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2.
Biosci. j. (Online) ; 35(4): 1143-1152, july/aug. 2019. tab, ilus
Artigo em Inglês | LILACS | ID: biblio-1048848

RESUMO

The objective of this study was to evaluate the physiological quality allied to biochemical quality of lettuce seeds by germination and enzymes expression at 20, 25, 30, 35, 40 and 42ºC. Germination speed index and percentage of germination were estimated. Isoenzyme expressions were assessed by alcohol dehydrogenase (ADH), malate dehydrogenase (MDH), catalase (CAT), esterase (EST), pyruvate decarboxylase (PDC) and glutamate oxaloacetate transferase (GOT). The experiment consisted of a completely randomized design in a factorial scheme 4x6, with four cultivars and six different temperatures, with four replications. The highest germination and vigor were observed for cv. 'Everglades' at 35°C, which proved that this cultivar is thermotolerant. Catalase can be considered a genetic marker for the identification ofthermotolerant lettuce cultivars. Cultivar 'Everglades' has potential to be used in lettuce breeding programs aimed at cultivars tolerant to high temperatures during germination.


O objetivo deste estudo foi avaliar a qualidade fisiológica e bioquímica de sementes de alface por meio da germinação e expressão de enzimas a 20, 25, 30, 35, 40 e 42ºC. As variáveis velocidade de germinação e o índice de velocidade de germinação foram estimadas. As expressões das enzimas alcool desidrogenase (ADH), malato desidrogenase (MDH), catalase (CAT), esterase (EST), piruvate descarboxilase (PDC) e glutamato oxaloacetato transferase (GOT) foram avaliadas. Para análise dos genótipos foi empregado o delineamento inteiramente casualizado em esquema fatorial 4x6, testando quatro cultivares e seis diferentes temperaturas, com quatro repetições. A maior germinação e vigor foram observadas para a cv. 'Everglades' a 35°C, o que prova que esta cultivar é termotolerante. A catalase pode ser considerada um marcador para a identificação de cultivares de alface termotolerantes. A cultivar 'Everglades' tem potential para uso em programas de melhoramento visando tolerância à alta temperatura durante a germinação.


Assuntos
Sementes , Catalase , Alface , Esterases , Termotolerância , Isoenzimas , Oxirredutases
3.
Biosci. j. (Online) ; 35(1): 166-176, jan./fev. 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1048570

RESUMO

The toxicity of some insecticides to Helicoverpa armigera was studied through sublethal effects, evaluating the enzymatic activity of esterase and the behavioral response. The commercial formulation of insecticides selected were chlorpyrifos, spinosad, indoxacarb, chlorantraniliprole, lambda-cyhalothrinand Bacillus thuringiensis, corresponding the most used by farmers to control of H. armigera. To determine the esterase activity, the larvae were fed with soybean leaf discs dipped in insecticide solution using the lethal concentration (LC50). For the behavioral response, filter paper were immersed in three concentrations of insecticides (LC50, LC95 and recommend dose.), then the behavior of the larvae observed in Videomex-One. The results for the enzymatic activity showed an increase in the activity of the esterase, with variation along the treatments and the time of exposure of the insects to the insecticides. With exception of spinosad, other insecticides showed an increase in EST-α activity, 6 and 24 hours after contact of caterpillars with insecticide.Different behavioral patterns of walking (walking distance, walking speed and resting time) were observed for H. armigera exposed to different insecticides. H. armigera exposed to chlorpyrifos, lambda-cyhalothrin and B. thuringiensis insecticides show greater esterase activity. Regarding walking behavior, the results confirms enzymatic activity, where H. armigera have behavioral alteration after exposure to insecticide.


A toxicidade de alguns inseticidas a Helicoverpa armigera foi estudada através de efeitos subletais, avaliando a atividade enzimática da esterase e a resposta comportamental. A formulação comercial dos inseticidas selecionados foram clorpirifos, espinosad, indoxacarb, clorantraniliprole, lambda-cialotrina eBacillus thuringiensis utilizados pelos agricultores para controle de H. armigera. Para determinar a atividade da esterase, as larvas foram alimentadas com discos de folha de soja mergulhados em solução de inseticida usando a concentração letal (CL50). Para os testes de características comportamentais, papel filtro foi imerso em três concentrações de inseticidas (CL50, CL95 e dose recomendada), posteriormente o comportamento das larvas foi observado em Videomex-One. Os resultados para a atividade enzimática mostraram aumento da atividade daesterase, com variação ao longo dos tratamentos e do tempo de exposição dos insetos aos inseticidas. Com exceção do spinosad, outros inseticidas mostraram um aumento na atividade EST-α, 6 e 24 horas após o contato das lagartas com inseticida. Diferentes padrões comportamentais de caminhada (distância percorrida, velocidade de caminhada e tempo de repouso) para H. armigera exposto a diferentes classes de inseticidas. H. armigera exposta aos inseticidas clorpirifos, lambda-cialotrina e B. thuringiensis mostraram maior atividade da esterase. Em relação ao comportamento ambulante, os resultados confirmam a atividade enzimática, onde H. armigera teve alteração comportamental após exposição ao inseticida.


