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Mem. Inst. Oswaldo Cruz ; 111(11): 670-675, Nov. 2016. graf
Artigo em Inglês | LILACS | ID: biblio-829246


Nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD) performs essential functions in cell metabolism and energy production due to its redox properties. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC enzyme catalyses the key step in the biosynthesis of NAD+. Previously, the enzyme NMNAT was identified in Trypanosoma cruzi (TcNMNAT), a pathogenic agent with epidemiological importance in Latin America. To continue with the functional characterisation of this enzyme, its subcellular location and its possible post-translational modifications were examined in this study. For this, polyclonal antibodies were generated in mice, with soluble and denatured recombinant protein being used to detect the parasite’s NMNAT. Immunodetection assays were performed on whole extracts of T. cruzi, and an approximation of its intracellular location was determined using confocal microscopy on wild and transgenic parasites, which revealed the cytosol distribution patterns. This localisation occurs according to the needs of the dinucleotides that exist in this compartment. Additionally, a bioinformatics study was performed as a first approach to establish the post-translational modifications of the enzyme. Possible phosphorylation events were experimentally analysed by western blot, highlighting TcNMNAT as a potential target for serine kinases.

Animais , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Citosol/parasitologia , Interações Hospedeiro-Parasita , Camundongos , Camundongos Endogâmicos BALB C , Nicotinamida-Nucleotídeo Adenililtransferase/isolamento & purificação , Fosforilação , Proteínas Serina-Treonina Quinases , Proteínas de Protozoários/isolamento & purificação
Braz. j. microbiol ; 46(2): 631-637, Apr-Jun/2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-749708


This work addresses the production of prodigiosin from ram horn peptone (RHP) using MO-1, a local isolate in submerged culture. First, a novel gram-negative and rod-shaped bacterial strain, MO-1, was isolated from the body of the grasshopper (Poecilemon tauricola Ramme 1951), which was collected from pesticide-contaminated fields. Sequence analysis of 16S rDNA classified the microbe as Serratia marcescens. The substrate utilization potential (BIOLOG) and fatty acid methyl ester profile (FAME) of S. marcescens were also determined. The effect of RHP on the production of prodigiosin by S. marcescens MO-1 was investigated, and the results showed that RHP supplementation promoted the growth of MO-1 and increased the production of prodigiosin. A concentration of 0.4% (w/v) RHP resulted in the greatest yield of prodigiosin (277.74 mg/L) after 48 h when mannitol was used as the sole source of carbon. The pigment yield was also influenced by the types of carbon sources and peptones. As a result, RHP was demonstrated to be a suitable substrate for prodigiosin production. These results revealed that prodigiosin could be produced efficiently by S. marcescens using RHP.

Animais , Meios de Cultura/química , Peptonas/metabolismo , Prodigiosina/metabolismo , Serratia marcescens/crescimento & desenvolvimento , Serratia marcescens/metabolismo , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Gafanhotos/microbiologia , Dados de Sequência Molecular , Filogenia , Pigmentos Biológicos/metabolismo , /genética , Análise de Sequência de DNA , Serratia marcescens/classificação , Serratia marcescens/isolamento & purificação
Arq. bras. cardiol ; 104(5): 366-374, 05/2015. tab, graf
Artigo em Inglês | LILACS | ID: lil-748157


Background: Physiological reflexes modulated primarily by the vagus nerve allow the heart to decelerate and accelerate rapidly after a deep inspiration followed by rapid movement of the limbs. This is the physiological and pharmacologically validated basis for the 4-s exercise test (4sET) used to assess the vagal modulation of cardiac chronotropism. Objective: To present reference data for 4sET in healthy adults. Methods: After applying strict clinical inclusion/exclusion criteria, 1,605 healthy adults (61% men) aged between 18 and 81 years subjected to 4sET were evaluated between 1994 and 2014. Using 4sET, the cardiac vagal index (CVI) was obtained by calculating the ratio between the duration of two RR intervals in the electrocardiogram: 1) after a 4-s rapid and deep breath and immediately before pedaling and 2) at the end of a rapid and resistance-free 4-s pedaling exercise. Results: CVI varied inversely with age (r = -0.33, p < 0.01), and the intercepts and slopes of the linear regressions between CVI and age were similar for men and women (p > 0.05). Considering the heteroscedasticity and the asymmetry of the distribution of the CVI values according to age, we chose to express the reference values in percentiles for eight age groups (years): 18–30, 31–40, 41–45, 46–50, 51–55, 56–60, 61–65, and 66+, obtaining progressively lower median CVI values ranging from 1.63 to 1.24. Conclusion: The availability of CVI percentiles for different age groups should promote the clinical use of 4sET, which is a simple and safe procedure for the evaluation of vagal modulation of cardiac chronotropism. .

