RESUMEN
Trypanosomatids are a monophyletic group of parasitic flagellated protists belonging to the order Kinetoplastida. Their cytoskeleton is primarily made up of microtubules in which no actin microfilaments have been detected. Although all these parasites contain actin, it is widely thought that their actin cytoskeleton is reduced when compared to most eukaryotic organisms. However, there is increasing evidence that it is more complex than previously thought. As in other eukaryotic organisms, trypanosomatids encode for a conventional actin that is expected to form microfilament-like structures, and for members of three conserved actin-related proteins probably involved in microfilament nucleation (ARP2, ARP3) and in gene expression regulation (ARP6). In addition to these canonical proteins, also encode for an expanded set of actins and actin-like proteins that seem to be restricted to kinetoplastids. Analysis of their amino acid sequences demonstrated that, although very diverse in primary sequence when compared to actins of model organisms, modelling of their tertiary structure predicted the presence of the actin fold in all of them. Experimental characterization has been done for only a few of the trypanosomatid actins and actin-binding proteins. The most studied is the conventional actin of Leishmania donovani (LdAct), which unusually requires both ATP and Mg2+ for polymerization, unlike other conventional actins that do not require ATP. Additionally, polymerized LdAct tends to assemble in bundles rather than in single filaments. Regulation of actin polymerization depends on their interaction with actin-binding proteins. In trypanosomatids, there is a reduced but sufficient core of actin-binding proteins to promote microfilament nucleation, turnover and stabilization. There are also genes encoding for members of two families of myosin motor proteins, including one lineage-specific. Homologues to all identified actin-family proteins and actin-binding proteins of trypanosomatids are also present in Paratrypanosoma confusum (an early branching trypanosomatid) and in Bodo saltans (a closely related free-living organism belonging to the trypanosomatid sister order of Bodonida) suggesting they were all present in their common ancestor. Secondary losses of these genes may have occurred during speciation within the trypanosomatids, with salivarian trypanosomes having lost many of them and stercorarian trypanosomes retaining most.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/química , Proteínas de Microfilamentos/química , Miosinas/química , Proteínas Protozoarias/química , Trypanosomatina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/clasificación , Actinas/genética , Actinas/metabolismo , Animales , Sitios de Unión , Expresión Génica , Humanos , Proteínas de Microfilamentos/clasificación , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Miosinas/clasificación , Miosinas/genética , Miosinas/metabolismo , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosomatina/clasificación , Trypanosomatina/genéticaRESUMEN
Cytokinin forchlorfenuron (FCF), a synthetic cytokinin, has been used specifically for the characterization of septins. In spite of genomic evidence of their existence, nothing is known about septin filaments in taeniid cestodes. The aim of this work was to determine the presence of a septin-like protein in cysticerci of Taenia crassiceps and Taenia solium using the deduced amino acid sequence of T. solium septin 4 (SEPT4_Tsm), to design and synthesize a derived immunogenic peptide (residues 88 to 103), to prepare a specific rabbit polyclonal antibody, and to examine the effects of FCF at different concentrations and exposure times on an in vitro culture of T. crassiceps cysticerci. In vitro, FCF altered the morphology and motility of T. crassiceps cysticerci, and its effects were reversible under specific concentrations. In addition, we observed by ultrastructural observation that FCF alters the cellular subunit of the protonephridial system of cestodes, where disruption of the axoneme pattern of flame cells was observed. The rabbit polyclonal antibody prepared against the synthetic peptide recognized a major band of 41 kDa in both parasites. Our results establish the importance of SEPT4_Tsm in the dynamics and survival of taeniid cysticerci, as well as their susceptibility to FCF. This is also the first report that a septin is present in the cytoskeleton of taeniids.
RESUMEN
Mesenchymal stem cells isolated from different tissues should share associated markers and the capability to differentiate to mesodermal lineages. However, their behavior varies in specific microenvironments. Herein, adhesion and fibrinolytic activity of mesenchymal stem cells from placenta, bone marrow, and Wharton's jelly were evaluated in fibrin hydrogels prepared with nonpurified blood plasma and compared with two-dimensional cultures. Despite the source, mesenchymal stem cells adhered through focal adhesions positive for vinculin and integrin αV in two dimensions, while focal adhesions could not be detected in fibrin hydrogels. Moreover, some cells could not spread and stay rounded. The proportions of elongated and round phenotypes varied, with placenta mesenchymal stem cells having the lowest percentage of elongated cells (~10%). Mesenchymal stem cells degraded fibrin at distinct rates, and placenta mesenchymal stem cells had the strongest fibrinolytic activity, which was achieved principally through the plasminogen-plasmin axis. These findings might have clinical implications in tissue engineering and wound healing therapy.