Assuntos
Pragas da Agricultura , Inseticidas , Controle de Pragas , Esterases
4.
Electron. j. biotechnol ; 31: 10-16, Jan. 2018. graf, tab, ilust
Artigo em Inglês | LILACS | ID: biblio-1022030

RESUMO

Background: Biodegradation is a reliable approach for efficiently eliminating persistent pollutants such as chlorpyrifos. Despite many bacteria or fungi isolated from contaminated environment and capable of degrading chlorpyrifos, limited enzymes responsible for its degradation have been identified, let alone the catalytic mechanism of the enzymes. Results: In present study, the gene cpd encoding a chlorpyrifos hydrolase was cloned by analysis of genomic sequence of Paracoccus sp. TRP. Phylogenetic analysis and BLAST indicated that CPD was a novel member of organophosphate hydrolases. The purified CPD enzyme, with conserved catalytic triad (Ser155-Asp251-His281) and motif Gly-Asp-Ser-Ala-Gly, was significantly inhibited by PMSF, a serine modifier. Molecular docking between CPD and chlorpyrifos showed that Ser155 was adjacent to chlorpyrifos, which indicated that Ser155 may be the active amino acid involved in chlorpyrifos degradation. This speculation was confirmed by site-directed mutagenesis of Ser155Ala accounting for the decreased activity of CPD towards chlorpyrifos. According to the key role of Ser155 in chlorpyrifos degradation and molecular docking conformation, the nucleophilic catalytic mechanism for chlorpyrifos degradation by CPD was proposed. Conclusion: The novel enzyme CPD was capable of hydrolyze chlorpyrifos and Ser155 played key role during degradation of chlorpyrifos.


Assuntos
Paracoccus/enzimologia , Clorpirifos/metabolismo , Esterases/metabolismo , Organofosfatos/metabolismo , Biodegradação Ambiental , Catálise , Mutagênese , Clonagem Molecular , Análise de Sequência , Esterases/isolamento & purificação , Esterases/genética , Hidrólise , Metais/metabolismo
5.
Einstein (Säo Paulo) ; 15(1): 34-39, Jan.-Mar. 2017. tab, graf
Artigo em Inglês | LILACS | ID: biblio-840287

RESUMO

ABSTRACT Objective To investigate whether the urine dipstick screening test can be used to predict urine culture results. Methods A retrospective study conducted between January and December 2014 based on data from 8,587 patients with a medical order for urine dipstick test, urine sediment analysis and urine culture. Sensitivity, specificity, positive and negative predictive values were determined and ROC curve analysis was performed. Results The percentage of positive cultures was 17.5%. Nitrite had 28% sensitivity and 99% specificity, with positive and negative predictive values of 89% and 87%, respectively. Leukocyte esterase had 79% sensitivity and 84% specificity, with positive and negative predictive values of 51% and 95%, respectively. The combination of positive nitrite or positive leukocyte esterase tests had 85% sensitivity and 84% specificity, with positive and negative predictive values of 53% and 96%, respectively. Positive urinary sediment (more than ten leukocytes per microliter) had 92% sensitivity and 71% specificity, with positive and negative predictive values of 40% and 98%, respectively. The combination of nitrite positive test and positive urinary sediment had 82% sensitivity and 99% specificity, with positive and negative predictive values of 91% and 98%, respectively. The combination of nitrite or leukocyte esterase positive tests and positive urinary sediment had the highest sensitivity (94%) and specificity (84%), with positive and negative predictive values of 58% and 99%, respectively. Based on ROC curve analysis, the best indicator of positive urine culture was the combination of positives leukocyte esterase or nitrite tests and positive urinary sediment, followed by positives leukocyte and nitrite tests, positive urinary sediment alone, positive leukocyte esterase test alone, positive nitrite test alone and finally association of positives nitrite and urinary sediment (AUC: 0.845, 0.844, 0.817, 0.814, 0.635 and 0.626, respectively). Conclusion A negative urine culture can be predicted by negative dipstick test results. Therefore, this test may be a reliable predictor of negative urine culture.


RESUMO Objetivo Verificar se a triagem de urina por fitas reativas é capaz de predizer a cultura de urina. Métodos Estudo retrospectivo realizado entre janeiro e dezembro de 2014 com 8.587 pacientes, com solicitação médica de triagem de urina (fita), sedimento urinário e cultura de urina. Foram analisados sensibilidade, especificidade, valor preditivo positivo, valor preditivo negativo e curva ROC. Resultados Foram positivas 17,5% das culturas. O nitrito apresentou sensibilidade de 28% e especificidade de 99%. O valor preditivo positivo foi de 89% e o valor preditivo negativo de 87%. Esterase apresentou sensibilidade de 79% e especificidade de 84%. Valor preditivo positivo e valor preditivo negativo foram de 51% e 95%, respectivamente. A combinação de nitrito ou esterase positivos apresentou sensibilidade de 85% e especificidade de 84%. Valor preditivo positivo e valor preditivo negativo foram, respectivamente, 53% e 96%. O sedimento positivo (mais de dez leucócitos por microlitro) apresentou sensibilidade de 92% e especificidade de 71%. O valor preditivo positivo foi 40% e o negativo, 98%. A combinação de nitrito e sedimento urinário positivos apresentou sensibilidade de 82% e especificidade de 99%. Os valores preditivos positivo e negativo foram 91% e 98%, respectivamente. Para o nitrito ou esterase positivos mais os leucócitos positivos, a sensibilidade foi de 94% e a especificidade de 84%. O valor preditivo positivo foi de 58% e o negativo foi de 99%. Com base na curva ROC, o melhor indicador de urocultura positiva foi a associação entre a esterase ou nitrito positivos na fita mais os leucócitos positivos no sedimento, seguido por nitrito e esterase positivos, sedimento urinário positivo isolado, esterase positiva isolada, nitrito positivo isolado e, finalmente, pela associação entre nitrito e sedimento urinário positivos (AUC: 0,845, 0,844, 0,817, 0,814, 0,635 e 0,626, respectivamente). Conclusão Uma urocultura negativa pode ser prevista com resultados negativos na fita. Portanto, este teste pode ser um preditor confiável de urocultura negativa.


