Myosin V and iNOS expression is enhanced in J774 murine macrophages treated with IFN-gamma

Actin-based motor protein requirements and nitric oxide (NO) production are important features of macrophage activity during phagocytosis or microbicidal processes. Different classes of myosins contribute directly or indirectly to phagocytosis by providing mechanical force for phagosome closure or organelle movement. Recent data have shown the presence of myosins IC, II, V and IXb in phagosomes of bone marrow-derived murine macrophages. In our investigation we demonstrated the presence of different classes of myosins in J774 macrophages. We also analyzed the effect of gamma interferon (IFN-gamma), with or without calcium ionophore or cytochalasin B, on myosins as well as on inducible nitric oxide synthase (iNOS) expression and NO production. Myosins IC, II, Va, VI and IXb were identified in J774 macrophages. There was an increase of myosin V expression in IFN-gamma-treated cells. iNOS expression was increased by IFN-gamma treatment, while calcium ionophore and cytochalasin B had a negative influence on both myosin and iNOS expression, which was decreased. The increases in NO synthesis were reflected by increased iNOS expression. Macrophages activated by IFN-gamma released significant amounts of NO when compared to control groups. In contrast, NO production by calcium ionophore- and cytochalasin B-treated cells was similar to that of control cells. These results suggest that IFN-gamma is involved in macrophage activation by stimulating protein production to permit both phagocytosis and microbicidal activity.

Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase) used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50) and alkaline incubation (pH=10.50), at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue), type IIA (oxidative-glycolytic, intermediate blue) and type IIX (glycolytic, dark blue). There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding) and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

Molecular mimicry between cardiac myosin and Trypanosoma cruzi antigen B13: identification of a B13-driven human T cell clone that recognizes cardiac myosin

Previous reports from our group have demonstrated the association of molecular mimicry between cardiac myosin and the immunodominant Trypanosoma cruzi protein B13 with chronic Chagas' disease cardiomyopathy at both the antibody and heart-infiltrating T cell level. At the peripheral blood level, we observed no difference in primary proliferative responses to T. cruzi B13 protein between chronic Chagas' cardiopathy patients, asymptomatic chagasics and normal individuals. In the present study, we investigated whether T cells sensitized by T. cruzi B13 protein respond to cardiac myosin. T cell clones generated from a B13-stimulated T cell line obtained from peripheral blood of a B13-responsive normal donor were tested for proliferation against B13 protein and human cardiac myosin. The results showed that one clone responded to B13 protein alone and the clone FA46, displaying the highest stimulation index to B13 protein (SI = 25.7), also recognized cardiac myosin. These data show that B13 and cardiac myosin share epitopes at the T cell level and that sensitization of a T cell with B13 protein results in response to cardiac myosin. It can be hypothesized that this also occurs in vivo during T. cruzi infection which results in heart tissue damage in chronic Chagas' disease cardiomyopathy

Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

Lead (Pb2+) poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g) before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM) to the bath. Changes in rate of stimulation (0.1-1.5 Hz), relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM), and the effect of isoproterenol (20 ng/mL) were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz) and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

Differential patterns of myosin Va expression during the ontogenesis of the rat hippocampus

Myosin Va is an actin-based, processive molecular motor protein highly enriched in the nervous tissue of vertebrates. It has been associated with processes of cellular motility, which include organelle transport and neurite outgrowth. The in vivo expression of myosin Va protein in the developing nervous system of mammals has not yet been reported. We describe here the immunolocalization of myosin Va in the developing rat hippocampus. Coronal sections of the embryonic and postnatal rat hippocampus were probed with an affinity-purified, polyclonal anti-myosin Va antibody. Myosin Va was localized in the cytoplasm of granule cells in the dentate gyrus and of pyramidal cells in Ammon's horn formation. Myosin Va expression changed during development, being higher in differentiating rather than already differentiated granule and pyramidal cells. Some of these cells presented a typical migratory profile, while others resembled neurons that were in the process of differentiation. Myosin Va was also transiently expressed in fibers present in the fimbria. Myosin Va was not detected in germinative matrices of the hippocampus proper or of the dentate gyrus. In conclusion, myosin Va expression in both granule and pyramidal cells showed both position and time dependency during hippocampal development, indicating that this motor protein is under developmental regulation.

Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.

Myosin light chain kinase is necessary for post-shock mesenteric lymph drainage enhancement of vascular reactivity and calcium sensitivity in hemorrhagic-shocked rats

Vascular hyporeactivity is an important factor in irreversible shock, and post-shock mesenteric lymph (PSML) blockade improves vascular reactivity after hemorrhagic shock. This study explored the possible involvement of myosin light chain kinase (MLCK) in PSML-mediated vascular hyporeactivity and calcium desensitization. Rats were divided into sham (n=12), shock (n=18), and shock+drainage (n=18) groups. A hemorrhagic shock model (40±2 mmHg, 3 h) was established in the shock and shock+drainage groups. PSML drainage was performed from 1 to 3 h from start of hypotension in shock+drainage rats. Levels of phospho-MLCK (p-MLCK) were determined in superior mesenteric artery (SMA) tissue, and the vascular reactivity to norepinephrine (NE) and sensitivity to Ca2+ were observed in SMA rings in an isolated organ perfusion system. p-MLCK was significantly decreased in the shock group compared with the sham group, but increased in the shock+drainage group compared with the shock group. Substance P (1 nM), an agonist of MLCK, significantly elevated the decreased contractile response of SMA rings to both NE and Ca2+ at various concentrations. Maximum contractility (Emax) in the shock group increased with NE (from 0.179±0.038 to 0.440±0.177 g/mg, P<0.05) and Ca2+ (from 0.515±0.043 to 0.646±0.096 g/mg, P<0.05). ML-7 (0.1 nM), an inhibitor of MLCK, reduced the increased vascular response to NE and Ca2+ at various concentrations in the shock+drainage group (from 0.744±0.187 to 0.570±0.143 g/mg in Emax for NE and from 0.729±0.037 to 0.645±0.056 g/mg in Emax for Ca2+, P<0.05). We conclude that MLCK is an important contributor to PSML drainage, enhancing vascular reactivity and calcium sensitivity in rats with hemorrhagic shock.

Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain

O-GlcNAcylation is a modification that alters the function of numerous proteins. We hypothesized that augmented O-GlcNAcylation levels enhance myosin light chain kinase (MLCK) and reduce myosin light chain phosphatase (MLCP) activity, leading to increased vascular contractile responsiveness. The vascular responses were measured by isometric force displacement. Thoracic aorta and vascular smooth muscle cells (VSMCs) from rats were incubated with vehicle or with PugNAc, which increases O-GlcNAcylation. In addition, we determined whether proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation. PugNAc enhanced phenylephrine (PE) responses in rat aortas (maximal effect, 14.2±2 vs 7.9±1 mN for vehicle, n=7). Treatment with an MLCP inhibitor (calyculin A) augmented vascular responses to PE (13.4±2 mN) and abolished the differences in PE-response between the groups. The effect of PugNAc was not observed when vessels were preincubated with ML-9, an MLCK inhibitor (7.3±2 vs 7.5±2 mN for vehicle, n=5). Furthermore, our data showed that differences in the PE-induced contractile response between the groups were abolished by the activator of AMP-activated protein kinase (AICAR; 6.1±2 vs 7.4±2 mN for vehicle, n=5). PugNAc increased phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and protein kinase C-potentiated inhibitor protein of 17 kDa (CPI-17), which are involved in RhoA/Rho-kinase-mediated inhibition of myosin phosphatase activity. PugNAc incubation produced a time-dependent increase in vascular phosphorylation of myosin light chain and decreased phosphorylation levels of AMP-activated protein kinase, which decreased the affinity of MLCK for Ca2+/calmodulin. Our data suggest that proteins that play an important role in the regulation of MLCK and MLCP activity are directly affected by O-GlcNAcylation, favoring vascular contraction.