RESUMEN
Resumen El virus sincitial respiratorio humano (VSRh) es considerado como el principal agente causal de infecciones del tracto respiratorio en niños. Su presentación clínica varía en cuanto a la gravedad: desde infecciones no complicadas de la vía aérea superior en adultos y niños sanos, hasta bronquiolitis y bronconeumonía en niños con factores de riesgo y menores de 2 años. Perteneciente a la familia Pneumoviridae y al género Orthopneumovirus, el VSRh es un virus envuelto que contiene un genoma de ácido ribonucleico (RNA) monocatenario de polaridad negativa, que codifica para 7 proteínas estructurales (G, F, SH, M, P, N y L) y 4 no estructurales (NS1, NS2, M1, M2). La presencia del virus se ha considerado como factor de riesgo para el desarrollo de asma infantil, que es una enfermedad inflamatoria de la vía aérea caracterizada por episodios recurrentes de obstrucción de la vía aérea inferior ante estímulos ambientales generalmente inocuos. El riesgo de desarrollar asma aumenta si la primoinfección sucede a edad temprana y si hay factores de riesgo como prematuridad y broncodisplasia pulmonar. En México, debido a la morbilidad y mortalidad asociada al VSRh, y como profilaxis en pacientes de alto riesgo; desde el año 2008, se recomienda el uso del biofármaco Pavilizumab. El objetivo de la presente revisión es describir los factores asociados a la patogénesis VSRh que podrían estar implicados en el desarrollo del asma infantil y, con ello, plantear que población está en riesgo. Para estos fines, se presenta un breve análisis de la biología del virus, la respuesta inmune que se induce durante la infección, así como aquellos fármacos aprobados en México para el tratamiento y profilaxis de infecciones asociadas al VRSh.
Abstract The human respiratory syncytial virus (hRSV) is the main pathogen of respiratory tract infections in children. The severity of the infection is depending of its clinical presentation that is moving from uncomplicated upper airway infections, in healthy adults and children, to bronchiolitis and bronchopneumonia that could be developed, in presence of risk factors, in children younger than 2 years. The virus belongs to the Pneumoviridae family and Orthopneumovirus genus, it is an enveloped virus with a single-stranded RNA genome of negative polarity that is codifying 7 structural proteins (G, F, SH, M, P, N and L) and four non-structural proteins (NS1, NS2, M1, M2). The viral infection has been considered as a risk factor for the development of childhood asthma, which is the most common airway inflammatory disease in children and characterized, by recurrent episodes of lower airway obstruction, by harmless environmental stimuli. The risk increases if primary infection occurs at an early age and in risk factors as prematurity and pulmonary broncho-dysplasia. Due to the morbidity and mortality associated with hRSV, since 2008 it has been approved the use of biopharmaceuticals as Palivizumab for prophylaxis in high-risk patients. In the present review, the aim is to present those factors that could be involved in the development of childhood asthma and their possible link to the presence of hRSV. In addition, it is an intention for presenting the possible facts of the risks in the potentially infected population. For a better comprehension of the virus, it is presented a briefly analysis of the viral structure, the induced immune response against the viral infection and those drugs that are approved in Mexico for the treatment and prophylaxis against hRSV.
RESUMEN
Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.
Asunto(s)
Bencimidazoles/farmacología , Citoesqueleto/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Actinas/aislamiento & purificación , Flagelos/efectos de los fármacos , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/ultraestructura , Tubulina (Proteína)/aislamiento & purificaciónRESUMEN
Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.
Asunto(s)
Bencimidazoles/farmacología , Citoesqueleto/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacos , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Actinas/aislamiento & purificación , Flagelos/efectos de los fármacos , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/ultraestructura , Tubulina (Proteína)/aislamiento & purificaciónRESUMEN
We examined the effects of oestradiol (E2) and progesterone (P4) on cytoskeletal protein expression in the helminth Taenia crassiceps - specifically actin, tubulin and myosin. These proteins assemble into flame cells, which constitute the parasite excretory system. Total protein extracts were obtained from E2- and P4-treated T. crassiceps cysticerci and untreated controls, and analysed by one- and two-dimensional protein electrophoresis, flow cytometry, immunofluorescence and videomicroscopy. Exposure of T. crassiceps cysticerci to E2 and P4 induced differential protein expression patterns compared with untreated controls. Changes in actin, tubulin and myosin expression were confirmed by flow cytometry of parasite cells and immunofluorescence. In addition, parasite morphology was altered in response to E2 and P4 versus controls. Flame cells were primarily affected at the level of the ciliary tuft, in association with the changes in actin, tubulin and myosin. We conclude that oestradiol and progesterone act directly on T. crassiceps cysticerci, altering actin, tubulin and myosin expression and thus affecting the assembly and function of flame cells. Our results increase our understanding of several aspects of the molecular crosstalk between host and parasite, which might be useful in designing anthelmintic drugs that exclusively impair parasitic proteins which mediate cell signaling and pathogenic reproduction and establishment.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Progesterona/farmacología , Taenia/clasificación , Taenia/citología , Animales , Células Cultivadas , Proteínas del Citoesqueleto/genética , Ratones , Ratones Endogámicos BALB CRESUMEN
Cysticercosis, caused by the larval stage of Taenia solium, is a zoonotic disease affecting pigs and humans that is endemic to developing countries in Latin America, Africa and South East Asia. The prevalence of infection in pigs, the intermediate host for T. solium, has been used as an indicator for monitoring disease transmission in endemic areas. However, accurate and specific diagnostic tools for porcine cysticercosis remain to be established. Using proteomic approaches and the T. solium genome sequence, seven antigens were identified as specific for porcine cysticercosis, namely, tropomyosin 2, alpha-1 tubulin, beta-tubulin 2, annexin B1, small heat-shock protein, 14-3-3 protein, and cAMP-dependent protein kinase. None of these proteins were cross-reactive when tested with sera from pigs infected with Ascaris spp., Cysticercus tenuicollis and hydatid cysts of Echinococcus spp. or with serum from a Taenia saginata-infected cow. Comparison with orthologues, indicated that the amino acid sequences of annexin B1 and cAMP-dependent protein kinase possessed highly specific regions, which might make them suitable candidates for development of a specific diagnostic assay for porcine cysticercosis.