Fundamento: Reflexos fisiológicos modulados primariamente pelo vago permitem que a frequência cardíaca diminua e aumente rapidamente, após uma inspiração profunda seguida da movimentação rápida dos membros. Essa é a base fisiológica e farmacologicamente validada do teste de exercício de 4 segundos (T4s) para a avaliação da modulação vagal sobre o cronotropismo cardíaco. Objetivo: Apresentar os dados de referência do T4s em adultos saudáveis. Métodos: Após aplicação de rígidos critérios clínicos de inclusão/exclusão foram estudados 1.605 adultos saudáveis (61% homens), entre 18 e 81 anos de idade, submetidos ao T4s, entre 1994 e 2014. No T4s, o Índice Vagal Cardíaco (IVC) foi obtido pela razão entre as durações de dois intervalos RR no traçado eletrocardiográfico: 1) após 4 segundos de inspiração rápida e profunda e imediatamente antes da pedalada; e 2) ao final da pedalada livre e rápida de 4 segundos. Resultados: IVC varia inversamente com a idade (r = - 0,33; p < 0,01) e os interceptos e as inclinações das regressões lineares entre IVC e idade são similares para homens e mulheres (p > 0,05). Considerando a heterocedasticidade e a assimetria da distribuição de IVC em função da idade, optou-se por expressar os valores de referência em percentis para oito faixas etárias (anos): 18-30, 31-40, 41-45, 46-50, 51-55, 56-60, 61-65 e 66+, obtendo-se medianas de IVC progressivamente menores de 1,63 a 1,24. Conclusão: A disponibilização dos percentis de IVC por diferentes faixas etárias deverá facilitar a maior utilização clínica do T4s, um procedimento simples e seguro, para a avaliação da modulação vagal do cronotropismo cardíaco. .

Animais , Humanos , Camundongos , Bactérias , Caspases , Citosol/microbiologia , Inflamassomos , Bactérias/imunologia , Bactérias/patogenicidade
Braz. j. phys. ther. (Impr.) ; 18(6): 513-520, 09/01/2015. graf
Artigo em Inglês | LILACS | ID: lil-732354


BACKGROUND: Knowing the potential for and limitations of information generated using different evaluation instruments favors the development of more accurate functional diagnoses and therapeutic decision-making. OBJECTIVE: To investigate the relationship between the number of compensatory movements when climbing up and going down stairs, age, functional classification and time taken to perform a tested activity (TA) of going up and down stairs in boys with Duchenne muscular dystrophy (DMD). METHOD: A bank of movies featuring 30 boys with DMD performing functional activities was evaluated. Compensatory movements were assessed using the climbing up and going down stairs domain of the Functional Evaluation Scale for Duchenne Muscular Dystrophy (FES-DMD); age in years; functional classification using the Vignos Scale (VS), and TA using a timer. Statistical analyses were performed using the Spearman correlation test. RESULTS: There is a moderate relationship between the climbing up stairs domain of the FES-DMD and age (r=0.53, p=0.004) and strong relationships with VS (r=0.72, p=0.001) and TA for this task (r=0.83, p<0.001). There were weak relationships between the going down stairs domain of the FES-DMD-going down stairs with age (r=0.40, p=0.032), VS (r=0.65, p=0.002) and TA for this task (r=0.40, p=0.034). CONCLUSION: These findings indicate that the evaluation of compensatory movements used when climbing up stairs can provide more relevant information about the evolution of the disease, although the activity of going down stairs should be investigated, with the aim of enriching guidance and strengthening accident prevention. Data from the FES-DMD, age, VS and TA can be used in a complementary way to formulate functional diagnoses. Longitudinal studies and with broader age groups may supplement this information. .

CONTEXTUALIZAÇÃO: Conhecer as potencialidades e limitações das informações geradas por diferentes instrumentos de avaliação favorece o desenvolvimento mais preciso do diagnóstico funcional e da tomada de decisão terapêutica. OBJETIVO : Investigar a relação entre o número de movimentos compensatórios ao subir e descer escadas, idade, classificação funcional e tempo de realização de atividade (TA) em meninos com Distrofia Muscular de Duchenne (DMD). MÉTODO : Foi utilizado banco de filmes de 30 meninos com DMD realizando atividades funcionais. Os movimentos compensatórios foram avaliados pela Escala de Avaliação Funcional para Distrofia Muscular de Duchenne (FES-DMD), domínio subir e descer escada; a idade, mensurada em anos; a classificação funcional foi pesquisada pela Escala de Vignos (EV), e o TA foi cronometrado. Foi utilizado o teste de correlação de Spearman. RESULTADOS : Existe moderada relação entre a FES-DMD-subir escada e a idade (r=0,53, p=0,004) e forte relação com a EV (r=0,72, p=0,001) e TA dessa tarefa (r=0,83, p<0,001). Houve fraca relação entre a FES-DMD-descer escada e a idade (r=0,40, p=0,032), EV (r=0,65, p=0,002) e o TA dessa tarefa (r=0,40, p=0,034). CONCLUSÃO : Esses achados indicam que a avaliação da tarefa de subir escada pode trazer informações mais relevantes sobre a evolução da doença, embora a atividade de descer escada deva ser pesquisada visando à orientação e prevenção de acidentes. A utilização conjunta de dados provenientes da FES-DMD, da idade e do TA pode se complementar para formulação do diagnóstico funcional. Estudos longitudinais e com outras faixas etárias mais amplas podem complementar tal informação. .

