BACKGROUND Leishmania (Viannia) braziliensis Thor strain exhibits a heterogeneous composition comprised of subpopulations with varying levels of infectivity. Clonal subpopulations were previously obtained from the strain Thor by sorting single-parasites and proceeding cultivation. The subpopulations used in this study are named Thor03, Thor 10 and Thor22. OBJECTIVES Phenotypic characteristics of the parasite, specially focusing on virulence factors and resistance to the antimicrobial mechanisms of macrophages, were investigate in these subpopulations. METHODS Cellular and molecular biology, as well as biochemistry approaches were applied to obtain the data analysed in this study. FINDINGS Relative quantification of gene expression was measured for calpain, cysteine protease B (CPB), and subtilisin proteases but no significant differences in these genes’ expression among subpopulations was observed. However, subtilisin and CPB proteins were assessed as more abundant in Thor03 by fluorescence-labelled flow cytometry technique. Western Blotting assays, as semi-quantitative analysis in gel, showed higher concentrations of subtilisin (110 to 50 kDa) and CPB (40 to 18 kDa) in extract of intracellular amastigotes from subpopulations Thor03 and Thor10 and calpain (60 to 25 kDa) showed no significant differences among subpopulations. Complementary, higher trypanothione reductase activity was observed in Thor10 intracellular amastigotes and assays of susceptibility to hydrogen peroxide-inducing agents and nitric oxide donors conducted with promastigotes revealed greater resistance to in vitro oxidative stress induction for Thor10, followed by Thor03. MAIN CONCLUSIONS The data obtained for the virulence factors explored here suggest how multiple coexisting phenotypic-distinct subpopulations may contribute in adaptability of a single L. (V.) braziliensis strain during infection in the host cells. Viannia (Viannia infectivity singleparasites parasites cultivation 1 Thor22 parasite macrophages biology CPB, , (CPB) genes However Thor0 fluorescencelabelled fluorescence labelled technique semiquantitative semi quantitative gel 110 (11 5 kDa 40 (4 Thor1 60 (6 2 Complementary peroxideinducing peroxide inducing phenotypicdistinct phenotypic distinct L V. V (V. cells Thor2 (CPB 11 (1 4 ( 6 (V

This perspective presents and supports arguments for a new formulation of epoxy-α-lapachone loaded microemulsion (ELAP-ME), a nanosystem, as a prototype drug for the treatment of leishmaniasis. The benefits of ELAP as a multitarget compound, with properties that affect key physiological pathways of Leishmania spp. are discussed. ELAP-ME demonstrated efficacy in murine infection models, particularly with the binomial BALB/c-Leishmania (Leishmania) amazonensis. Furthermore, it is proposed that the technological maturity of ELAP-ME be classified as Technology Readiness Level 4 (TLR 4) within the context of innovative drugs for American Cutaneous Leishmaniasis (ACL). epoxyαlapachone epoxy α lapachone ELAPME, ELAPME ME , (ELAP-ME) nanosystem leishmaniasis compound spp discussed models BALB/cLeishmania BALBcLeishmania BALB/c BALB c (Leishmania amazonensis Furthermore TLR ACL. ACL . (ACL) (ELAP-ME cLeishmania BALBc (ACL

ABSTRACT Natural products and their derivatives have been sources of search and research for new drugs for the treatment of neglected diseases. Naphthoquinones, a special group of quinones, are products of natural metabolites with a wide spectrum of biological activities and represent a group of interesting molecules for new therapeutic propositions. Among these compounds, lapachol stands out as a molecule from the heartwood of Tabebuia sp. whose structural changes resulted in compounds considered promising, such as epoxy-a-lapachone (ELAP). The biological activity of ELAP has been demonstrated, so far, for parasitic protozoa such as Leishmania spp., Trypanosoma cruzi and Plasmodium spp., species causing diseases needing new drug development and adequate health policy. This work gathers in vitro and in vivo studies on these parasites, as well as the toxicity profile, and the probable mechanisms of action elucidated until then. The potential of ELAP-based technology alternatives for a further drug is discussed here. Naphthoquinones quinones propositions sp promising epoxyalapachone epoxy lapachone ELAP. . (ELAP) demonstrated far spp spp. policy parasites profile then ELAPbased based here (ELAP