Assuntos
Humanos , Masculino , Pré-Escolar , Adulto , Pessoa de Meia-Idade , Bacteriúria/urina , Urinálise/instrumentação , Urinálise/métodos , Padrões de Referência , Valores de Referência , Infecções Urinárias/urina , Urina/microbiologia , Contagem de Colônia Microbiana , Estudos Retrospectivos , Análise de Variância , Sensibilidade e Especificidade , Esterases/urina , Leucócitos , Nitritos/urina
6.
Braz. j. microbiol ; 47(4): 846-852, Oct.-Dec. 2016. tab, graf
Artigo em Inglês | LILACS | ID: biblio-828191

RESUMO

Abstract Copper mine drainages are restricted environments that have been overlooked as sources of new biocatalysts for bioremediation and organic syntheses. Therefore, this study aimed to determine the enzymatic activities (esterase, epoxide hydrolase and monooxygenase) of 56 heterotrophic bacteria isolated from a neutral copper mine drainage (Sossego Mine, Canaã dos Carajás, Brazil). Hydrolase and monooxygenase activities were detected in 75% and 20% of the evaluated bacteria, respectively. Bacterial strains with good oxidative performance were also evaluated for biotransformation of organic sulfides. Fourteen strains with good enzymatic activity were identified by 16S rRNA gene sequencing, revealing the presence of three genera: Bacillus, Pseudomonas and Stenotrophomonas. The bacterial strains B. megaterium (SO5-4 and SO6-2) and Pseudomonas sp. (SO5-9) efficiently oxidized three different organic sulfides to their corresponding sulfoxides. In conclusion, this study revealed that neutral copper mine drainages are a promising source of biocatalysts for ester hydrolysis and sulfide oxidation/bioremediation. Furthermore, this is a novel biotechnological overview of the heterotrophic bacteria from a copper mine drainage, and this report may support further microbiological monitoring of this type of mine environment.


Assuntos
Bactérias/classificação , Bactérias/enzimologia , Cobre , Microbiologia Ambiental , Oxirredução , Filogenia , Sulfetos/metabolismo , Bactérias/isolamento & purificação , Bactérias/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Enzimas , Esterases/genética , Esterases/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Mineração
7.
Braz. j. microbiol ; 47(2): 327-336, Apr.-June 2016. tab, graf
Artigo em Inglês | LILACS | ID: lil-780820

RESUMO

Abstract Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13) and adults (n = 12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.


Assuntos
Animais , Masculino , Feminino , Oxazinas/metabolismo , Bactérias/enzimologia , Carboxilesterase/metabolismo , Esterases/metabolismo , Microbioma Gastrointestinal , Inseticidas/metabolismo , Mariposas/microbiologia , Filogenia , Bactérias/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Trato Gastrointestinal/microbiologia , Carboxilesterase/genética , Esterases/genética , Índia
8.
Acta sci., Biol. sci ; 37(4): 463-469, Oct.-Dec. 2015. ilus
Artigo em Inglês | LILACS | ID: biblio-875832

RESUMO

Current analysis characterizes the effect of different fungicides often applied for pest control on α−and ß-esterase patterns of four economically important table-wine grape cultivars (Italia, Rubi, Benitaka and Brasil) of Vitis vinifera. The α- and ß-esterase patterns in bud leaves of the cultivars were assessed by native PAGE analysis. Cabrio Top® compound inhibited EST-2, EST-5, EST-6, EST-7, EST-8, EST-9 and EST-10 carboxylesterases, whereas EST-4, EST-11, EST-12, EST-13, EST-14 acetylesterases and EST-16 carboxylesterase were detected as weakly stained bands. Carboxylesterases and acetylesterases were also detected as weakly stained bands when exposed to fungicides Orthocide 500®, Positron Duo® and Folicur PM®. No changes in α- and ß-esterase patterns were reported when the vines were exposed to the fungicides Rovral SC®, Kumulus DF®, Curzate M®, Score® or Cuprogarb 500®. The evidence of functional changes in carboxylesterase and acetylesterase levels in current study is a warning to grape producers on the dangers inherent in the indiscriminate use of potent and modern fungicides extensively used in agriculture. The inhibition effect of fungicides on esterase isozyme molecules seems to be independent of the fungicide chemical.


O presente estudo caracterizou o efeito de diferentes fungicidas comumente aplicados como medidas de controle de pragas sobre padrões de α- e ß-esterases de quatro importantes cultivares de uva de mesa (Itália, Rubi, Benitaka e Brasil) de Vitis vinifera. Os padrões de α- e ß-esterases de brotos foliares das cultivares foram avaliados por PAGE. O composto Cabrio Top® inibiu as carboxilesterases EST-2, EST-5, EST-6, EST-7, EST-8, EST-9 e EST-10, enquanto as acetilesterases EST-4, EST-11, EST-12, EST-13, EST-14 e a carboxilesterase EST-16 foram detectadas como bandas fracamente coradas. As carboxilesterases e acetilesterases também foram detectadas como bandas fracamente coradas quando expostas aos fungicidas Orthocide 500®, Positron Duo® e Folicur PM®. Não foram observadas alterações nos padrões de α- e ß-esterases quando as videiras foram expostas aos fungicidas Rovral SC®, Kumulus DF®, Curzate M®, Score® ou Cuprogarb 500®. A evidência de alterações em nível funcional em carboxilesterases e acetilesterases, apresentada neste estudo, pode servir como um alerta aos produtores de uva dos perigos inerentes ao uso indiscriminado de fungicidas potentes e modernos amplamente utilizados hoje na agricultura. O efeito dos fungicidas sobre as enzimas esterases parece ser independente do grupo químico ao qual pertence o fungicida.


