RESUMO

The S. mansoni adult worm n-butanol extract (Sm-AWBE) has been previously shown to contain specific S. mansoni antigens that have been used for immunodiagnosis of schistosomiasis in solid phase alkaline phosphatase immunoassay (APIA) and western blot (WB) analyses. Sm-AWBE was also used in immunoprotection studies against a fatal live-cercariae challenge in experimental mouse vaccination (~43% protection). The Sm-AWBE fraction was prepared by mixing adult worm membranous suspensions with aqueous-saturated n-butanol, centrifuging and recovering n-butanol-resistant proteins in the aqueous phase. Here we report a preliminary identification of Sm-AWBE protein components as revealed from a qualitative proteomic study after processing Sm-AWBE by 1D-gel electrophoresis, in-gel and in-solution tryptic digestions, and mass spectrometry analyses. We identified 33 proteins in Sm-AWBE, all previously known S. mansoni proteins and antigens; among them, immunomodulatory proteins and proteins mostly involved in host-parasite interactions. About 81.8% of the identified Sm-AWBE proteins are antigenic. STRING analysis showed a set of Sm-AWBE proteins configuring a small network of interactive proteins and a group of proteins without interactions. Functional groups of proteins included muscle contraction, antioxidant, GPI-anchored phosphoesterases, regulatory 14-3-3, various enzymes and stress proteins. The results widen the possibilities to design novel antigen combinations for better diagnostic and immunoprotective strategies for schistosomiasis control.

RESUMO

Tarantula's leg muscle thick filament is the ideal model for the study of the structure and function of skeletal muscle thick filaments. Its analysis has given rise to a series of structural and functional studies, leading, among other things, to the discovery of the myosin interacting-heads motif (IHM). Further electron microscopy (EM) studies have shown the presence of IHM in frozen-hydrated and negatively stained thick filaments of striated, cardiac, and smooth muscle of bilaterians, most showing the IHM parallel to the filament axis. EM studies on negatively stained heavy meromyosin of different species have shown the presence of IHM on sponges, animals that lack muscle, extending the presence of IHM to metazoans. The IHM evolved about 800 MY ago in the ancestor of Metazoa, and independently with functional differences in the lineage leading to the slime mold Dictyostelium discoideum (Mycetozoa). This motif conveys important functional advantages, such as Ca2+ regulation and ATP energy-saving mechanisms. Recent interest has focused on human IHM structure in order to understand the structural basis underlying various conditions and situations of scientific and medical interest: the hypertrophic and dilated cardiomyopathies, overfeeding control, aging and hormone deprival muscle weakness, drug design for schistosomiasis control, and conditioning exercise physiology for the training of power athletes.

RESUMO

Adult Schistosoma mansoni parasites have the capacity to degrade ingested host hemoglobin and other host plasma proteins by using a series of gut proteolytic enzymes, including cathepsin B; this enzyme is released to the host intravascular environment during regurgitations of adult worms. Cathepsin B becomes thus a circulating parasite component that has been shown to be specifically recognized as the Sm31 antigen by antibodies present in most S. mansoni infected patients. Taking advantage of this immunological property, we attempted here to immunocapture Sm31 from sera of infected patients using specific polyclonal rabbit antibodies raised against a highly enriched preparation of Sm31 and detect its intrinsic proteolytic activity using a previously described solid-phase procedure called Cysteine Protease Immuno Assay (CPIA). To produce highly specific anti-Sm31/cathepsin B antibodies, cathepsin B (Sm31 or SmCB) was enriched more than 3000-folds from an adult worm preparation using a series of conventional biochemical steps including ion exchange and affinity chromatography. Anti-cathepsin B antibodies were generated by immunizing rabbits with the enriched cathepsin B fraction; these antibodies recognized a band of Mr.~31 kDa in Western-blot (WB) analysis of this fraction and were able to capture, in a modified CPIA procedure, Sm31/SmCB present in sera from infected Venezuelan patients living in low endemic areas for schistosomiasis. CPIA showed 100% sensitivity and 100% specificity; representing a new diagnostic tool to detect circulating Sm31 antigen in actual infections.