Humanos , Masculino , Hiperplasia Prostática/metabolismo , Receptores Androgênicos/metabolismo , Ligação Competitiva , Tampões (Química) , Carvão Vegetal , Citosol/metabolismo , Dextranos , Di-Hidrotestosterona/metabolismo , Eletroforese em Gel de Ágar , Ativação Enzimática/efeitos dos fármacos , Estrenos/metabolismo , Metribolona , Molibdênio/farmacologia , Progesterona/metabolismo , Inibidores de Proteases/farmacologia , Temperatura , Tartaratos/farmacologia , Congêneres da Testosterona/metabolismo
Rev. latinoam. enferm ; 22(6): 902-910, 16/12/2014. tab
Artigo em Inglês | LILACS, BDENF - Enfermagem | ID: lil-732951


METHOD: one hundred (n=100) elderly outpatients with diabetic retinopathy taking antihypertensives and/or oral antidiabetics/insulin were interviewed. Adherence was evaluated by the adherence proportion and its association with the care taken in administrating medications and by the Morisky Scale. The National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25) was used to evaluate HRQoL. RESULTS: most (58%) reported the use of 80% or more of the prescribed dose and care in utilizing the medication. The item "stopping the drug when experiencing an adverse event", from the Morisky Scale, explained 12.8% and 13.5% of the variability of adherence proportion to antihypertensives and oral antidiabetics/insulin, respectively. CONCLUSION: there was better HRQoL in the Color Vision, Driving and Social Functioning domains of the NEI VFQ-25. Individuals with lower scores on the NEI VFQ-25 and higher scores on the Morisky Scale presented greater chance to be nonadherent to the pharmacological treatment of diabetes and hypertension. .

OBJETIVO: investigar os fatores relacionados à adesão medicamentosa e sua relação com a qualidade de vida relacionada à saúde em idosos com retinopatia diabética. MÉTODO: foram entrevistados 100 idosos, em acompanhamento ambulatorial, em uso de anti-hipertensivos e/ou antidiabéticos orais/insulina. A adesão foi avaliada pela proporção de adesão e sua associação com os cuidados no uso dos medicamentos e pela Escala de Morisky. O National Eye Institute Visual Funcioning Questionnaire foi utilizado para avaliar a qualidade de vida relacionada à saúde. RESULTADOS: A maioria (58%) relatou o uso de 80% ou mais das doses prescritas e os cuidados na tomada dos medicamentos. O item "interromper o uso dos medicamentos por se sentir pior", da Escala de Morisky, explicou 12,8 e 13,5% da variabilidade da proporção de adesão aos anti-hipertensivos e aos antidiabéticos orais/insulina, respectivamente. CONCLUSÃO: observou-se melhor qualidade de vida relacionada à saúde nos domínios visão de cores, dirigir automóvel e apectos sociais do National Eye Institute Visual Funcioning Questionnaire. Indivíduos com menor pontuação na National Eye Institute Visual Funcioning Questionnaire e maiores escores na Escala de Morisky apresentaram maiores chances de serem não aderentes aos medicamentos do diabetes e da hipertensão arterial. .

OBJETIVO: investigar los factores relacionados a la adhesión a la medicación y su relación con la Calidad de Vida Relacionada a la Salud (CVRS) de ancianos con retinopatía diabética. MÉTODO: fueron entrevistados cien (n=100) pacientes ancianos de ambulatorio con retinopatía diabética que toman medicamentos antihipertensivos y/o antidiabéticos orales/insulina. La adhesión fue evaluada mediante la proporción de adhesión y su asociación con el cuidado tomado en la administración de medicamentos y mediante la Escala de Morisky. El National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25) fue usado para evaluar la CVRS. RESULTADOS: la mayoría (58%) relató el uso de 80% o más de la dosis prescrita y cuidado con el uso de la medicación. El ítem "suspender la droga cuando vivencia un evento adverso", de la Escala de Morisky, explicó 12.8% y 13.5% de la variabilidad en la proporción de adhesión a los antihipertensivos y antidiabéticos orales/insulina, respectivamente. CONCUSIÓN: fue encontrada mejor CVRS en los dominios de Visión Cromática, Dirección y Funcionamiento Social del NEI VFQ-25. Individuos con puntuaciones menores en el NEI VFQ-25 y puntuaciones mayores en la Escala de Morisky revelaron mayor chance de no adhesión al tratamiento farmacológico de la diabetes y hipertensión. .

