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Rev. chil. infectol ; 36(3): 392-395, jun. 2019. tab, graf
Article in Spanish | LILACS | ID: biblio-1013799


Resumen Presentamos un caso de bacteriemia por Vibrio cholerae no-O1/ no-O139 en una mujer de 81 años con un cuadro de dolor abdominal, fiebre, vómitos, diarrea, coluria e ictericia, mientras visitaba una zona rural sin acceso a agua potable. La identificación se realizó por la técnica de espectrometría de masa MALDI-TOF, confirmándose una cepa no toxigénica no-O1/no-139. La caracterización molecular del aislado demostró la ausencia del gen de la toxina del cólera (CTX), y pilus TCP; sin embargo, presentó cinco de los seis genes de virulencia presentes en la isla de patogenicidad homóloga denominada VPaI-7 del V. parahaemolyticus (vcs N2+, vcs C2+, vcs V2+,toxR-, vspD+, T vopF+). Además, el aislado presentó los genes de virulencia hylA y rtxA. Este es el primer caso reportado en Chile de una cepa clínica de V. cholerae no-O1, no-O139 aislada de hemocultivos portador de un segmento homólogo de la isla de patogenicidad denominada VPaI-7 de V. parahaemolyticus, el cual codifica para un sistema de secreción tipo III (TTSS), que probablemente contribuye a su virulencia.

We report a case of V. cholerae non-O1 / non-O139 bacteremia in an 81-year-old woman with abdominal pain, fever, vomiting, liquid stools, choluria and jaundice, while visiting a rural area without access to potable water. The identification was made by the MALDI-TOF mass spectrometry technique and subsequently the non-toxigenic non-O1 / non-139 strain was confirmed in the national reference laboratory. The molecular characterization demonstrated the absence of the cholera toxin gene (CTX), and the TCP pilus, however, presented 5 of 6 virulence genes present in an island of homologous pathogenicity named VPaI-7 of V. parahaemolyticus (vcs N2 +, vcs C2 +, vcs V2 +, toxR-, vspD +, T vopF +) and in addition it was positive for hylAy rtxA virulence genes recognized outside the island. This is the first case reported in Chile of a clinical strain of V. cholerae non-O1, non-O139 isolated from blood culture that carries in its genome a homologous segment of the pathogenicity island named VPaI-7 of V. parahaemolyticus, which codifies for a type III secretion system (TTSS) that probably contributes to his virulence.

Humans , Female , Aged, 80 and over , Bacterial Proteins/chemistry , Vibrio cholerae/chemistry , Bacteremia/etiology , Vibrio cholerae non-O1/chemistry , Bacterial Proteins/isolation & purification , Vibrio cholerae/isolation & purification , Vibrio cholerae/pathogenicity , Virulence , Cholera/complications , Cholera/microbiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Vibrio cholerae non-O1/isolation & purification , Vibrio cholerae non-O1/pathogenicity , Genomic Islands
Braz. j. infect. dis ; 22(2): 129-136, Mar.-Apr. 2018. tab, graf
Article in English | LILACS | ID: biblio-951633


ABSTRACT Introduction: Biofilm production is an important mechanism for the survival of Pseudomonas aeruginosa and its relationship with antimicrobial resistance represents a challenge for patient therapeutics. P. aeruginosa is an opportunistic pathogen frequently associated to nosocomial infections, especially in imunocompromised hosts. Objectives: Analyze the phenotypic biofilm production in P. aeruginosa isolates, describe clonal profiles, and analyze quorum sensing (QS) genes and the occurrence of mutations in the LasR protein of non-biofilm producing isolates. Methods: Isolates were tested for biofilm production by measuring cells adherence to the microtiter plates. Clonal profile analysis was carried out through ERIC-PCR, QS genes were by specific PCR. Results: The results showed that 77.5% of the isolates were considered biofilm producers. The results of genotyping showed 38 distinct genetic profiles. As for the occurrence of the genes, 100% of the isolates presented the lasR, rhlI and rhlR genes, and 97.5%, presented the lasI gene. In this study nine isolates were not biofilm producers. However, all presented the QS genes. Amplicons related to genes were sequenced in three of the nine non-biofilm-producing isolates (all presenting different genetic similarity profile) and aligned to the sequences of those genes in P. aeruginosa strain PAO1 (standard biofilm-producing strain). Alignment analysis showed an insertion of three nucleotides (T, C and G) causing the addition of an amino acid valine in the sequence of the LasR protein, in position 53. Conclusion: The modeling of the resulting LasR protein showed a conformational change in its structure, suggesting that this might be the reason why these isolates are unable to produce biofilm.