BACKGROUND Leishmania parasites carry a double-stranded RNA virus (Leishmania RNA virus - LRV) that has been divided in LRV1 and LRV2. OBJECTIVES Leishmania (Viannia) braziliensis clinical isolates were assessed in order to determine LRV presence. METHODS Two-round polymerase chain reaction (PCR and nested PCR) was performed to detect LRV1 or LRV2 in L. (V.) braziliensis clinical isolates (n = 12). FINDINGS LRV1 was detected in three clinical isolates which was phylogenetically related to other sequences reported from other American tegumentary leishmaniasis (ATL) endemic areas of Brazil. Patients infected with L. (V.) braziliensis LRV-negative showed only cutaneous lesions while LRV-positive reported different manifestations. MAIN CONCLUSION Data presented here show for the first time that LRV1 is circulating in L. (V.) braziliensis clinical isolates from Rio de Janeiro State in Brazil.

ABSTRACT Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14 kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500 ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n = 114), including suspect (n = 30) and positive (n = 50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n = 34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density = 0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction.

BACKGROUND Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

Resumo Objetivo A obesidade está associada ao metabolismo da glicose anormal que antecede o diabetes mellitus tipo 2. Assim, uma investigação adicional sobre a predição desse resultado letal deve ser antecipada. O objetivo era a avaliação do perfil glicêmico de crianças e adolescentes obesos assintomáticos de Salvador, Brasil. Método Uma amostra de sangue venoso em jejum foi obtida de 90 indivíduos obesos consecutivos entre 8-18 anos, de ambos os sexos, para determinações laboratoriais de hemoglobina glicosilada, insulina basal e Índice do Modelo de Avaliação da Homeostase de Resistência à Insulina (HOMA-IR). A avaliação clínica incluiu peso, estatura, circunferência da cintura, avaliação do desenvolvimento puberal e pesquisa sobre a acantose nigricans. Utilizamos o indicador de índice de massa corporal/idade referente à gravidade da avaliação de sobrepeso. Resultados Alterações glicêmicas foram comprovadas clínica e bioquimicamente, apesar de esses indivíduos não apresentarem queixas ou sintomas relacionados a níveis de açúcar no sangue. Variáveis quantitativas e qualitativas foram, respectivamente, medidas expressas de tendência central/dispersão e amostra/frequência relativa, com o software Pacote Estatístico para as Ciências Sociais, versão 20.0. O valor de p < 0,05 foi considerado significativo. Conclusão Observamos, contudo, alta prevalência de distúrbios de glicose e insulina em crianças e adolescentes obesos assintomáticos.

Abstract Objective Obesity is associated with the abnormal glucose metabolism preceding type 2 diabetes mellitus. Thus, further investigation on the prediction of this lethal outcome must be sought. The objective was the profile glycemic assessment of asymptomatic obese children and adolescents from Salvador, Brazil. Method A fasting venous blood sample was obtained from 90 consecutive obese individuals aged 8-18 years, of both sexes, for laboratory determinations of glycated hemoglobin, basal insulin, and the Homeostasis Model Assessment Insulin Resistance index. The clinical evaluation included weight, height, waist circumference, assessment of pubertal development, and acanthosis nigricans research. The body mass index/age indicator was used for the severity of overweight assessment. Results Glycemic alterations were evidenced clinically and biochemically, although these individuals had no complaints or symptoms related to blood sugar levels. Quantitative and qualitative variables were respectively expressed measures of central tendency/dispersion and simple/relative frequency, using the SPSS, version 20.0. A p-value <0.05 was considered significant. Conclusion Notably, this study found a high prevalence of glucose and insulin disorders in asymptomatic obese children and adolescents.