Animais , Bovinos , Arsenitos , DNA , Proteínas de Ligação a DNA/fisiologia , Receptores de Glucocorticoides/metabolismo , Compostos de Sódio , Timo/metabolismo , Arsênico/farmacologia , Fenômenos Químicos , Química , Cromatografia em Gel , Citosol/metabolismo , Dextranos , Metanossulfonato de Metila/análogos & derivados , Metanossulfonato de Metila/farmacologia , Molibdênio/farmacologia
Braz. j. microbiol ; 45(4): 1239-1245, Oct.-Dec. 2014. ilus, graf
Artigo em Inglês | LILACS | ID: lil-741273


A bacterial isolate capable of utilizing tributyltin chloride (TBTCl) as sole carbon source was isolated from estuarine sediments of west coast of India and identified as Pseudomonas stutzeri based on biochemical tests and Fatty acid methyl ester (FAME) analysis. This isolate was designated as strain DN2. Although this bacterial isolate could resist up to 3 mM TBTCl level, it showed maximum growth at 2 mM TBTCl in mineral salt medium (MSM). Pseudomonas stutzeri DN2 exposed to 2 mM TBTCl revealed significant alteration in cell morphology as elongation and shrinkage in cell size along with roughness of cell surface. FTIR and NMR analysis of TBTCl degradation product extracted using chloroform and purified using column chromatography clearly revealed biotransformation of TBTCl into Dibutyltin dichloride (DBTCl2) through debutylation process. Therefore, Pseudomonas stutzeri strain DN2 may be used as a potential bacterial strain for bioremediation of TBTCl contaminated aquatic environmental sites.

Pseudomonas stutzeri/metabolismo , Compostos de Trialquitina/metabolismo , Técnicas de Tipagem Bacteriana , Biotransformação , Cromatografia Líquida , Carbono/metabolismo , Citosol/química , Ácidos Graxos/análise , Sedimentos Geológicos , Índia , Espectroscopia de Ressonância Magnética , Pseudomonas stutzeri/classificação , Pseudomonas stutzeri/crescimento & desenvolvimento , Pseudomonas stutzeri/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier
Rev. latinoam. enferm ; 22(6): 1026-1033, 16/12/2014.
Artigo em Inglês | LILACS, BDENF - Enfermagem | ID: lil-732940


OBJECTIVE: to identify the perceptions of professionals working in a facility connected with the Brazilian Unified Health System - SUS in regard to what they know, think and talk about public health policy. METHOD: this exploratory-descriptive study with a qualitative nature was conducted with 28 professionals working in a facility connected with the SUS. Data were collected through interviews with guiding questions and analyzed through the thematic content analysis technique. RESULTS: coded and interpreted data resulted in three thematic axes: The SUS - perfect web that does not work in practice; The recurrent habit of complaining about the SUS; The need to rethink the way of thinking about, acting in and managing the SUS. CONCLUSION: the professionals working for the SUS are aware of the principles and guidelines that govern the Brazilian health system, however, they reproduce a dichotomous and linear model of conception and practice strongly linked to the thinking of society in general. .

OBJETIVO: conhecer a percepção de profissionais que atuam em uma instituição conveniada com o Sistema Único de Saúde sobre o que sabem, pensam e falam dessa política pública de saúde. MÉTODO: trata-se de estudo exploratório-descritivo, de caráter qualitativo, realizado com 28 profissionais que atuam em uma instituição conveniada com o Sistema Único de Saúde. Os dados foram coletados por meio de entrevistas com questões norteadoras e analisados pela técnica de análise de conteúdo temática. RESULTADOS: os dados codificados e interpretados resultaram em três eixos temáticos: Sistema Único de Saúde - teia perfeita que não funciona na prática; o recorrente hábito de reclamar do Sistema Único de Saúde; a necessidade de repensar o modo de pensar, atuar e gerir o Sistema Único de Saúde. CONCLUSÃO: os profissionais que atuam no Sistema Único de Saúde têm conhecimento dos princípios e diretrizes que regem o sistema de saúde nacional, no entanto, reproduzem um modelo de concepção e atuação dicotômico, pontual e linear ainda fortemente vigente no pensar da sociedade em geral. .