Humans , Pseudomonas aeruginosa/physiology , Pseudomonas Infections/microbiology , Bacterial Proteins/genetics , Trans-Activators/genetics , Biofilms/growth & development , Biofilms/drug effects , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/chemistry , Pseudomonas Infections/drug therapy , Bacterial Proteins/chemistry , Trans-Activators/chemistry , Polymerase Chain Reaction/methods , Cross Infection , Drug Resistance, Multiple, Bacterial , Anti-Infective Agents/pharmacology , Anti-Bacterial Agents/pharmacology
Braz. j. microbiol ; 49(1): 169-176, Jan.-Mar. 2018. tab, graf
Article in English | LILACS | ID: biblio-889211


ABSTRACT Major health challenges as the increasing number of cases of infections by antibiotic multiresistant microorganisms and cases of Alzheimer's disease have led to searching new control drugs. The present study aims to verify a new way of obtaining bioactive extracts from filamentous fungi with potential antimicrobial and acetylcholinesterase inhibitory activities, using epigenetic modulation to promote the expression of genes commonly silenced. For such finality, five filamentous fungal species (Talaromyces funiculosus, Talaromyces islandicus, Talaromyces minioluteus, Talaromyces pinophilus, Penicillium janthinellum) were grown or not with DNA methyltransferases inhibitors (procainamide or hydralazine) and/or a histone deacetylase inhibitor (suberohydroxamic acid). Extracts from T. islandicus cultured or not with hydralazine inhibited Listeria monocytogenes growth in 57.66 ± 5.98% and 15.38 ± 1.99%, respectively. Increment in inhibition of acetylcholinesterase activity was observed for the extract from P. janthinellum grown with procainamide (100%), when compared to the control extract (39.62 ± 3.76%). Similarly, inhibition of acetylcholinesterase activity increased from 20.91 ± 3.90% (control) to 92.20 ± 3.72% when the tested extract was obtained from T. pinophilus under a combination of suberohydroxamic acid and procainamide. Concluding, increases in antimicrobial activity and acetylcholinesterase inhibition were observed when fungal extracts in the presence of DNA methyltransferases and/or histone deacetylase modulators were tested.

Anti-Bacterial Agents/pharmacology , Cholinesterase Inhibitors/pharmacology , Penicillium/chemistry , Talaromyces/chemistry , Acetylcholinesterase/chemistry , Acetylcholinesterase/metabolism , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/metabolism , Chromatin/metabolism , Listeria monocytogenes/drug effects , Listeria monocytogenes/enzymology , Listeria monocytogenes/growth & development , Penicillium/metabolism , Talaromyces/metabolism
Braz. j. microbiol ; 49(supl.1): 166-177, 2018. tab, graf
Article in English | LILACS | ID: biblio-974336


Abstract In the previous study, we used genome shuffling to improve fengycin production of the original strain Bacillus amyloliquefaciens ES-2-4. After two rounds of genome shuffling, a high-yield recombinant FMB72 strain that exhibited 8.30-fold increase in fengycin production was obtained. In this study, comparative proteomic analysis of the parental ES-2-4 and genome-shuffled FMB72 strains was conducted to examine the differentially expressed proteins. In the shuffled strain FMB72, 50 differently expressed spots (p < 0.05) were selected to be excised and analyzed using Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry, and finally 44 protein spots were confidently identified according to NCBI database. According to clusters of orthologous groups (COG) functional category analysis and related references, the differentially expressed proteins could be classified into several functional categories, including proteins involved in metabolism, energy generation and conversion, DNA replication, transcription, translation, ribosomal structure and biogenesis, cell motility and secretion, signal transduction mechanisms, general function prediction. Of the 44 identified proteins, signaling proteins ComA and Spo0A may positively regulate fengycin synthesis at transcriptional level. Taken together, the present study will be informative for exploring the exact roles of ComA and Spo0A in fengycin synthesis and explaining the molecular mechanism of fengycin synthesis.