Abstract Introduction: Non-alcoholic fatty liver disease is characterized by the intrahepatic deposition of fat. It is the most prevalent liver disease in the world, affecting obese children and adolescents. Its pathophysiology is not fully understood, although it is often related to insulin resistance. This in turn would be due to an inflammatory condition common to obesity. Thus, the objective of this study was to describe the behavior of proinflammatory cytokines in obese children and adolescents, with and without non-alcoholic fatty liver disease. Method: A fasting venous blood sample was obtained of consecutive 90 obese individuals aged 8-18 years, of both sexes, for laboratory determinations of glycaemia, basal insulin and homeostasis model assessment insulin-resistance index, and the inflammatory markers tumor necrosis factor-alpha (TNF- α), interleukins 2 and 6 (IL-2 and IL-6), interferon-gamma and high sensitive C-reactive protein. The clinical evaluation included weight, height and waist circumference. We used the body mass index/age indicator for the severity of overweight assessment. The degrees of steatosis were determined by ultrasonography. Quantitative and qualitative variables were respectively expressed by measures of central tendency/dispersion and simple/relative frequency, using Statistical Program for Social Sciences, version 20.0. A p-value < 0.05 was considered as significant. Results: A total of 90 individuals were studied, with a mean age of 11.98 (2.72) years, of which 48 (53%) were male. The body mass index (BMI) for age (BMI/i) and sex (z-score) classified 38 (42.2%) participants as obese and 52 (57.7%) as severe obese; Hepatic steatosis was identified in 56 (62.2%) participants and approximately 90% of them presented grade I steatosis. The inflammatory markers TNF- α, and C-reactive protein were increased in the studied sample and correlated in a positive and statistically significant way with the index of body mass/age and sex. Conclusion: Hepatic steatosis was prevalent in the group of children and adolescents studied, but was not related to obesity degrees

Resumen Introducción: la enfermedad hepática grasa no alcohólica se caracteriza por la deposición intrahepática de grasa. Es la enfermedad hepática más prevalente en el mundo y afecta a niños y adolescentes obesos. Su fisiopatología no se entiende completamente, aunque a menudo se relaciona con la resistencia a la insulina. Esto, a su vez, sería debido a una condición inflamatoria común a la obesidad. Así, el objetivo de este estudio fue describir el comportamiento de las citocinas proinflamatorias en niños y adolescentes obesos, con y sin enfermedad hepática grasa no alcohólica. Método: se obtuvo una muestra de sangre venosa en ayuno de 90 individuos obesos consecutivos, de 8 a 18 años, de ambos sexos, para las determinaciones de laboratorio de glucosa, insulina basal, resistencia a la insulina (homeostasis model assessment insulin-resistance index) y marcadores inflamatorios como TNF-alfa, interleucinas 2 y 6, interferón-gamma y proteína C-reactiva ultrasensible. La evaluación clínica incluyó peso, altura y circunferencia de la cintura. El indicador del índice de masa corporal para la edad (IMC/e) y sexo (z-score) evaluó la gravedad del exceso de peso. Los grados de esteatosis se determinaron por ecografía. Las variables cuantitativas y cualitativas se expresaron respectivamente mediante tendencia/dispersión central y frecuencia simple/relativa, utilizando el Programa Estadístico para Ciencias Sociales, versión 20.0. Se consideró significativo un valor p < 0,05. Resultados: fueron estudiados 90 pacientes con una media de edad de 11,98 (2,72) años, de los cuales 48 (53%) eran varones. Según IMC/e y sexo, 38 (42.2%) participantes fueron clasificados como obesos y 52 (57,7%), como obesos graves. La esteatosis hepática fue identificada en 56 (62,2%) participantes y aproximadamente el 90% de ellos presentaron esteatosis grado I. Los marcadores inflamatorios TNF-alfa y proteína C-reactiva estaban aumentados en la muestra estudiada y se correlacionaron de forma positiva y estadísticamente significativa con el IMC/e y sexo. Conclusión: la esteatosis hepática fue predominante en el grupo de niños y adolescentes estudiados, pero no se relacionó con los grados de obesidad.