Assuntos
Esterases , Isoenzimas , Vitis
9.
Rev. Soc. Bras. Med. Trop ; 47(5): 579-582, Sep-Oct/2014. tab
Artigo em Inglês | LILACS | ID: lil-728904

RESUMO

Introduction The effects of piperonyl butoxide (PBO) on the toxicity of the organophosphate temephos (TE) and the role of esterases in the resistance of Aedes aegypti to this insecticide were evaluated. Methods A. aegypti L4 larvae susceptible and resistant to TE were pre-treated with PBO solutions in acetone at concentrations of 0.125, 0.25, 0.5, 1, and 2% for 24h and subsequently exposed to a diagnostic concentration of 0.02mg/L aqueous TE solution. The esterase activity of the larvae extracts pre-treated with varying PBO concentrations and exposed to TE for three time periods was determined. Results At concentrations of 0.25, 0.5, 1, and 2%, PBO showed a significant synergistic effect with TE toxicity. High levels of esterase activity were associated with the survival of A. aegypti L4 larvae exposed to TE only. Conclusions The results of the biochemical assays suggest that PBO has a significant inhibitory effect on the total esterase activity in A. aegypti larvae. .


Assuntos
Animais , Aedes/efeitos dos fármacos , Aedes/enzimologia , Esterases/fisiologia , Resistência a Inseticidas , Sinergistas de Praguicidas/farmacologia , Butóxido de Piperonila/farmacologia , Temefós/toxicidade , Larva/efeitos dos fármacos , Organofosfatos
10.
Recife; s.n; 2014. 73 p. ilus, mapas, tab.
Tese em Português | LILACS | ID: lil-720615

RESUMO

Em Cabo Verde, arquipélago situado na Costa Ocidental Africana, os primeiros casos de dengue ocorreram em 2009, com a notificação de mais de 21.000 casos, a maioria desses registrados na Ilha de Santiago. O mosquito Aedes aegypti foi identificado como vetor, e ações para seu controle, usando os inseticidas temephos (larvicida) e a deltametrina (adulticida), têm sido implementadas. Objetiva-se com esse trabalho avaliar o atual status de suscetibilidade a inseticidas e caracterizar os mecanismos de resistência nessa população. Amostras de A. aegypti da ilha de Santiago foram coletadas através de armadilhas de oviposição, para o estabelecimento de uma população a ser analisada. Foram realizados bioensaios do tipo dose diagnóstica, usando garrafas impregnadas com doses únicas dos adulticidas malathion (organofosforado), deltametrina (piretróide) e cipermetrina (piretróide), e bioensaios do tipo dose resposta, usando múltiplas concentrações dos inseticidas temephos (organofosforado), Bacillus thuringiensis sorovariedade israelensis (bactéria entomopatogênica) e diflubenzuron (inibidor de síntese de quitina). Para a investigação dos mecanismos de resistências, foram realizados testes bioquímicos com substratos específicos para quantificar a atividade das enzimas glutationa S-transferases, esterases (alfa, beta e PNPA) e oxidases de função mista, ligadas a detoxificação de xenobióticos, e a taxa de inibição da acetilcolinesterase ligada a insensibilidade do sítio alvo...


Cape Verde, an archipelago located on the West African Coast, recorded the first cases of dengue in 2009 in an epidemic with more than 21,000 reportedcases. The worst affected area was Santiago Island...


Assuntos
Animais , Aedes , Enzimas/toxicidade , Insetos Vetores/virologia , Resistência a Inseticidas , África Ocidental , Bacillus thuringiensis/patogenicidade , Esterases/análise , Glutationa Transferase/análise , Malation/toxicidade , Testes de Toxicidade , Temefós/toxicidade
11.
Mem. Inst. Oswaldo Cruz ; 108(8): 1031-1036, 6/dez. 2013. tab, graf
Artigo em Inglês | LILACS | ID: lil-697150

RESUMO

The aim of the present work was to study the deltamethrin susceptibility of eggs from Triatoma infestans populations and the contribution of pyrethroid esterases to deltamethrin degradation. Insects were collected from sylvatic areas, including Veinte de Octubre and Kirus-Mayu (Bolivia) and from domiciliary areas, including El Palmar (Bolivia) and La Pista (Argentina). Deltamethrin susceptibility was determined by dose-response bioassays. Serial dilutions of deltamethrin (0.0005-1 mg/mL) were topically applied to 12-day-old eggs. Samples from El Palmar had the highest lethal dose ratio (LDR) value (44.90) compared to the susceptible reference strain (NFS), whereas the Veinte de Octubre samples had the lowest value (0.50). Pyrethroid esterases were evaluated using 7-coumaryl permethrate (7-CP) on individually homogenised eggs from each population and from NFS. The El Palmar and La Pista samples contained 40.11 and 36.64 pmol/min/mg protein, respectively, and these values were statistically similar to NFS (34.92 pmol/min/mg protein) and different from Kirus-Mayu and Veinte de Octubre (27.49 and 22.69 pmol/min/mg protein, respectively). The toxicological data indicate that the domestic populations were resistant to deltamethrin, but no statistical contribution of 7-CP esterases was observed. The sylvatic populations had similar LDR values to NFS, but lower 7-CP esterase activities. Moreover, this is the first study of the pyrethroid esterases on T. infestans eggs employing a specific substrate (7-CP).