OBJETIVO: conocer la percepción de profesionales que actúan en una institución que tiene convenio con el Sistema Único de Salud - SUS sobre lo que saben, piensan y hablan de esta política pública de salud. MÉTODO: se trata de un estudio exploratorio descriptivo, de carácter cualitativo, realizado con 28 profesionales que actúan en una institución que tiene convenio con el SUS. Los datos fueron recolectados por medio de entrevistas con preguntas orientadoras y analizados con la técnica de análisis de contenido temático. RESULTADOS: los datos codificados y interpretados resultaron en tres ejes temáticos: SUS - red perfecta que no funciona en la práctica; el recurrente hábito de reclamar del SUS; y la necesidad de repensar el modo de pensar, actuar y administrar el SUS. CONCLUSIÓN: los profesionales que actúan en el SUS tienen conocimiento de los principios y directrices que gobiernan el sistema de salud nacional, sin embargo, reproducen un modelo de concepción y actuación dicotómico, puntual, linear y además fuertemente vigente en el pensar de la sociedad en general. .

Animais , Masculino , Ratos , Dexametasona/análogos & derivados , Peso Corporal/efeitos dos fármacos , Corticosterona/sangue , Citosol/metabolismo , Citosol/ultraestrutura , Dexametasona/farmacologia , Fígado/metabolismo , Fígado/ultraestrutura , Molibdênio/farmacologia , Ratos Endogâmicos , Receptores de Glucocorticoides/metabolismo , Timo/metabolismo , Timo/ultraestrutura , Trítio
Braz. j. microbiol ; 45(3): 985-993, July-Sept. 2014. mapas, tab
Artigo em Inglês | LILACS | ID: lil-727030


In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

Bactérias/classificação , Bactérias/isolamento & purificação , Raízes de Plantas/microbiologia , Microbiologia do Solo , Técnicas de Tipagem Bacteriana , Bactérias/genética , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Genes , Glucose 1-Desidrogenase/genética , Dados de Sequência Molecular , Paquistão , Filogenia , Plantas , Quinonas/análise , Rizosfera , /genética , Análise de Sequência de DNA
Braz. j. microbiol ; 45(2): 603-611, Apr.-June 2014. ilus, tab
Artigo em Inglês | LILACS | ID: lil-723124


Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p < 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities.

Enterobacteriaceae/enzimologia , Enterobacteriaceae/fisiologia , Glucose 1-Desidrogenase/genética , Fatores de Crescimento Neural , Phaseolus/crescimento & desenvolvimento , Phaseolus/microbiologia , Análise por Conglomerados , Biologia Computacional , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Glucose 1-Desidrogenase/química , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Estrutura Terciária de Proteína , Fósforo/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/crescimento & desenvolvimento , Quinonas/análise , Análise de Sequência de DNA , Homologia de Sequência
Biol. Res ; 42(1): 111-119, 2009. ilus, tab
Artigo em Inglês | LILACS | ID: lil-519090


Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca2+ dependent exocitosis of vasoactive compounds. Using the Ca2+ indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs) respond to acetylcholine (ACh) with a cytosolic Ca2+ increase and depolarization of the membrane potential (20.3±0.9 mV; n=23). The increase in cytosolic Ca2+ induced by 10µM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 µM of hexamethonium. On the other hand, the increase in cytosolic Ca2+ could be depressed by nifedipine (0.01 -100 µM) or withdrawal of extracellular Ca2+. Taken together, these results give evidence for functional nicotinic receptors (nAChRs) in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal gland's microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca2+ channels, influx of external Ca2+ and liberation of vasoactive compounds.

Animais , Bovinos , Medula Suprarrenal/efeitos dos fármacos , Canais de Cálcio/efeitos dos fármacos , Citosol/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Nicotina/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Acetilcolina/farmacologia , Medula Suprarrenal/irrigação sanguínea , Medula Suprarrenal/citologia , Canais de Cálcio/metabolismo , Capilares/citologia , Capilares/efeitos dos fármacos , Citosol/metabolismo , Potenciais Evocados/efeitos dos fármacos , Hexametônio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Muscarina/farmacologia , Receptores Nicotínicos/metabolismo
Rev. invest. clín ; 57(4): 555-562, jul.-ago. 2005. ilus, tab
Artigo em Inglês | LILACS | ID: lil-632429


