of phosphate cycle in higher organisms.