Abstract Objective: Although the nonalcoholic fatty liver disease was first identified in 1980, it presents multifactorial and unclear pathophysiology. In this review, we intend to update the pathophysiological mechanisms of a high morbidity and mortality associated disease that is affecting obese children worldwide. Data sources: The PubMed and the Cochrane Library databases were used in the search strategy for articles related to nonalcoholic fatty liver disease and published in the last three decades. Data summary: This review describes the current knowledge on the different mechanisms related to the pathophysiology of nonalcoholic fatty liver disease focused on histological, anatomical and biochemical aspects involved in triggering steatohepatitis and leading to cirrhosis. Conclusions: The clinical research and advanced technological resources demonstrated several determinants pathophysiological mechanisms of nonalcoholic fatty liver disease trying to assist in their treatment and change its natural course.

Resumen Objetivo: aunque la enfermedad de hígado graso no alcohólico se identificó por primera vez en 1980, presenta una fisiopatología multifactorial y mal definida. En esta revisión, tenemos la intención de actualizar los mecanismos fisiopatológicos de esta enfermedad con alta morbilidad y mortalidad asociadas que está afectando a niños obesos en todo el mundo. Fuentes de datos: las bases de datos PubMed y la Biblioteca Cochrane se utilizaron en la estrategia de búsqueda de los artículos relacionados con la enfermedad de hígado graso no alcohólico publicados en las últimas tres décadas. Resumen de datos: esta revisión describe el conocimiento actual acerca de los diferentes mecanismos relacionados con la fisiopatología de la enfermedad de hígado graso no alcohólico, con especial énfasis en aspectos histológicos, anatómicos y bioquímicos implicados en el desencadenamiento de la esteatohepatitis y conducentes a la cirrosis. Conclusiones: la investigación clínica y los recursos tecnológicos avanzados han demostrado diversos mecanismos fisiopatológicos determinantes de la enfermedad de hígado graso no alcohólico, tratando de ayudar en su tratamiento y cambiar su curso.

O curso clínico da infecção pelo HIV é determinado por complexas interações entre características virais e o hospedeiro. Variações no hospedeiro, a exemplo das mutações CCR5Δ32 e CCR264I, são importantes para a vulnerabilidade e progressão do HIV/aids. Atualmente, observa-se um aumento do número de casos da infecção entre os segmentos da sociedade com menor nível de escolaridade e pior condição socioeconômica. Com o objetivo de estimar a ancestralidade e verificar a sua associação com renda, escolaridade vulnerabilidade e progressão ao HIV/aids foram analisados 517 indivíduos infectados pelo HIV-1, sendo 289 homens e 224 mulheres. Os pacientes foram classificados segundo a ancestralidade genômica avaliada por 10 AIMs e pela vulnerabilidade e progressão ao HIV/aids através das mutações CCR5Δ32 e CCR264I. Os indivíduos infectados pelo HIV-1 apresentaram contribuição africana de 47%. As mutações CCR5Δ32 e CCR264I foram mais frequentes nos indivíduos brancos (3%) e negros (18%) respectivamente, e essas mutações mostraram frequência mais elevada nos tipicamente progressores (TP), quando comparados com os rapidamente progressores (RP) para aids. Não foi encontrada associação entre ancestralidade e vulnerabilidade ao HIV na análise para o grau de instrução. A pauperização da infecção pelo HIV-1 nessa população foi confirmada pela relação inversa entre renda e ancestralidade africana, pois quanto menor a renda maior a ancestralidade africana. Os resultados deste estudo sugerem associação entre as condições socioeconômicas e vulnerabilidade ao HIV/aids da população afrodescendente.