Objective. To determine the processing pathways used by peripheral blood mononuclear cells (PBMC) and present the rHSP60Kp, and the T cell subpopulations involved in the response, in patients with ankylosing spondylitis (AS) Methods. The lymphoproliferative response to the rHSP60Kp in PBMC from 14 HLA-B27 + AS patients and 15 B27 healthy controls was assessed by ³H-TdR incorporation. The processing pathways for the rHSP60Kp were analyzed by ³H-TdR incorporation in fresh PBMC from patients using homologous PBMC preincubated with the antigen and specific inhibitors: chloroquine, N-acetyl-L-leucil-L-leucil-L-nor-leucinal (LLnL) or brefeldin A (BFA), fixed with p-formaldehyde (fixed APC). The CD4+/CD8+ T cell subpopulation activated with the antigen was determined by three colours flow cytometry in PBMC from patients. Results. Eight out of fourteen patients showed positive lymphoproliferative responses to the rHSP60Kp while none of the healthy controls responded (p < 0.012). In five patients S.I. was above 4.0. In these patients lymphoproliferation was lower when chloroquine and LLnL was used and it became negative with BFA, indicating that both pathways are used. CD4+ and CD8+ T cells populations expressed CD69 when activated by the rHSP60Kp. Conclusions. Our results suggest that CD4 and CD8 T cells participate in the response to the rHSP60Kp in B27+ AS patients.

Objetivo.Determinar las vías utilizadas por las células mononucleares de sangre periférica (CMSP) de pacientes con espondilitis anquilosante para procesar a la rHSPGO de Klebsiella pneumoniae (rHSPGOKp) y las subpoblaciones de linfocitos T involucrados en la activación. Métodos. Se determinó la respuesta linfoproliferativa, por incorporación de ³H-TdR en CMSP, en presencia de la rHSPGOKp, en 14 pacientes con EA HLA-B27+y en 15 sujetos sanos HLA-B27-. La ruta de procesamiento y presentación de la rHSPGOKp se determinó por incorporación de ³H-TdR en las CMSP de los pacientes utilizando como células presentadoras a las CMSP homologas, preíncubadas con el antígeno y los inhibidores específicos: cloroquína, brefeldína A y N-acetil-L-leucil-L-leucil-L-nor-leucinal (LLnL), y fijadas con p-formaldehído. Se evaluaron las subpoblaciones de linfocitos T CD4+ y CD8+ que expresaron CD69, frente al antígeno, por citometría de flujo. Resultados. Ocho de los 14 pacientes y ninguno de los sujetos sanos, tuvo respuesta linfoproliferativa positiva (IE > 3.0) contra la rHSPGOKp (p < 0.012). En cinco de los pacientes el I.E. fue superior a 4.0. En estos pacientes la linfoproliferación disminuyó cuando se utilizó cloroquína y LLnL, y se hizo negativa cuando se utilizó BFA, lo que indica que ambas vías son empleadas. Las subpoblaciones de linfocitos T (CD4+ y CD8+) expresaron CD69 frente al antigeno. Conclusiones. Nuestros resultados sugieren que ambas poblaciones de linfocitos T: CD4+ y CD8+ participan en la respuesta a la rHSPGOKp.

Humanos , Apresentação do Antígeno , Antígenos de Bactérias/imunologia , Doenças Autoimunes/imunologia , /imunologia , /imunologia , /imunologia , Klebsiella pneumoniae/imunologia , Ativação Linfocitária , Espondilite Anquilosante/imunologia , Subpopulações de Linfócitos T/imunologia , Apresentação do Antígeno/efeitos dos fármacos , Doenças Autoimunes/sangue , Doenças Autoimunes/genética , Brefeldina A/farmacologia , /efeitos dos fármacos , /efeitos dos fármacos , Cloroquina/farmacologia , Citosol/imunologia , Endocitose , Citometria de Fluxo , /análise , /genética , Klebsiella pneumoniae/química , Leucócitos Mononucleares/imunologia , Leupeptinas/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Espondilite Anquilosante/sangue , Espondilite Anquilosante/genética , Subpopulações de Linfócitos T/efeitos dos fármacos
Braz. j. med. biol. res ; 38(1): 5-10, Jan. 2005. tab, graf
Artigo em Inglês | LILACS | ID: lil-405548


An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ß-ATP, and ADP failed to do so in a 1- to 200-æm con- centration. The EC50 obtained for the compounds tested was 41.77 æM for UTP, 48.11 æM for GTP, 53.11 æM for UDP, and 30.78 æM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.

Animais , Cálcio/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Eritrócitos/citologia , Lagartos/sangue , /fisiologia , /agonistas , Espectrometria de Fluorescência
Mem. Inst. Oswaldo Cruz ; 99(2): 223-226, Mar. 2004. graf
Artigo em Inglês | LILACS | ID: lil-360980


Cholinesterase and acid phosphatase (AP), but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of ivermectin resistant and susceptible Haemonchus contortus infective-stage larvae. Some differences in acetylcholinesterase activity of cytosolic fractions and in the AP activity of these fractions as well as in the response to AP inhibitors by membrane-bound fractions were detected. Data are discussed.