Bacterial Proteins/metabolism , Lipopeptides/biosynthesis , Bacillus amyloliquefaciens/genetics , Bacillus amyloliquefaciens/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Genome, Bacterial , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , DNA Shuffling , Proteomics , Bacillus amyloliquefaciens/chemistry
Braz. j. microbiol ; 48(4): 801-808, Oct.-Dec. 2017. tab, graf
Article in English | LILACS | ID: biblio-889172


ABSTRACT The various types of lignocellulosic biomass found in plants comprise the most abundant renewable bioresources on Earth. In this study, the ruminal microbial ecosystem of black goats was explored because of their strong ability to digest lignocellulosic forage. A metagenomic fosmid library containing 115,200 clones was prepared from the black-goat rumen and screened for a novel cellulolytic enzyme. The KG35 gene, containing a novel glycosyl hydrolase family 5 cellulase domain, was isolated and functionally characterized. The novel glycosyl hydrolase family 5 cellulase gene is composed of a 963-bp open reading frame encoding a protein of 320 amino acid residues (35.1 kDa). The deduced amino acid sequence showed the highest sequence identity (58%) for sequences from the glycosyl hydrolase family 5 cellulases. The novel glycosyl hydrolase family 5 cellulase gene was overexpressed in Escherichia coli. Substrate specificity analysis revealed that this recombinant glycosyl hydrolase family 5 cellulase functions as an endo-β-1,4-glucanase. The recombinant KG35 endo-β-1,4-glucanase showed optimal activity within the range of 30-50 °C at a pH of 6-7. The thermostability was retained and the pH was stable in the range of 30-50 °C at a pH of 5-7.

Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacteria/enzymology , Cellulase/chemistry , Cellulase/genetics , Rumen/microbiology , Bacterial Proteins/metabolism , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Cellulase/metabolism , Cloning, Molecular , Enzyme Stability , Gastrointestinal Microbiome , Goats , Hydrogen-Ion Concentration , Metagenome , Metagenomics
Braz. j. microbiol ; 47(3): 647-657, July-Sept. 2016. tab, graf
Article in English | LILACS | ID: lil-788974


ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Organic Chemicals , Solvents , Bacterial Proteins/isolation & purification , Bacterial Proteins/biosynthesis , Acinetobacter/enzymology , Lipase/isolation & purification , Lipase/biosynthesis , Organic Chemicals/chemistry , Solvents/chemistry , Substrate Specificity , Temperature , Bacterial Proteins/chemistry , Enzyme Stability , Kinetics , Chromatography, Ion Exchange , Enzyme Activation , Extracellular Space/enzymology , Hydrogen-Ion Concentration , Ions , Lipase/chemistry , Lipolysis , Metals , Molecular Weight
Braz. j. med. biol. res ; 49(4): e5178, 2016. graf
Article in English | LILACS | ID: biblio-951664


A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.

Animals , Organic Chemicals/metabolism , Peptide Hydrolases/biosynthesis , Bacterial Proteins/biosynthesis , Brevibacillus/metabolism , Peptide Hydrolases/chemistry , Soil Microbiology , Solvents , Temperature , Time Factors , Bacterial Proteins/chemistry , Enzyme Stability , Cattle , Culture Media , Electrophoresis, Polyacrylamide Gel , Brevibacillus/isolation & purification , Hydrogen-Ion Concentration
Article in English | WPRIM | ID: wpr-48338


BACKGROUND: Acinetobacter baumannii has a greater clinical impact and exhibits higher antimicrobial resistance rates than the non-baumannii Acinetobacter species. Therefore, the correct identification of Acinetobacter species is clinically important. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has recently become the method of choice for identifying bacterial species. The purpose of this study was to evaluate the ability of MALDI-TOF MS (Bruker Daltonics GmbH, Germany) in combination with an improved database to identify various Acinetobacter species. METHODS: A total of 729 Acinetobacter clinical isolates were investigated, including 447 A. baumannii, 146 A. nosocomialis, 78 A. pittii, 18 A. ursingii, 9 A. bereziniae, 9 A. soli, 4 A. johnsonii, 4 A. radioresistens, 3 A. gyllenbergii, 3 A. haemolyticus, 2 A. lwoffii, 2 A. junii, 2 A. venetianus, and 2 A. genomospecies 14TU. After 212 isolates were tested with the default Bruker database, the profiles of 63 additional Acinetobacter strains were added to the default database, and 517 isolates from 32 hospitals were assayed for validation. All strains in this study were confirmed by rpoB sequencing. RESULTS: The addition of the 63 Acinetobacter strains' profiles to the default Bruker database increased the overall concordance rate between MALDI-TOF MS and rpoB sequencing from 69.8% (148/212) to 100.0% (517/517). Moreover, after library modification, all previously mismatched 64 Acinetobacter strains were correctly identified. CONCLUSIONS: MALDI-TOF MS enables the prompt and accurate identification of clinically significant Acinetobacter species when used with the improved database.