Assuntos
Animais , Esterases/análise , Nitrilos/farmacologia , Óvulo/efeitos dos fármacos , Piretrinas/farmacologia , Triatoma/efeitos dos fármacos , Bioensaio , Óvulo/enzimologia , Triatoma/enzimologia
12.
Braz. j. microbiol ; 44(3): 923-926, July-Sept. 2013. ilus, tab
Artigo em Inglês | LILACS | ID: lil-699782

RESUMO

A sensitive and efficient colorimetric method was optimized for detection of esterase enzymes produced by endophytic fungi for development of High-Throughput Screening (HTS). The fungi were isolated and obtained previously from plant species of Cerrado and Atlantic Forest located in areas of environmental preservation in the State of Sao Paulo / Brazil, as part of the project "Chemical and biological prospecting endophytic fungi associated to plant species of Cerrado and Atlantic Forest". The compounds ethyl butyrate, ethyl acetate and methyl propionate were used as standards esters which were hydrolyzed by extracellular enzyme from endophytic fungi (EC. 3.1.1.1 -carboxylesterases) for production of carboxylic acids. Thus, the reduction of the pH increases the protonated indicator concentration (bromothymol blue), changing the color of the reaction medium (from blue to yellow), that can be observed and measured by spectrophotometry at 616 nm. The methodology with acid-base indicator was performed on 13 microorganisms, aiming Periconia atropurpurea asapotential source of esterase for biotransformation of short chain esters. The results also evidenced that this methodology showed to be efficient, fast, cheap, having low consumption of reagents and easy development, and can be applied to screen carboxylic-ester hydrolases in a large number of microorganisms.


Assuntos
Colorimetria/métodos , Endófitos/enzimologia , Esterases/análise , Fungos/enzimologia , Acetatos/metabolismo , Brasil , Butiratos/metabolismo , Fungos/isolamento & purificação , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Plantas/microbiologia , Propionatos/metabolismo
13.
Rev. Soc. Bras. Med. Trop ; 46(2): 178-184, Mar-Apr/2013. tab, graf
Artigo em Inglês | LILACS | ID: lil-674659

RESUMO

INTRODUCTION: The need for studies that describe the resistance patterns in populations of Aedes aegypti (Linnaeus) in function of their region of origin justified this research, which aimed to characterize the resistance to temephos and to obtain information on esterase activity in populations of Aedes aegypti collected in municipalities of the State of Paraíba. METHODS: Resistance to temephos was evaluated and characterized from the diagnostic dose of 0.352mg i.a./L and multiple concentrations that caused mortalities between 5% and 99%. Electrophoresis of isoenzymes was used to verify the patterns of esterase activity among populations of the vector. RESULTS: All populations of Aedes aegypti were resistant to temephos, presenting a resistance rate (RR) greater than 20. The greatest lethal dose 50% of the sample (CL50) was found for the municipality of Lagoa Seca, approximately forty-one times the value of CL50 for the Rockefeller population. The populations characterized as resistant showed two to six regions of α and β-esterase, called EST-1 to EST-6, while the susceptible population was only seen in one region of activity. CONCLUSIONS: Aedes aegypti is widely distributed and shows a high degree of resistance to temephos in all municipalities studied. In all cases, esterases are involved in the metabolism and, consequently, in the resistance to temephos. .


Assuntos
Animais , Aedes/enzimologia , Esterases/metabolismo , Inseticidas , Temefós , Eletroforese , Resistência a Inseticidas
14.
Rev. argent. microbiol ; 45(1): 3-12, mar. 2013. graf, tab
Artigo em Inglês | LILACS | ID: lil-672048

RESUMO

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.


Assuntos
Esterases/isolamento & purificação , Lipase/isolamento & purificação , Metagenômica , Sequência de Aminoácidos , Bacillaceae/enzimologia , Proteínas de Bactérias/química , Butiratos/metabolismo , Sequência Conservada , DNA , Esterases/classificação , Alemanha , Concentração de Íons de Hidrogênio , Hidrólise , Lipólise , Lipase/classificação , Dados de Sequência Molecular , Concentração Osmolar , Filogenia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Microbiologia do Solo , Especificidade por Substrato , Sais/farmacologia , Solventes/farmacologia , Temperatura , Árvores , Triglicerídeos/metabolismo
15.
Bol. malariol. salud ambient ; 53(1): 46-55, ene. 2013. ilus, graf, tab
Artigo em Espanhol | LILACS | ID: lil-690368

