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Mem. Inst. Oswaldo Cruz ; 110(8): 1003-1009, Dec. 2015. tab, graf
Article in English | LILACS | ID: lil-769825


An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Humans , Carbapenems/metabolism , Drug Resistance, Multiple, Bacterial/genetics , Mutation , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/genetics , beta-Lactam Resistance/genetics , beta-Lactamases/metabolism , Aminoglycosides/metabolism , Amphotericin B/analogs & derivatives , Amphotericin B/metabolism , Antifungal Agents/metabolism , Brazil , Cephalosporinase/classification , Cephalosporinase/metabolism , Codon, Nonsense/metabolism , Enzyme Activation/genetics , Frameshift Mutation/genetics , Gene Expression Regulation, Bacterial/genetics , Membrane Transport Proteins/metabolism , Methyltransferases/metabolism , Nucleotidyltransferases/metabolism , Point Mutation/genetics , Porins/metabolism , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/isolation & purification , Repetitive Sequences, Nucleic Acid , beta-Lactamases/genetics
Experimental & Molecular Medicine ; : e153-2015.
Article in English | WPRIM | ID: wpr-57313


Lysosomal dysfunction is a common pathological feature of neurodegenerative diseases. GTP-binding protein type A1 (GBA1) encodes beta-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase. Homozygous mutations in GBA1 cause Gaucher disease, the most common lysosomal storage disease, while heterozygous mutations are strong risk factors for Parkinson's disease. However, whether loss of GCase 1 activity is sufficient for lysosomal dysfunction has not been clearly determined. Here, we generated human neuroblastoma cell lines with nonsense mutations in the GBA1 gene using zinc-finger nucleases. Depending on the site of mutation, GCase 1 activity was lost or maintained. The cell line with GCase 1 deficiency showed indications of lysosomal dysfunction, such as accumulation of lysosomal substrates, reduced dextran degradation and accumulation of enlarged vacuolar structures. In contrast, the cell line with C-terminal truncation of GCase 1 but with intact GCase 1 activity showed normal lysosomal function. When alpha-synuclein was overexpressed, accumulation and secretion of insoluble aggregates increased in cells with GCase 1 deficiency but did not change in mutant cells with normal GCase 1 activity. These results demonstrate that loss of GCase 1 activity is sufficient to cause lysosomal dysfunction and accumulation of alpha-synuclein aggregates.

Humans , Cell Line , Enzyme Activation/genetics , Gene Knockout Techniques , Gene Order , Genetic Loci , Glucosylceramidase/genetics , Lysosomes/metabolism , Mutation , Protein Aggregation, Pathological/genetics , Protein Binding , Zinc Fingers , alpha-Synuclein/chemistry
Salud colect ; 10(3): 325-337, sep.-dic. 2014. tab
Article in Spanish | LILACS | ID: lil-733293


En el marco de la creciente feminización de la profesión médica en México, el artículo indaga sobre las características de este proceso para el caso de la ginecobstetricia. Considerando la feminización como un proceso de cambio, que se analiza cuantitativa y cualitativamente, el artículo se detiene en especial en las experiencias de las mujeres ginecobstetras, experiencias que se dan en el seno de una especialidad que, desde sus orígenes, funcionó como un dispositivo de control del cuerpo de las mujeres. Basado en una investigación etnográfica, el artículo combina fuentes estadísticas, de archivo y de observación de campo. El material que surge de las entrevistas muestra las experiencias y tensiones que viven las ginecobstetras en este contexto.

In the framework of an increasing feminization of the medical profession in Mexico, this article explores the characteristics of this process in the obstetrics and gynecology specialty. Understanding feminization as a process of change to be analyzed both quantitatively and qualitatively, the article focuses special attention on the experiences of female obstetrician-gynecologists within a medical specialty that has since its origins functioned as a mechanism of control over women's bodies. Based on ethnographic research, the article combines statistical and archival sources and field observation. The interviews reveal the experiences and tensions women obstetrician-gynecologists encounter in this context.

Alcohol Oxidoreductases/chemistry , Alcohol Oxidoreductases/metabolism , Arginine/chemistry , Pseudomonas putida/enzymology , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/isolation & purification , Binding, Competitive/genetics , Catalysis , Enzyme Activation/genetics , Flavin Mononucleotide/metabolism , Kinetics , Ligands , Mandelic Acids/metabolism , Mutagenesis, Site-Directed , Phenylacetates/metabolism , Protein Binding/genetics , Pseudomonas putida/genetics , Substrate Specificity/genetics , Sulfites/metabolism
Braz. j. microbiol ; 41(1): 201-208, Jan.-Mar. 2010. ilus, graf
Article in English | LILACS | ID: lil-531753


The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100 percent similarity, according to ARDRA results.

Enzyme Activation/genetics , In Vitro Techniques , Rhizobiaceae/cytology , Rhizobiaceae/genetics , DNA Repair Enzymes/genetics , DNA Repair Enzymes/isolation & purification , Genes, Plant , Genetic Variation , Polymerase Chain Reaction , Rhizobiaceae/growth & development
Experimental & Molecular Medicine ; : 385-390, 2002.
Article in English | WPRIM | ID: wpr-160901


Change in fibrin stabilizing activity of factor XIII A subunit (FXIII-A) caused by a specific mutation, Val34Leu, is recently implicated to incidences of pathophysiology of thrombosis. In an effort to understand the effect of Val34Leu on enhanced catalytic role of FXIII-A, wild type human factor XIII A (HFXIII-A) and mutant HFXIII-A: HFXIII-A (V34L), HFXIII-A (V35L) and HFXIII-A (V34L/V35L) cDNA were expressed in E.coli system where the purified recombinant FXIII-A (rFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet. Using these rFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated rFXIII-A were characterized. rFXIII-A (V34L) and rFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type rFXIII-A and V35L variant. However, the activated rFXIII-A and mutants showed the identical catalytic efficiency as measured by in vitro assay. These results suggest that ready activation caused by a specific mutation of neighboring thrombin cleavage site(s) in the activation peptide of FXIII-A like V34L resulted in the real-time amount of the activated factor XIII-A that could influence the outcome of fibrin stabilization in vivo such as alpha2- plasmin inhibitor crosslinking to fibrin, a reaction known to be dependent on the initial concentration of active factor-XIII-A.

Humans , Blood Coagulation Tests , Catalysis , Enzyme Activation/genetics , Escherichia coli/genetics , Factor XIII/genetics , Fibrin/metabolism , Immunoblotting , Leucine/genetics , Mutagenesis, Site-Directed , Polymorphism, Genetic , Recombinant Proteins/genetics , Thrombin/metabolism , Valine/genetics