Acinetobacter Infections/microbiology , Acinetobacter baumannii/chemistry , Bacterial Proteins/chemistry , Databases, Factual , Humans , Phylogeny , RNA, Ribosomal, 16S/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Rev. panam. salud pública ; 38(6): 442-449, nov.-dic. 2015. tab
Article in English | LILACS | ID: lil-788101


OBJECTIVE:To describe the volume and patterns of alcohol consumption up to and including 2012, and to estimate the burden of disease attributable to alcohol consumption as measured in deaths and disability-adjusted life years (DALYs) lost in the Americas in 2012. METHODS: Measures of alcohol consumption were obtained from the World Health Organization (WHO) Global Information System on Alcohol and Health (GISAH). The burden of alcohol consumption was estimated in both deaths and DALYs lost based on mortality data obtained from WHO, using alcohol-attributable fractions. Regional groupings for the Americas were based on the WHO classifications for 2004 (according to child and adult mortality). RESULTS: Regional variations were observed in the overall volume of alcohol consumed, the proportion of the alcohol market attributable to unrecorded alcohol consumption, drinking patterns, prevalence of drinking, and prevalence of heavy episodic drinking, with inhabitants of the Americas consuming more alcohol (8.4 L of pure alcohol per adult in 2012) compared to the world average. The Americas also experienced a high burden of disease attributable to alcohol consumption (4.7% of all deaths and 6.7% of all DALYs lost), especially in terms of injuries attributable to alcohol consumption. CONCLUSIONS: Alcohol is consumed in a harmful manner in the Americas, leading to a high burden of disease, especially in terms of injuries. New cost-effective alcohol policies, such as increasing alcohol taxation, increasing the minimum legal age to purchase alcohol, and decreasing the maximum legal blood alcohol content while driving, should be implemented to decrease the harmful consumption of alcohol and the resulting burden of disease.

OBJETIVO:Describir el volumen y los modelos de consumo de alcohol hasta el año 2012 incluido, y calcular la carga de morbilidad atribuible al consumo de alcohol medida según el número de defunciones y los años de vida ajustados en función de la discapacidad (AVAD) perdidos en la Región de las Américas en el 2012. MÉTODOS: Los datos sobre el consumo de alcohol se obtuvieron a partir del Sistema Mundial de Información sobre el Alcohol y la Salud (GISAH, por sus siglas en inglés) de la Organización Mundial de la Salud (OMS). La carga del consumo de alcohol se calculó según la mortalidad y según los AVAD perdidos con base en los datos de mortalidad obtenidos de la OMS, tomando en consideración las fracciones atribuibles al alcohol. La división en subregiones se basó en las clasificaciones de la OMS del año 2004 (según la mortalidad en niños y adultos). RESULTADOS: Se observaron variaciones regionales en el volumen total de alcohol consumido, la proporción del mercado del alcohol atribuible al consumo de alcohol no registrado, los hábitos de consumo, la prevalencia del consumo y la prevalencia de los episodios de consumo excesivo de alcohol. Los habitantes de la Región de las Américas consumieron más alcohol (8,4 litros de alcohol puro por adulto en el 2012) en comparación con el promedio mundial. La Región también experimentó una alta carga de morbilidad atribuible al consumo de alcohol (4,7% de las defunciones y 6,7% de los AVAD perdidos), especialmente en forma de lesiones atribuibles al consumo de alcohol. CONCLUSIONES: El alcohol se consume de una manera perjudicial en la Región de las Américas y ello comporta una alta carga de morbilidad, especialmente en forma de lesiones. Con objeto de disminuir el consumo perjudicial de bebidas alcohólicas y la carga de morbilidad resultante, es preciso introducir nuevas políticas en materia de consumo de alcohol que sean eficaces en función de los costos, tales como el incremento de los impuestos sobre el alcohol, el aumento de la edad mínima legal para adquirir alcohol, y la disminución de la concentración máxima legal de alcohol en sangre mientras se conduce.