RESUMO

En Venezuela, el malation ha sido ampliamente usado en forma continua en programas de control de aedes aegypti. Por tal motivo se realizó un estudio en mosquitos provenientes de zonas urbanas con alta casuística de dengue de los estados: Amazonas, Aragua, Bolívar, Lara, Mérida y Zulia, para determinar el status de susceptibilidad en este vector al malatión, en comparación con la cepa susceptible referencial, Rockefeller (Rock). Se hicieron bioensayos en botellas tratadas con el insecticida malatión evaluando la dosis diagnóstica 100ug/mL en 30 minutos y ensayos bioquímicos en microplacas para determinar mecanismos metabólicos asociados al status frente al insecticida. Los resultados de los bioensayos mostraron que existe susceptibilidad a malatión, lo cual fue confirmado por pruebas bioquímicas. Sin embargo, se encontraron diferencias significativas entre todas las cepas evaluadas con valores de P<0,005 para esterasas alfa (α), esterasas beta (β) y acetilcolinesterasa normal (Ache) y acetilcolinesterasa inhibida (Achei). La prueba de comparación de medias de Bonferroni encontró similitud entre la cepa Rock, mazonas y Lara para esterasas α y β. Se encontró similitud de la cepa Rock con las cepas de Bolívar y Zulia para las pruebas con Ache y Achei. Este estudio concluye que el malatión mostró su potencial de uso en el control del vector del dengue de las localidades evaluadas.


In Venezuela, malathion has been widely used continuously in control programs for Aedes aegypti. Therefore, a study in mosquitoes from urban areas with high dengue casuistry in the states of Amazonas, Aragua, Bolivar, Lara, Merida and Zulia was conducted to determine the status of this vector susceptibility to malathion, compared with the reference susceptible strain, Rockefeller. Bioassays were done on bottles treated with the insecticide malathion, 100ug/mL evaluating the diagnostic doses in 30 minutes and biochemical assays in microplates were performed to determine metabolic mechanisms associated with status against insecticide. The bioassay results showed that there is malathion susceptibility, which was confirmed by biochemical tests. However, significant differences were found among all strains assessed values of P<0.005 for esterases alpha (α), beta esterases (β) and standard acetylcholinesterase (AChe) and inhibited acetylcholinesterase (Achei). The mean comparison test of Bonferroni showed similarity between the strains Rock, Amazon and Lara for esterases α and β. Similarity was found between the strains Rock, Bolivar and Zulia for the Ache and Achei tests. This study concludes that malathion showed its potential use in controlling the dengue vector in the locations evaluated.


Assuntos
Humanos , Masculino , Feminino , Aedes/crescimento & desenvolvimento , Esterases/administração & dosagem , Malation/análise , Dengue , Febre Amarela
16.
Biomédica (Bogotá) ; 32(3): 333-343, jul.-set. 2012. tab
Artigo em Espanhol | LILACS | ID: lil-663705

RESUMO

Introducción. La resistencia fisiológica de las poblaciones naturales de Aedes aegypti a los insecticidas de uso en salud pública, disminuye la eficacia del control químico utilizado para interrumpir la transmisión de dengue durante los brotes y epidemias. Objetivo. Determinar el estado de la susceptibilidad al carbamato propoxur, a los organofosforados adulticidas malatión y fenitrotión y al larvicida temefos, de 13 poblaciones naturales de Ae. aegypti en ocho municipios: una de Bucaramanga y dos de Sabana de Torres en el departamento de Santander; dos de Girardot y dos de La Mesa en Cundinamarca; dos de Villavicencio y dos de Puerto López en el Meta; una de San José del Guaviare, en Guaviare, y una de Florencia en Caquetá. Materiales y métodos. Las pruebas de susceptibilidad consistieron en bioensayos estandarizados de la Organización Mundial de la Salud (OMS), 1981, y botellas de los Centers for Disease Control and Prevention (CDC), 1998. Se aplicaron pruebas colorimétricas para determinar los niveles de enzimas como posibles responsables de la reducción de la susceptibilidad a insecticidas organofosforados y al carbamato propoxur. Resultados. Todas las poblaciones presentaron susceptibilidad a malatión y propoxur. Para el temefos, solo cuatro de las trece localidades evaluadas registraron susceptibilidad. Con respecto a los posibles mecanismos de desintoxicación enzimática, en siete de las once poblaciones naturales se registraron valores superiores al punto de corte para esterasas inespecíficas, solamente en Bucaramanga se presentó un incremento en las enzimas monooxigenasas del citocromo P450, pero en ninguna población se registró acetilcolinestarasa modificada. Conclusiones. La susceptibilidad generalizada a los organofosforados adulticidas evaluados, indica que el malatión, el insecticida más utilizado en Colombia para interrumpir la transmisión del dengue, sigue siendo eficaz. Las particularidades de los resultados de la resistencia fisiológica a insecticidas de las comunas de un mismo municipio, evidenciaron un fenómeno localizado de manera específica en espacio y tiempo.


Introduction. Physiological resistance of natural population of Aedes aegypti to insecticides contribute to the decreased efficacy of chemical control as a main control strategy during dengue outbreaks. Objective. The susceptibility status of Ae. aegypti was assessed for the carbamate propoxur, the adulticide malathion and the larvicide temephos on 13 natural populations of Ae. aegypti immature forms were taken from 8 Colombian localities. These included the following: Bucaramanga (1), Sabana de Torres (2), Girardot (2), La Mesa (2), Villavicencio (2), Puerto López (2), San José del Guaviare (1) and Florencia (1). Materials and methods. Susceptibility tests mainly consisted of the standardized bioassay outlined by WHO (1981) and CDC bottles (1998). Colorimetric tests were undertaken to determine enzyme levels possibly responsible for the reduction of susceptibility to organophosphate and carbamate insecticides. Results. All specimens demonstrated susceptibility to malathion and propoxur insecticides. Four of the 13 populations revealed susceptibility to the temephos larvicide. Seven of 11 populations showed a limited increase in values for nonspecific esterase enzymes. The Bucaramanga population was the only one which showed an increase in the cytochrome P450 monooxygenases enzymes. Neither population was found with modified acetilcolinesterase. Conclusions. The widespread susceptibility to organophosphates used as adulticides indicated that malathion, the most used insecticide in Colombia, remains effective in interrupting the transmission of dengue. Physiological resistance to insecticides occurring in communities of a single township proved to be a localized phenomenon.