Armazenamento de sementes de cerejas-do-mato (Eugenia involucrata) DC. submetidas ao recobrimento com biofilmes e embalagem a vácuo

Fruteiras como Eugenia involucrata ainda continuam inexploradas, necessitando-se de informações técnicas que incentivem o agricultor a utilizá-las. O método de propagação por sementes é o normalmente adotado; porém, elas devem ser imediatamente semeadas, pois corre-se o risco de perda de sua viabilidade com o armazenamento. O objetivo deste trabalho foi avaliar o hidrocondicionamento e as técnicas de conservação (vácuo e biofilme), durante armazenamento, aos cinco e aos 30 dias, de sementes de cerejeira-do-mato. O trabalho foi realizado na Universidade Tecnológica Federal do Paraná - Câmpus Dois Vizinhos. O delineamento experimental foi em blocos ao acaso, em fatorial 2 × 4 × 2 (pré-hidrocondicionamento x técnica de armazenamento x tempo de armazenamento), com quatro repetições, de 50 sementes por unidade experimental. As sementes extraídas foram separadas em dois lotes, sendo um submetido ao pré-hidrocondicionamento, em água destilada, durante 24 horas, e, outro, não. Sementes hidrocondicionadas, ou não, foram submetidas a quatro técnicas de armazenamento, sendo, estas, a embalagem plástica a vácuo, o revestimento com biofilme de fécula de mandioca (3% m/v), a embalagem plástica a vácuo + biofilme de fécula de mandioca e sem tratamento (controle). Posteriormente, as sementes foram mantidas em câmara fria, em temperatura de 5 ºC e UR 85%, durante cinco e 30 dias. Aos 110 dias após a semeadura, avaliou-se a percentagem de germinação, o índice de velocidade de emergência e a massa da matéria seca total das plântulas. Para o armazenamento das sementes de cerejeira-do-mato, devem-se utilizar técnicas a vácuo, isoladamente, ou com revestimento de biofilme.

Aspectos ecológicos da tripanossomose americana: V- observações sobre colonização espontânea de triatomíneos silvestres em ecótopos artificiais, com especial referência ao Triatoma sordida

Relatam-se os resultados obtidos com investigações destinadas a detectar o poder invasivo de triatomíneos silvestres em relação a ecótopos artificiais. Estes foram representados por galinheiros experimentais instalados em áreas habitadas por populações extradomiciliares de Triatoma sordida e T. arthurneivai. Ambos revelaram tendência invasiva, mas a capacidade de colonização no ambiente artificial só foi demonstrada pelo primeiro. As colônias obtidas de T. sordida monstraram-se bastante estáveis de acordo com a permanência da população silvestre. A presença desta última foi periodicamente detectada graças à idealização e emprego de artefatos que servissem de esconderijos tipo "calha". As observações no ambiente natural confirmam a ação favorável que as alterações nele introduzidas exercem sobre a densidade desse triatomíneo. Assim sendo, é de se prever que ele venha a invadir e ocupar com relativa rapidez os nichos ecológicos domiciliares deixados vagos pela eliminação de outros triatomíneos.