Animais , Fosfatase Ácida , Anti-Helmínticos , Colinesterases , Haemonchus , Ivermectina , Fosfatase Ácida , Inibidores da Colinesterase , Colinesterases , Citosol , Resistência a Medicamentos , Haemonchus , Larva
Biol. Res ; 37(4): 593-602, 2004. graf
Artigo em Inglês | LILACS | ID: lil-437513


Calcium release via intracellular Ca2+ release channels is a central event underpinning the generation of numerous, often divergent physiological processes. In electrically non-excitable cells, this Ca2+ release is brought about primarily through activation of inositol 1,4,5-trisphosphate receptors and typically takes the form of calcium oscillations. It is widely believed that information is carried in the temporal and spatial characteristics of these signals. Furthermore, stimulation of individual cells with different agonists can generate Ca2+ oscillations with dramatically different spatial and temporal characteristics. Thus, mechanisms must exist for the acute regulation of Ca2+ release such that agonist-specific Ca2+ signals can be generated. One such mechanism by which Ca2+ signals can be modulated is through simultaneous activation of multiple second messenger pathways. For example, activation of both the InsP3 and cAMP pathways leads to the modulation of Ca2+ release through protein kinase A mediated phosphoregulation of the InsP3R. Indeed, each InsP3R subtype is a potential substrate for PKA, although the functional consequences of this phosphorylation are not clear. This review will focus on recent advances in our understanding of phosphoregulation of InsP3R, as well as the functional consequences of this modulation in terms of eliciting specific cellular events.

Animais , Canais de Cálcio/metabolismo , Citosol/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Sinalização do Cálcio , Sinalização do Cálcio/fisiologia , Técnicas de Cultura de Células , Glândula Parótida , Glândula Parótida/fisiologia , Fosforilação , Pâncreas/citologia , Pâncreas
Biol. Res ; 37(4): 665-674, 2004. ilus, graf
Artigo em Inglês | LILACS | ID: lil-437524


In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca2+-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+]I-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.

Animais , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canais de Cálcio/metabolismo , Neurônios/fisiologia , Neurônios/metabolismo , Retículo Endoplasmático/fisiologia , Citosol/fisiologia , Citosol/metabolismo , /fisiologia
Mem. Inst. Oswaldo Cruz ; 98(4): 487-493, June 2003. ilus, graf
Artigo em Inglês | LILACS | ID: lil-344240


Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1 percent while A23187 (0.5 æM) alone did not induce significant effects (17 percent); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC

Animais , Camundongos , Cálcio , Trypanosoma cruzi , Células Cultivadas , Quelantes , Citosol , Ácido Egtázico , Microscopia de Fluorescência , Sarcolema , Fatores de Tempo , Trypanosoma cruzi
Acta cient. venez ; 52(supl.1): 30-32, 2001.
Artigo em Espanhol | LILACS | ID: lil-305346


En esta revisión se muestran las ventajas y desventajas de algunos de los métodos dirigidos al diagnóstico de la tuberculosis y la identificación de micobacterias

Humanos , Masculino , Feminino , Antígenos , Citosol , Hibridização de Ácido Nucleico/métodos , Tuberculose , Medicina , Ciência , Venezuela
Braz. j. med. biol. res ; 33(11): 1387-91, Nov. 2000. ilus, graf
Artigo em Inglês | LILACS | ID: lil-273213


Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1) interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP) have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5 percent) followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia

Humanos , Feminino , Citosol/metabolismo , Endométrio/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Ciclo Menstrual/metabolismo , Marcadores de Afinidade , Autorradiografia , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Endométrio/metabolismo , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteínas de Membrana , Sensibilidade e Especificidade
Acta physiol. pharmacol. ther. latinoam ; 48(3): 125-36, 1998. ilus, tab
Artigo em Espanhol | LILACS | ID: lil-216880