The clinical course of HIV infection is determined by complex/ interactions between viral and host's characteristics./ Host variations, such as CCR5δ32 and CCR264I mutations, are important/ to vulnerability and progression of HIV/AIDS./ Currently, the number of cases among patients with lower educational level and lower social and economic status is/ increasing./ Aiming to/ estimate the ancestry and verify its association with income,/ education, vulnerability and progression of HIV/AIDS, 517 individuals infected with HIV-1 were studied (55.9% men and 43.3% women). The/ patients were/ classified according to/ genomic ancestry evaluated by 10 AIMs and by vulnerability and/ progression of HIV/AIDS through CCR5δ32 and CCR264I mutations./ The/ individuals infected with HIV-1 showed 47% of African contribution./ CCR5δ32 and CCR264I mutations were more frequent in white/ (3%) and black (18%) individuals, respectively, and these same mutations/ showed higher frequency in the typically progressive HIV-infected individuals (TP), when compared to the rapidly progressive (RP)./ There was no association between ancestry and/ vulnerability to HIV in the analysis of level of education./ The pauperization of the HIV-1 infection in this population was confirmed by/ the inverse relationship between income and African ancestry, because the lower/ the income, the greater the African ancestry./ The results suggest that there is an association between socioeconomic status and vulnerability to HIV/AIDS in the Afro-descendant population.

O curso clínico da infecção pelo HIV é determinado por complexas interações entre características virais e o hospedeiro. Variações no hospedeiro, a exemplo das mutações CCR5Δ32 e CCR264I, são importantes para a vulnerabilidade e progressão do HIV/aids. Atualmente, observa-se um aumento do número de casos da infecção entre os segmentos da sociedade com menor nível de escolaridade e pior condição socioeconômica. Com o objetivo de estimar a ancestralidade e verificar a sua associação com renda, escolaridade vulnerabilidade e progressão ao HIV/aids foram analisados 517 indivíduos infectados pelo HIV-1, sendo 289 homens e 224 mulheres. Os pacientes foram classificados segundo a ancestralidade genômica avaliada por 10 AIMs e pela vulnerabilidade e progressão ao HIV/aids através das mutações CCR5Δ32 e CCR264I. Os indivíduos infectados pelo HIV-1 apresentaram contribuição africana de 47%. As mutações CCR5Δ32 e CCR264I foram mais frequentes nos indivíduos brancos (3%) e negros (18%) respectivamente, e essas mutações mostraram frequência mais elevada nos tipicamente progressores (TP), quando comparados com os rapidamente progressores (RP) para aids. Não foi encontrada associação entre ancestralidade e vulnerabilidade ao HIV na análise para o grau de instrução. A pauperização da infecção pelo HIV-1 nessa população foi confirmada pela relação inversa entre renda e ancestralidade africana, pois quanto menor a renda maior a ancestralidade africana. Os resultados deste estudo sugerem associação entre as condições socioeconômicas e vulnerabilidade ao HIV/aids da população afrodescendente.

The clinical course of HIV infection is determined by complex/ interactions between viral and host's characteristics./ Host variations, such as CCR5δ32 and CCR264I mutations, are important/ to vulnerability and progression of HIV/AIDS./ Currently, the number of cases among patients with lower educational level and lower social and economic status is/ increasing./ Aiming to/ estimate the ancestry and verify its association with income,/ education, vulnerability and progression of HIV/AIDS, 517 individuals infected with HIV-1 were studied (55.9% men and 43.3% women). The/ patients were/ classified according to/ genomic ancestry evaluated by 10 AIMs and by vulnerability and/ progression of HIV/AIDS through CCR5δ32 and CCR264I mutations./ The/ individuals infected with HIV-1 showed 47% of African contribution./ CCR5δ32 and CCR264I mutations were more frequent in white/ (3%) and black (18%) individuals, respectively, and these same mutations/ showed higher frequency in the typically progressive HIV-infected individuals (TP), when compared to the rapidly progressive (RP)./ There was no association between ancestry and/ vulnerability to HIV in the analysis of level of education./ The pauperization of the HIV-1 infection in this population was confirmed by/ the inverse relationship between income and African ancestry, because the lower/ the income, the greater the African ancestry./ The results suggest that there is an association between socioeconomic status and vulnerability to HIV/AIDS in the Afro-descendant population.