Assuntos
Animais , Feminino , Masculino , Aedes , Vírus da Dengue , Insetos Vetores , Resistência a Inseticidas , Inseticidas , Malation , Controle de Mosquitos , Propoxur , Temefós , Acetilcolinesterase/análise , Aedes/enzimologia , Aedes/crescimento & desenvolvimento , Colômbia , /análise , Resistência a Múltiplos Medicamentos , Esterases/análise , Proteínas de Insetos/análise , Larva , Saúde da População Urbana
17.
Rev. cuba. med. trop ; 64(3): 256-267, jul.-sep. 2012.
Artigo em Espanhol | LILACS | ID: lil-653844

RESUMO

Introducción: las enzimas esterasas han sido identificadas como mecanismo de resistencia a temefos en Aedes aegypti de Cuba, larvicida más utilizado en el mundo. Objetivo: caracterizar parcialmente la actividad de esterasas en larvas expuestas y no expuestas a dosis subletales de temefos en una cepa de Aedes aegypti resistente a este insecticida. Métodos: se utilizó una cepa de Aedes aegypti de referencia susceptible (Rockefeller) y otra resistente a temefos (SANtemF11). Se expusieron las larvas de la cepa SANtemF11 a la concentración letal 90 (CL90) de temefos (1 ppm), 10 % de larvas sobrevivientes a las 24 h (SANtem [24 h]) se transfirieron a agua limpia y sin exposición a insecticidas por otras 24 h (SANtem [48 h]). Se caracterizó de modo parcial, en estas larvas, la actividad de esterasas a través de ensayos bioquímicos y electroforesis en gel de poliacrilamida. Se estimó por duodecil sulfato de sodio (SDS-PAGE) el peso molecular de la esterasa (Est. A4). Resultados: la actividad de esterasas en la cepa SANtemF11 resultó significativamente mayor que en Rockefeller. Se observó una disminución significativa de la actividad de esterasas en las larvas sobrevivientes (SANtemF11 [24 h]), la cual se recuperó 24 h después sin exposición a temefos. En el zimograma se observó que en 10 % de las larvas sobrevivientes a temefos, solo apareció incrementada la banda de esterasa A4, en comparación con las observadas en SANtemF11. El peso molecular estimado de la esterasa A4 fue de 58 kDa. Conclusiones: la presencia de una banda específica de esterasa (58 kDa), en las larvas sobrevivientes a la selección con temefos, confirma su papel en la resistencia a este insecticida. Diagnosticar la función de las esterasas en la resistencia a temefos, a través de ensayos bioquímicos, no debe realizarse en larvas expuestas a dosis subletales de este insecticida, para evitar falsos negativos.


Introduction: the esterase enzymes have been defined as the mechanism of resistance to temephos in Aeges aegypti in Cuba, which is the most used larvacide worldwide. Objective: to partially characterize the activity of esterases in exposed and non-exposed larvae at sublethal doses of temephos in an Aedes aegypti strain that is resistant to this product. Methods: a susceptible reference Aedes aegypti strain (Rockefeller) and another temephos-resistant strain (SANtemFII) were used. The larvae from SANtemF11 strain were exposed to lethal concentration 90 (LC90) of temephos (1 ppm); 10 % of the surviving larvae after 24 hours (SANtem[24 h] was moved to clean water, with no exposure to insecticide for 24 hours (SANtem [48 h]). The activity of esterases was partially characterized in these larvae through biochemical assays and gel-polyacrylamide electrophoresis. The molecular weight of esterase A 4 (ESt. A4) was estimated with the support of sodium duodecyl sulophate (SDS-PAGE). Results: the activity of esterases in SANtemF11 was significantly higher than in Rockefeller strain. Significant reduction of the activity of esterases in surviving larvae was observed (SANtemF11 [24 h], but it increased 24 h later without exposure to temephos. The zymogram showed that 10% of larvae that survived from temephos action, just the esterase A4 band increased if compared with those of SAntemF11. The estimated molecular weight of esterase A4 was 58 kDa. Conclusions: the presence of a specific band of esterase (58 kDa) in surviving larvae confirmed the role of these enzymes in insecticidal resistance. The diagnosis of the function of the esterases in resistance to temephos through biochemical tests should not be made in larvae exposed to sublethal doses of this insecticide, in order to avoid false negatives.