El hexaclorobenceno (HCB) es un tóxico ampliamente distribuído en la biosfera. La exposición crónica de animales de laboratorio al HCB provoca disfunciones tiroideas. Previamente hemos demostrado que el HCB incrementa la actividad de enzimas hepáticas reguladas por hormonas tiroideas (HT) tales como: enzima málica (EM) y glucosa-6fosfato de dehidrogenasa (G6PD) sin alterar la actividad de la alpha-glicerol fosfato deshidrogenasa mitocondrial (alpha-GPD). En éste estudio hemos investigado si el HCB afectaba: a) la concentración del receptor de hormonas tiroideas (RT3) y su afinidad por el ligando, b) la expresión del gen de EM y de otras enzimas HT-dependientes, c) los complejos proteína/DNA formados sobre el elemento de respuesta a hormonas tiroideas (TRE). Se utilizaron hígados de ratas hembras Wistar intoxicadas con HCB (100 mg/100 g P.C.), por 9 y 15 días. El análisis de Scatchard mostró que ni la afinidad ni el número de sitios RT3 estaban alterados luego de 9 y 15 días de tratamiento con HCB (Control, Ka: 1,9 nM, Bmáx:3.9 fmol/100mug DNA; HCB9díasKa2.1nM, Bmáx4.5 fmol/100mug DNA; HCB15 días Ka 1.9nM, Bmáx5.1 fmol/100mug DNA). Tampoco los niveles de RNAm de TRbeta1 medidos por ensayos de protección a RNasa fueron afectados por HCB. Ensayos de Northern Blot han demostrado que los niveles de RNAm de EM se incrementaban 4 veces y 2 veces con respecto al control después de 9 y 15 días de intoxicación respectivamente, sin observarse alteraciones en los niveles de RNAm de otras enzimas cuya expresión es regulada por HT como gliceraldehído - 3 - fosfato deshidrogenasa (GAPDH) y fosfoenolpiruvatocarboxiquinasa (PEPCK) ni tampoco en la alpha-GPD mitocondrial. Ensayos de retardo en gel mostraron que el HCB no modificó la afinidad de las proteínas presentes en extractos nucleares por el TRE presente en el promotor de EM. Nuestros resultados sugieren que el RT3 no está involucrado en forma directa en la inducción de la expresión del gen de EM por HCB, sin embargo podría interaccionar con otros factores de transcripción en la sobreexpresión del gen de EM.

Ratos , Animais , Fungicidas Industriais/toxicidade , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hexaclorobenzeno/toxicidade , Fígado/enzimologia , Malato Desidrogenase/genética , Receptores dos Hormônios Tireóideos/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Tiroxina/farmacologia , Tri-Iodotironina/farmacologia , Northern Blotting , Citosol/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/efeitos dos fármacos , Glicerolfosfato Desidrogenase/efeitos dos fármacos , Fígado/efeitos dos fármacos , Mitocôndrias Hepáticas/enzimologia , Fosfoenolpiruvato Carboxilase/efeitos dos fármacos , Ratos Wistar , Receptores dos Hormônios Tireóideos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sensibilidade e Especificidade , Fatores de Tempo , Transcrição Genética
Braz. j. med. biol. res ; 30(7): 827-36, July 1997. graf
Artigo em Inglês | LILACS | ID: lil-197232


The influence of Ca2+ on hepatic gluconeogenesis was measured in the isolated perfused rat liver at different cytosolic NAD+-NADH potentials. Lactate and pyruvate were the gluconeogenic substrates and the cytosolic NAD+-NADH potentials were changed by varying the lactate to pyruvate rations from 0.01 to 100. The following results were obtained: a) gluconeogenesis from lactate plus pyruvate was not affected by Ca2+-free perfusion (no Ca2+ in the perfusion fluid combined with previous depletion of the intracellular pools); gluconeogenesis was also poorly dependent on the lactate to pyruvate rations in the range of 0.1 to 100; only for a ratio equal to 0.01 was a significantly smaller gluconeogenic activity observed in comparison to the other rations. b) In the presence of Ca2+, the increase in oxygen uptake caused by the infusion of lactate plus pyruvate at a ratio equal to 10 was the most pronounced one; in Ca2+-free perfusion the increase in oxygen uptake caused by lactate plus pyruvate infusion tended to be higher for all lactate to pyruvate ratios; the most pronounced difference was observed for a lactate/pyruvate ratio equal to 1.c) In the presence of Ca2+ the effects of glucagon on gluconeogenesis showed a positive correlation with the lactate to pyruvate rations; for a ratio equal to 0.01 no stimulation ocurred, but in the 0.1 to 100 range stimulation increased progressively, producing a clear parabolic dependence between the effects of glucagon and the lactate to pyruvate ratio. d) In the absence of Ca2+ the relationship between the changes caused by glucagon in gluconeogenesis and the lactate to pyruvate ratio was substantially changed; the dependence curve was no longer parabolic but sigmoidal in shape with a plateau beginning at a lactate/pyruvate ratio equal to 1; there was inhibition at the lactate to pyruvate ratios of 0.01 and 0.1 and a constant stimulation starting with a ratio equal to 1; for the lactate to pyruvate ratios of 10 and 100, stimulation caused by glucagon was much smaller than that found when Ca2+ was present. e) The effects of glucagon on oxygen uptake in the presence of Ca2+ showed a parabolic relationship with the lactate to pyruvate ratios which was closely similar to that found in the case of gluconeogenesis.

Ratos , Animais , Cálcio/fisiologia , Citosol , Glucagon/fisiologia , Gluconeogênese/fisiologia , Técnicas In Vitro , NAD/fisiologia , Oxirredução , Fígado , Perfusão