Bacterial Proteins/chemistry , Neuraminidase/chemistry , Streptococcus pneumoniae/enzymology , Virulence Factors/chemistry , Binding Sites , Bacterial Proteins/metabolism , Lactose/analogs & derivatives , Lactose/metabolism , Models, Molecular , Neuraminidase/metabolism , Protein Binding , Protein Folding , Protein Structure, Tertiary , Sialic Acids/metabolism , Streptococcus pneumoniae/chemistry , Virulence Factors/metabolism
Braz. j. microbiol ; 46(4): 1065-1076, Oct.-Dec. 2015. tab, graf
Article in English | LILACS | ID: lil-769637


Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques.

Bacillus/chemistry , Bacillus/classification , Bacillus/enzymology , Bacillus/genetics , Bacillus/growth & development , Bacillus/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/classification , Bacterial Proteins/enzymology , Bacterial Proteins/genetics , Bacterial Proteins/growth & development , Bacterial Proteins/metabolism , Enzyme Stability/chemistry , Enzyme Stability/classification , Enzyme Stability/enzymology , Enzyme Stability/genetics , Enzyme Stability/growth & development , Enzyme Stability/metabolism , Genetic Variation/chemistry , Genetic Variation/classification , Genetic Variation/enzymology , Genetic Variation/genetics , Genetic Variation/growth & development , Genetic Variation/metabolism , Genotype/chemistry , Genotype/classification , Genotype/enzymology , Genotype/genetics , Genotype/growth & development , Genotype/metabolism , Hot Temperature/chemistry , Hot Temperature/classification , Hot Temperature/enzymology , Hot Temperature/genetics , Hot Temperature/growth & development , Hot Temperature/metabolism , Hydrogen-Ion Concentration/chemistry , Hydrogen-Ion Concentration/classification , Hydrogen-Ion Concentration/enzymology , Hydrogen-Ion Concentration/genetics , Hydrogen-Ion Concentration/growth & development , Hydrogen-Ion Concentration/metabolism , Lipase/chemistry , Lipase/classification , Lipase/enzymology , Lipase/genetics , Lipase/growth & development , Lipase/metabolism , Phylogeny/chemistry , Phylogeny/classification , Phylogeny/enzymology , Phylogeny/genetics , Phylogeny/growth & development , Phylogeny/metabolism
Braz. j. med. biol. res ; 48(8): 683-690, 08/2015. tab, graf
Article in English | LILACS | ID: lil-753056


NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Bacterial Proteins/genetics , Escherichia coli/genetics , Herbaspirillum/genetics , Transcription Factors/genetics , Bacterial Proteins/chemistry , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Herbaspirillum/metabolism , Nitrogen Fixation/genetics , Point Mutation , Protein Interaction Domains and Motifs , Transcription Factors/chemistry
Arch. endocrinol. metab. (Online) ; 59(3): 245-251, 06/2015. tab, graf
Article in English | LILACS | ID: lil-751309


Objective Evaluate the effect of glycemic index (GI) on biochemical parameters, food intake, energy metabolism, anthropometric measures and body composition in overweight subjects.Materials and methods Simple blind study, in which nineteen subjects were randomly assigned to consume in the laboratory two daily low GI (n = 10) or high GI (n = 9) meals, for forty-five consecutive days. Habitual food intake was assessed at baseline. Food intake, anthropometric measures and body composition were assessed at each 15 days. Energy metabolism and biochemical parameters were evaluated at baseline and the end of the study.Results Low GI meals increased fat oxidation, and reduced waist circumference and HOMA-IR, while high GI meals increased daily dietary fiber and energy intake compared to baseline. There was a higher reduction on waist circumference and body fat, and a higher increase on postprandial fat oxidation in response to the LGI meals than after high GI meals. High GI meals increased fasting respiratory coefficient compared to baseline and low GI meals.Conclusion The results of the present study showed that the consumption of two daily low GI meals for forty-five consecutive days has a positive effect on obesity control, whereas, the consumption of high GI meals result has the opposite effect. Arch Endocrinol Metab. 2015;59(3):245-51.