Assuntos
Animais , Aedes/enzimologia , Esterases/fisiologia , Inseticidas , Temefós , Resistência a Inseticidas/fisiologia
18.
Rev. panam. salud pública ; 32(1): 1-8, July 2012. ilus
Artigo em Inglês | LILACS | ID: lil-646445

RESUMO

OBJECTIVE: To examine the effects of increasing larval rearing temperatures on the resistance status of Trinidadian populations of Aedes aegypti to organophosphate (OP) insecticides. METHODS: In 2007-2008, bioassays and biochemical assays were conducted on A. aegypti larvae collected in 2006 from eight geographically distinct areas in Trinidad (Trinidad and Tobago). Larval populations were reared at four temperatures (28 ± 2ºC, 32ºC, 34ºC, and 36ºC) prior to bioassays with OP insecticides (fenthion, malathion, and temephos) and biochemical assays for esterase enzymes. RESULTS: Most larval populations reared at 28 ± 2ºC were susceptible to fenthion (>98% mortality) but resistant to malathion and temephos (< 80% mortality). A positive association was found between resistance to OP insecticides and increased activities of α- and β-esterases in larval populations reared at 28 ± 2ºC. Although larval populations reared at higher temperatures showed variations in resistance to OPs, there was a general increase in susceptibility. However, increases or decreases in activity levels of enzymes did not always correspond with an increase or decrease in the proportion of resistant individuals reared at higher temperatures. CONCLUSIONS: Although global warming may cause an increase in dengue transmission, based on the current results, the use of insecticides for dengue prevention and control may yet be effective if temperatures increase as projected.


OBJETIVO: Examinar los efectos del aumento de las temperaturas de desarrollo larvario sobre el estado de resistencia a los insecticidas organofosforados de las poblaciones de Aedes aegypti en Trinidad. MÉTODOS: En 2007 y 2008 se llevaron a cabo ensayos biológicos y bioquímicos en larvas de A. aegypti recogidas en el 2006 de ocho áreas geográficamente separadas en Trinidad (Trinidad y Tabago). Las poblaciones larvarias se desarrollaron en cuatro temperaturas (28 ± 2 ºC, 32 ºC, 34 ºC y 36 ºC) antes de los ensayos biológicos con insecticidas organofosforados (fentión, malatión y temefós) y los análisis bioquímicos para las enzimas de esterasa. RESULTADOS: La mayoría de las poblaciones larvarias que se desarrollaron a 28 ± 2 ºC fueron susceptibles al fentión (mortalidad > 98%) pero resistentes al malatión y al temefós (mortalidad < 80%). Se encontró una asociación positiva entre la resistencia a los insecticidas organofosforados y la mayor actividad de αy β-esterasas en las poblaciones larvarias que se desarrollaron a 28 ± 2 ºC. Aunque las poblaciones larvarias que se desarrollaron a temperaturas mayores mostraron variaciones en la resistencia a los organofosforados, hubo un aumento general de la sensibilidad. Sin embargo, los aumentos o las disminuciones en los niveles de actividad de las enzimas no siempre se correspondieron con un aumento o disminución en la proporción de individuos resistentes desarrollados a las temperaturas más altas. CONCLUSIONES: Aunque el recalentamiento del planeta puede causar un aumento de la transmisión del dengue, según los resultados de este estudio el uso de insecticidas para la prevención y el control del dengue todavía puede ser eficaz si las temperaturas aumentan según lo proyectado.


Assuntos
Animais , Aedes/efeitos dos fármacos , Fention/farmacologia , Resistência a Inseticidas , Insetos Vetores/efeitos dos fármacos , Inseticidas/farmacologia , Malation/farmacologia , Temperatura , Temefós/farmacologia , Aedes/enzimologia , Aedes/crescimento & desenvolvimento , Dengue/prevenção & controle , Esterases/análise , Esterases/fisiologia , Aquecimento Global , Temperatura Alta , Proteínas de Insetos/análise , Proteínas de Insetos/fisiologia , Insetos Vetores/enzimologia , Insetos Vetores/crescimento & desenvolvimento , Resistência a Inseticidas/fisiologia , Larva/efeitos dos fármacos , Larva/enzimologia , Especificidade da Espécie , Trinidad e Tobago
19.
Mem. Inst. Oswaldo Cruz ; 107(4): 437-449, June 2012. ilus
Artigo em Inglês | LILACS | ID: lil-626435

RESUMO

The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.


Assuntos
Animais , Esterases/classificação , Resistência a Inseticidas/genética , Inativação Metabólica/genética , Esterases/química , Esterases/genética , Filogenia
20.
Mem. Inst. Oswaldo Cruz ; 107(4): 458-465, June 2012. ilus, graf
Artigo em Inglês | LILACS | ID: lil-626437

RESUMO

Black flies, a non-target species of the insecticides used in fruit production, represent a severe medical and veterinary problem. Large increases in the level of resistance to the pyrethroids fenvalerate (more than 355-fold) and deltamethrin (162-fold) and a small increase in resistance to the organophosphate azinphos methyl (2-fold) were observed between 1996-2008 in black fly larvae under insecticide pressure. Eventually, no change or a slight variation in insecticide resistance was followed by a subsequent increase in resistance. The evolution of pesticide resistance in a field population is a complex and stepwise process that is influenced by several factors, the most significant of which is the insecticide selection pressure, such as the dose and frequency of application. The variation in insecticide susceptibility within a black fly population in the productive area may be related to changes in fruit-pest control. The frequency of individuals with esterase activities higher than the maximum value determined in the susceptible population increased consistently over the sampling period. However, the insecticide resistance was not attributed to glutathione S-transferase activity. In conclusion, esterase activity in black flies from the productive area is one mechanism underlying the high levels of resistance to pyrethroids, which have been recently used infrequently. These enzymes may be reselected by currently used pesticides and enhance the resistance to these insecticides.


Assuntos
Animais , Azinfos-Metil , Esterases/metabolismo , Inseticidas , Nitrilos , Piretrinas , Simuliidae/efeitos dos fármacos , Argentina , Bioensaio , Resistência a Inseticidas , Simuliidae/enzimologia
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