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RESUMO

BACKGROUND: Schistosomiasis continues to be one of the most prevalent parasitic diseases in the world. Despite the existence of a highly effective antischistosome drug, the disease is spreading into new areas, and national control programs do not arrive to complete their tasks particularly in low endemic areas. The availability of a vaccine could represent an additional component to chemotherapy. Experimental vaccination studies are however necessary to identify parasite molecules that would serve as vaccine candidates. In the present work, C57BL/6 female mice were subcutaneously immunized with an n-butanol extract of the adult worm particulate membranous fraction (AWBE) and its protective effect against a S. mansoni challenge infection was evaluated. METHODOLOGY AND FINDINGS: Water-saturated n-butanol release into the aqueous phase a set of membrane-associated (glyco)proteins that are variably recognized by antibodies in schistosome-infected patients; among the previously identified AWBE antigens there is Alkaline Phosphatase (SmAP) which has been associated with resistance to the infection in mice. As compared to control, a significantly lower number of perfuse parasites was obtained in the immunized/challenged mouse group (P<0.05, t test); and consequently, a lower number of eggs and granulomas (with reduced sizes), overall decreasing pathology. Immunized mice produced high levels of sera anti-AWBE IgG recognizing antigens of ∼190-, 130-, 98-, 47-, 28-23, 14-, and 9-kDa. The ∼130-kDa band (the AP dimer) exhibited in situ SmAP activity after addition of AP substrate and the activity was not apparently inhibited by host antibodies. A preliminary proteomic analysis of the 25-, 27-, and 28-kDa bands in the immunodominant 28-23 kDa region suggested that they are composed of actin. CONCLUSIONS: Immunization with AWBE induced the production of specific antibodies to various adult worm membrane molecules (including AP) and a partial (43%) protection against a challenging S. mansoni infection by mechanism(s) that still has to be elucidated.

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Myosin filaments from many muscles are activated by phosphorylation of their regulatory light chains (RLCs). To elucidate the structural mechanism of activation, we have studied RLC phosphorylation in tarantula thick filaments, whose high-resolution structure is known. In the relaxed state, tarantula RLCs are ~50% non-phosphorylated and 50% mono-phosphorylated, while on activation, mono-phosphorylation increases, and some RLCs become bi-phosphorylated. Mass spectrometry shows that relaxed-state mono-phosphorylation occurs on Ser35, while Ca(2+)-activated phosphorylation is on Ser45, both located near the RLC N-terminus. The sequences around these serines suggest that they are the targets for protein kinase C and myosin light chain kinase (MLCK), respectively. The atomic model of the tarantula filament shows that the two myosin heads ("free" and "blocked") are in different environments, with only the free head serines readily accessible to kinases. Thus, protein kinase C Ser35 mono-phosphorylation in relaxed filaments would occur only on the free heads. Structural considerations suggest that these heads are less strongly bound to the filament backbone and may oscillate occasionally between attached and detached states ("swaying" heads). These heads would be available for immediate actin interaction upon Ca(2)(+) activation of the thin filaments. Once MLCK becomes activated, it phosphorylates free heads on Ser45. These heads become fully mobile, exposing blocked head Ser45 to MLCK. This would release the blocked heads, allowing their interaction with actin. On this model, twitch force would be produced by rapid interaction of swaying free heads with activated thin filaments, while prolonged exposure to Ca(2+) on tetanus would recruit new MLCK-activated heads, resulting in force potentiation.

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El metabolismo oxidativo es el mecanismo de mayor relevancia involucrado en la producción de energía en los animales superiores. La falta de control en este proceso conduce a una sobreproducción de radicales libres de oxígeno, los cuales pueden ocasionar daño a las estructuras celulares. El malondialdehído (MDA) es un marcador de la peroxidación lipídica, cuya producción puede ser compensada por el óxido nítrico (NO), vitamina C y glutatión reducido (GSH) como integrantes del sistema antioxidante del organismo. El objetivo de este estudio fue establecer la influencia de la edad y el sexo en el sistema oxidante/antioxidante en un grupo de sujetos sanos. El MDA fue determinado como producto del ácido tiobarbitúrico, el NO como nitritos totales, la vitamina C oxidada y reducida, utilizando el método de Scharz y Williams y el GSH mediante kit colorimétrico del GSH-400 Assaytm. No se observaron diferencias significativas en las concentraciones de oxidante (MDA), así como tampoco en el sistema antioxidante (NO, vitamina C, GSH) cuando se compararon sujetos de 13 a 19 años con sujetos de 20 a 38 años. Sólo se encontró diferencia significativa al analizar los valores de NO entre sexos, presentando las mujeres un valor significativamente superior (p<0,012). Estos resultados sugieren que el MDA, NO, vitamina C y GSH no cambian en el intervalo de edad estudiado (13-38 años) y que la concentración de NO, sólo se ve afectada por el sexo

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