Bacterial Proteins/chemistry , Escherichia coli/enzymology , Membrane Proteins/chemistry , Phenylalanine/chemistry , Amino Acid Motifs , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chemotaxis , Conserved Sequence , Dimerization , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/physiology , Molecular Sequence Data , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Conformation , Phenylalanine/genetics , Phenylalanine/metabolism
Clinics ; 70(5): 363-368, 05/2015. tab, graf
Article in English | LILACS | ID: lil-748276


OBJECTIVES: To evaluate the clinical outcomes and identify the predictors of mortality in elderly patients undergoing peritoneal dialysis. METHODS: We conducted a retrospective study including all incident peritoneal dialysis cases in patients ≥65 years of age treated from 2001 to 2014. Demographic and clinical data on the initiation of peritoneal dialysis and the clinical events during the study period were collected. Infectious complications were recorded. Overall and technique survival rates were analyzed. RESULTS: Fifty-eight patients who began peritoneal dialysis during the study period were considered for analysis, and 50 of these patients were included in the final analysis. Peritoneal dialysis exchanges were performed by another person for 65% of the patients, whereas 79.9% of patients preferred to perform the peritoneal dialysis themselves. Peritonitis and catheter exit site/tunnel infection incidences were 20.4±16.3 and 24.6±17.4 patient-months, respectively. During the follow-up period, 40 patients were withdrawn from peritoneal dialysis. Causes of death included peritonitis and/or sepsis (50%) and cardiovascular events (30%). The mean patient survival time was 38.9±4.3 months, and the survival rates were 78.8%, 66.8%, 50.9% and 19.5% at 1, 2, 3 and 4 years after peritoneal dialysis initiation, respectively. Advanced age, the presence of additional diseases, increased episodes of peritonitis, the use of continuous ambulatory peritoneal dialysis, and low albumin levels and daily urine volumes (<100 ml) at the initiation of peritoneal dialysis were predictors of mortality. The mean technique survival duration was 61.7±5.2 months. The technique survival rates were 97.9%, 90.6%, 81.5% and 71% at 1, 2, 3 and 4 years, respectively. None of the factors analyzed were predictors of technique survival. CONCLUSIONS: Mortality was higher in elderly patients. Factors affecting mortality in elderly patients included advanced age, ...

Gene Expression Regulation , Bacterial Proteins/chemistry , Computational Biology , Kinetics , Ligands , Nucleic Acid Conformation , Nucleotides/chemistry , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA , Thermodynamics
Radiol. bras ; 48(2): 111-120, Mar-Apr/2015. graf
Article in English | LILACS | ID: lil-746615


Whole-body imaging in children was classically performed with radiography, positron-emission tomography, either combined or not with computed tomography, the latter with the disadvantage of exposure to ionizing radiation. Whole-body magnetic resonance imaging (MRI), in association with the recently developed metabolic and functional techniques such as diffusion-weighted imaging, has brought the advantage of a comprehensive evaluation of pediatric patients without the risks inherent to ionizing radiation usually present in other conventional imaging methods. It is a rapid and sensitive method, particularly in pediatrics, for detecting and monitoring multifocal lesions in the body as a whole. In pediatrics, it is utilized for both oncologic and non-oncologic indications such as screening and diagnosis of tumors in patients with genetic syndromes, evaluation of disease extent and staging, evaluation of therapeutic response and post-therapy follow-up, evaluation of non neoplastic diseases such as multifocal osteomyelitis, vascular malformations and syndromes affecting multiple regions of the body. The present review was aimed at describing the major indications of whole-body MRI in pediatrics added of technical considerations.

A avaliação de corpo inteiro em crianças era classicamente realizada com radiografias simples, cintilografia e tomografia por emissão de pósitrons combinada ou não à tomografia computadorizada, estes com a desvantagem de exposição à radiação ionizante. A ressonância magnética de corpo inteiro (RMCI), associada ao desenvolvimento de técnicas metabólicas e funcionais como difusão, trouxe a vantagem de uma avaliação global do paciente pediátrico sem os riscos da radiação ionizante habitualmente presente nos métodos radiológicos convencionais. A RMCI é um método rápido e sensível, com aplicação especial na área de pediatria na detecção e no monitoramento de lesões multifocais no corpo como um todo. Em pediatria, esta técnica é utilizada tanto em oncologia - no diagnóstico e rastreamento de tumores em pacientes portadores de síndromes genéticas, na avaliação da extensão de doenças e estadiamento oncológico, na avaliação da resposta terapêutica e no seguimento pós-terapêutico - como em lesões não neoplásicas - osteomielite multifocal, malformações vasculares e síndromes que comprometam múltiplas regiões do corpo. Esta revisão tem como objetivo mostrar as principais indicações do exame na população pediátrica e técnica de realização.