INTRODUÇÃO: A leishmaniose visceral (LV) é uma importante zoonose para os sistemas de saúde pública, sendo os cães o principal reservatório doméstico. Este estudo teve como objetivo investigar a ocorrência de LV canina (LVC) em Dias D'Ávila, Estado da Bahia, Brasil. MÉTODOS: A prevalência foi avaliada através de exames clínicos e laboratoriais em uma população de 312 cães domésticos de 23 localidades da cidade, utilizando-se imunofluorescência indireta e ensaio imunoenzimático. RESULTADOS: Dentre os animais examinados, 3.2% e 6.7% apresentaram sinais de LV, confirmados por imunofluorescência indireta e ensaio imunoenzimático, respectivamente, com distribuição de 29.9% (24 cães) na zona rural e 4.9% (288 cães) na área urbana (p=0,001). A avaliação clínica de cães soropositivos apresentou animais assintomáticos (2,4%) e sintomáticos (47,6%), além de outras alterações (e.g., anemia normocítica e normocrômica, com leucocitose e trombocitopenia). Observações relativas a características fenotípicas (e.g. sexo, idade, raça e pelo) não apresentaram significância estatística, embora uma soropositividade alta entre machos, animais de pelo curto e de raça indefinida tenha sido observada. CONCLUSÕES: Os achados indicam que a LV é uma zoonose predominantemente rural e que o convívio de aves e cães domésticos aumenta significativamente o risco da infecção canina na região.

INTRODUCTION: Visceral leishmaniasis (VL) is an important zoonosis in relation to public health systems. Dogs are the main domestic reservoir. This study aimed to investigate occurrences of canine VL in Dias D'Ávila, State of Bahia, Brazil. METHODS: The prevalence was evaluated by means of clinical and laboratory tests on a population of 312 domestic dogs from 23 localities in this municipality, using indirect immunofluorescence and immunoenzymatic assays. RESULTS: Among the animals examined, 3.2% and 6.7% showed signs of VL, confirmed by indirect immunofluorescence and immunoenzymatic assays, respectively, with a distribution of 29.9% (24 dogs) in the rural zone and 4.9% (288 dogs) in the urban zone (p = 0.001). The clinical evaluation on seropositive dogs showed both asymptomatic animals (2.4%) and symptomatic animals (47.6%), along with other abnormalities (e.g. normocytic and normochromic anemia, with leukocytosis and thrombocytopenia). Observations relating to phenotypic characteristics (e.g. sex, age, breed and hair) did not present statistical significance, although high seropositivity among male, short-haired and mixed-breed dogs was observed. CONCLUSIONS: The findings showed that VL was a predominantly rural zoonosis and that close contact between poultry and domestic dogs significantly increased the risk of canine infection in this region.

We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Este trabalho teve como objetivo avaliar a qualidade de painéis compensados de Pinus taeda e de Pinus oocarpa, com 20 e 24 anos de idade, respectivamente, utilizando três diferentes formulações de adesivo uréia-formaldeído. Foram produzidos 18 painéis, com três repetições por tratamento. As formulações com maior proporção relativa de resina não influenciaram de forma conclusiva as propriedades físico-mecânicas dos painéis.Os painéis de P. oocarpa apresentaram valores médios de resistência da linha de cola, módulos de elasticidade e de ruptura superiores àqueles dos painéis de P. taeda. Os resultados das propriedades físico-mecânicas dos painéis indicaram grande potencial de utilização de lâminas de P. oocarpa para produção de compensados.

The objective of this study was to evaluate the quality of both Pinus taeda and Pinus oocarpa plywood, 20 and 24-years-old, respectively, using three different formulations of urea formaldheyde resin. A total of 18 boards were produced, using three repetitions per treatment. The formulations containing a high relative proportion of the resin did not show a conclusive influence on the physical and mechanical properties of the boards. The boards made from P. oocarpa showed higher average values of the glue line strength, modulus of elasticity and modulus of rupture in comparison to boards of P. taeda. The results showed that the evaluation of the physical and mechanical properties of the board, indicate that the veneers of P. oocarpa have a high potentiality for plywood production.