Bioquímica da esquistossomose mansônica: V - atividade mitocondrial em fígados e rins de sagüis (Callitrix penicillata) infestados pelo Schistosoma mansoni

Foram estudadas mitocôndrias isoladas de fígados e rins de sagüis (Callitrix penicillata) infestados pela inoculação de 200 cercárias e entre 80 e 100 dias de infecção, nos aspectos relativos às suas atividades respiratórias endógenas, bem como frente ao succinato e ao alfa ceto glutarato de sódio. Cada experiência foi acompanhada de controle, usando-se mitocôndrias isoladas de fígados e rins de animais não infectados. As medidas das atividades respiratórias foram efetuadas polarograficamente e expressas em microlitros de oxigênio consumidos por miligrama de proteínas totais por minuto. Os resultados mostraram que as respirações endógenas das mitocôndrias isoladas dos animais infectados foram sempre maiores do que aquelas observadas nos controles. Detectou-se um estímulo para o fígado de 217% e para o rim de 84%. O succinato de sódio estimulou em 85% a respiração das mitocôndrias dos fígados dos animais controles, enquanto a inibiu de 39% nos infectados. Com referência aos rins, este mesmo substrato estimulou a referida respiração tanto nos controles quanto nos infectados, em 89% e 94% respectivamente. O alfa ceto glutarato estimulou as mitocôndrias hepáticas isoladas dos controles em 48% e as renais em 84%. Nos animais esquistossomóticos ou ele não modificou a capacidade respiratória mitocondrial como se observou para o rim, ou a inibiu de 58%, no caso do fígado. Os dados obtidos sugerem que o fígado sofre muito mais, em termos bioquímicos, com a esquistossomose mansônica do que os rins, pelo menos em nossas condições experimentais. As atividades do sistema enzimático succinato desidrogenase e do complexo alfa ceto glutarato desidrogenase permitiram identificar bioquimicamente, em nossos animais esquistossomóticos, vários graus de lesão celular, principalmente hepáticos, que vão desde os mais simples, decorrentes de modificações da físico-química dos sistemas de membranas, até os mais graves, tipo necrose.

Estudo das condições de saúde das crianças no Município de São Paulo, SP (Brasil), 1984-1985: V - Anemia

Dentro de estudo populacional sobre condições de saúde na infância, uma amostra representativa das crianças menores de cinco anos do Município de São Paulo, Brasil (n = 912) foi estudada quanto à prevalência e distribuição da anemia. A freqüência de crianças anêmicas (concentração de hemoglobina < 11g/dl) foi de 35,6%, sendo de 14,7% a freqüência de casos severos (hemoglobina < 9,5g/dl). As maiores prevalências de anemia se concentraram na faixa etária de 6 a 24 meses de idade, onde mais da metade das crianças se mostraram anêmicas e cerca de metade dos casos corresponderam a formas severas da deficiência. Embora a prevalência da anemia tenda a diminuir com a melhoria do nível socioeconômico da população, não se detectaram estratos da população imunes à deficiência. Em comparação à situação observada no Município há cerca de dez anos, verifica-se elevação expressiva da prevalência da anemia: mais de 50% para o total de casos e mais de 100% para as formas severas da deficiência. O conjunto das informações geradas pelo estudo epidemiológico realizado em 1984/85 sugere que a anemia na infância no Município de São Paulo esteja relacionada basicamente a características desfavoráveis da alimentação infantil, sendo menos relevante o papel que poderia ser atribuído às parasitoses intestinais.

Simuliidae (Diptera: Culicomorpha) no Brasil: V - Sobre o Simulium (Chirostilbia) friedlanderi sp.n. e a revisão do Simulium (C.) laneportoi Vargas, 1941

Uma espécie nova, Simulium friedlanderi, do subgênero Simulium (Chirostilbia) Enderlein é descrita, sendo a mesma procedente do norte da Província Hidrogeológica do Centro-Oeste, subprovíncia Alto Paraguai. Simulium (Chirostilbia) laneportoi Vargas, 1941 é revisada, sendo o Simulium (C.) dekeyseri Shelley e Py-Daniel, 1981 colocada em sua sinonimia, e apresentando uma distribuição geográfica tanto no norte da Província Hidrogeológica do Centro-Oeste, subprovíncia Alto Paraguai, como no sudeste da Província Hidrogeológica do Escudo Central. São apresentadas chaves diferenciativas para larvas e pupas do grupo pertinax.