Animals , Female , Mice , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Models, Molecular , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/pathogenicity , Phosphotransferases/chemistry , Phosphotransferases/metabolism , Signal Transduction/physiology , Amino Acid Sequence , Computer Simulation , Mice, Inbred BALB C , Molecular Sequence Data , Mutation , Mycobacterium tuberculosis/growth & development , Oxidative Stress , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Tuberculosis/microbiology
Indian J Biochem Biophys ; 2015 Apr; 52 (2): 179-188
Article in English | IMSEAR | ID: sea-158217


Lipases are the enzymes of choice for laundry detergent industries, owing to their triglyceride removing ability from the soiled fabric, which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In this study, a novel thermo-alkaline lipase-producing strain identified as Bacillus stearothermophilus was isolated from the soil samples of olive oil mill. Enhanced lipase production was observed at 55°C, pH 11 and after 48 h of incubation. Among the substrates tested, xylose (a carbon source), peptone (a nitrogen source) and olive oil at a concentration of 1% were suitable substrates for enhancing lipase production. MgSO4 and Tween-80 were suitable substrates for maximizing lipase production. The enzyme was purified to homogeneity by a single CM-Sephadex column chromatography and revealed molecular mass of 67 kDa. The enzyme (BL1) was active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 11.0, exhibited maximal activity at 55°C and retained more than 70% of its activity after incubation at 70°C or pH 13 for 0.5 h or 24 h, respectively. The enzyme hydrolyzed both short and long-chain triacylglycerols at comparable rates. BL1 was studied in a preliminary evaluation for use in detergent formulation solutions. This novel lipase showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and good stability towards oxidizing agents. Additionally, the enzyme showed excellent stability and compatibility with various commercial detergents, suggesting its potential as an additive in detergent formulations.

Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Detergents/chemistry , Geobacillus stearothermophilus/enzymology , Lipase/chemistry , Lipase/isolation & purification , Solvents/chemistry , Temperature
Salud colect ; 11(1): 115-128, ene.-mar. 2015.
Article in Spanish | LILACS | ID: lil-746688


Los antipsicóticos no parecen revertir las causas de la esquizofrenia y, aunque son fármacos que pueden aliviar los síntomas a corto y mediano plazo, a largo plazo pueden no ser beneficiosos e incluso ser contraproducentes. Su empleo debería limitarse a situaciones agudas con agitación y tensión incapacitante. Presentan considerables efectos adversos y, ante la negativa de una persona a seguir tomándolos, adoptar una estrategia de reducción de daños apoyando y supervisando la retirada puede ser preferible a la coerción. Existen alternativas a los neurolépticos. Los prescriptores deberían estar más atentos y considerar las valoraciones que los usuarios hacen de sus efectos. El apego a las guías de tratamiento es escaso, seguramente por basarse en ensayos clinicos de calidad deficente, que deben mejorar y prolongarse en el tiempo. La raíz del problema probablemente se encuentra en la tautología sobre la etiología y naturaleza biológica de lo que llaman esquizofrenia, que realmente no parece ser más que un constructo ideológico-comercial.

Antipsychotic drugs do not appear to reverse the causes of schizophrenia, and although they can relieve symptoms in the short to medium term, in the long term they may not be beneficial and could even be counterproductive. Their use should be limited to acute situations in which agitation and tension is disabling. The drugs have significant adverse effects, and given the refusal of a person to continue taking them, a harm reduction strategy to support and monitor the withdrawal may be preferable to coercion. There are alternatives to neuroleptics. Prescribers should be more vigilant and consider the assessments of users regarding the drugs' effects. Adherence to treatment guidelines is low, probably because the guidelines are based on clinical trials of deficient quality which consequently should be improved and extended over a greater period of time. The root of the problem is likely the tautology on the etiology and biological nature of what is known as schizophrenia, which in fact does not seem to be more than a commercial and ideological construct.

Bacterial Proteins/chemistry , Biophysics/methods , DNA-Binding Proteins/chemistry , Microscopy, Atomic Force/methods , Hydrogen Bonding , Kinetics , Models, Molecular , Models, Statistical , Monte Carlo Method , Protein Conformation , Protein Denaturation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Peptostreptococcus/metabolism , Proteins/chemistry , Stress, Mechanical , Temperature , Time Factors , Ubiquitin/chemistry
Salud pública Méx ; 56(6): 625-630, nov.-dic. 2014. tab
Article in Spanish | LILACS | ID: lil-733341


Objetivo. Estimar la prevalencia de parásitos potencialmente zoonóticos en heces caninas de Puerto Escondido. Material y métodos. La ciudad se dividió en diez zonas de estudio y éstas se categorizaron en hábitats natural, urbano y suburbano. Se colectaron muestras fecales caninas del piso. Se recuperaron los parásitos por medio de técnicas coproparasitológicas de flotación y frotis directo para su observación microscópica y posterior identificación. Se estimó la prevalencia parasitaria en las heces caninas. Resultados. Todas las zonas presentaron fecalismo canino. La prevalencia parasitaria fue de 73.33%. Los parásitos con mayor prevalencia fueron Toxocara canis (47.78%), Ancylostoma caninum (17.88%) y Dipylidium caninum (13.89%). Conclusión. El fecalismo canino proviene de perros errantes y con dueño. Del total de parásitos encontrados, 66.66% son zoonóticos. Los factores que favorecen la problemática son el hábitat suburbano, el manejo indeseable de la basura y la tenencia irresponsable de los cánidos.

Objective. To estimate the zoonotic parasites prevalence in feral dog feces in Puerto Escondido. Material and methods. The fecalism frecuency was estimated in ten zones. To identify the parasites parasitological flotation and direct smear methods were used. The parasitic prevalence was estimated in the canine feces. Results. All the zones presented canine fecalism. The parasitic prevalence in the feces was 73.33%. The parasites with the highest prevalence were Toxocara canis (47.78%), Ancylostoma caninum (17.88%), and Dipylidium caninum (13.89%). Conclusion. Canine fecalism comes from strayed and owned dogs. 66.66% of the parasites found in the dog feces are zoonotics. The factors associated to this problem are the suburban habitat, waste mishandling and nil tenure of stray dogs.

Penicillin Amidase/chemistry , Bacterial Proteins/chemistry , Escherichia coli/enzymology , Hot Temperature , Kinetics , Protein Denaturation , Phenylacetates/chemistry
Braz. j. microbiol ; 45(4): 1541-1550, Oct.-Dec. 2014. tab
Article in English | LILACS | ID: lil-741311


Lactic acid bacteria capable of producing bacteriocins and presenting probiotic potential open innovative technological applications in the dairy industry. In this study, a bacteriocinogenic strain (Lactococcus lactis subsp. lactis DF4Mi) was isolated from goat milk, and studied for its antimicrobial activity. The bacteriocin presented a broad spectrum of activity, was sensitive to proteolytic enzymes, resistant to heat and pH extremes, and not affected by the presence of SDS, Tween 20, Tween 80, EDTA or NaCl. Bacteriocin production was dependent on the components of the culture media, especially nitrogen source and salts. When tested by PCR, the bacteriocin gene presented 100% homology to nisin Z gene. These properties indicate that this L. lactis subsp. lactis DF4Mi can be used for enhancement of dairy foods safety and quality.

Animals , Anti-Bacterial Agents/metabolism , Bacteriocins/metabolism , Lactococcus lactis/metabolism , Milk/microbiology , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriocins/chemistry , Bacteriocins/genetics , Culture Media/chemistry , Detergents , DNA, Bacterial/genetics , Goats , Hydrogen-Ion Concentration , Lactococcus lactis/growth & development , Molecular Sequence Data , Polymerase Chain Reaction , Protein Stability , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sodium Chloride/metabolism , Temperature
Rev. argent. microbiol ; 45(1): 3-12, mar. 2013. graf, tab
Article in English | LILACS | ID: lil-672048


In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.

Esterases/isolation & purification , Lipase/isolation & purification , Metagenomics , Amino Acid Sequence , Bacillaceae/enzymology , Bacterial Proteins/chemistry , Butyrates/metabolism , Conserved Sequence , DNA , Esterases/classification , Germany , Hydrogen-Ion Concentration , Hydrolysis , Lipolysis , Lipase/classification , Molecular Sequence Data , Osmolar Concentration , Phylogeny , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Soil Microbiology , Substrate Specificity , Salts/pharmacology , Solvents/pharmacology , Temperature , Trees , Triglycerides/metabolism