Asunto(s)
Cisticercosis/diagnóstico , Electroforesis en Gel Bidimensional/métodos , Immunoblotting/métodos , Enfermedades de los Porcinos/diagnóstico , Taenia solium/aislamiento & purificación , Animales , Antígenos de Protozoos/sangre , Cisticercosis/parasitología , Electroforesis en Gel Bidimensional/veterinaria , Immunoblotting/veterinaria , Proteómica/métodos , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Porcinos , Enfermedades de los Porcinos/parasitología , Taenia solium/inmunologíaRESUMEN
Bovine bone matrix Nukbone® (NKB) is an osseous tissue-engineering biomaterial that retains its mineral and organic phases and its natural bone topography and has been used as a xenoimplant for bone regeneration in clinics. There are not studies regarding its influence of the NKB in the behavior of cells during the repairing processes. The aim of this research is to demonstrate that NKB has an osteoinductive effect in human mesenchymal stem cells from amniotic membrane (AM-hMSCs). Results indicated that NKB favors the AM-hMSCs adhesion and proliferation up to 7 days in culture as shown by the scanning electron microscopy and proliferation measures using an alamarBlue assay. Furthermore, as demonstrated by reverse transcriptase polymerase chain reaction, it was detected that two gene expression markers of osteoblastic differentiation: the core binding factor and osteocalcin were higher for AM-hMSCs co-cultured with NKB in comparison with cultivated cells in absence of the biomaterial. As the results indicate, NKB possess the capability for inducing successfully the osteoblastic differentiation of AM-hMSC, so that, NKB is an excellent xenoimplant option for repairing bone tissue defects.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Andamios del Tejido , Amnios , Animales , Bovinos , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Helminth ß-tubulins are the targets of benzimidazole (BZM) carbamate compounds. The specificity of the interactions between such compounds and their in vivo targets depends on the presence of specific amino acid residues in the target molecules. To discover new and effective anthelmintic drugs, we used a medicinal chemistry approach to synthesize a series of BZM derivatives that exploited the BZM moiety as a template. We have previously found that one compound, 2-(trifluoromethyl)-1H-benzimidazole (RCB20), has better in vitro and in vivo activity than albendazole sulfoxide (ABZSO). In the present study, the effect of RCB20 and ABZSO treatment on expression of Taenia crassiceps cysticerci cytoskeletal proteins such as actin, myosin II, and tubulin isoforms was examined. The effects of RCB20 and ABZSO after 11 days treatment of the parasites was evaluated by light, confocal, and electron microscopy, and by immunochemistry and immunohistochemistry. The RCB20-induced effects were more rapid than the ABZSO-induced effects on the parasites. In the RCB20-treated parasites, we observed gross-structural damage at the whole parasite level, particularly in the inner tissues and flame cells. Changes in the expression patterns of the cytoskeletal proteins, as assessed by immunohistochemistry and immunoblotting, revealed that the most important drug-induced effect on the parasites was a reduction in the expression level of tyrosinated α-tubulins. Our research findings suggest that RCB20 treatment affected posttranslational modification of parasite α-tubulin molecules, which involved removal of the α-tubulin carboxy-terminal tyrosine.
Asunto(s)
Antihelmínticos/farmacología , Bencimidazoles/farmacología , Expresión Génica/efectos de los fármacos , Taenia/efectos de los fármacos , Tubulina (Proteína)/biosíntesis , Actinas/biosíntesis , Albendazol/análogos & derivados , Albendazol/farmacología , Animales , Cysticercus/anatomía & histología , Cysticercus/efectos de los fármacos , Inmunoquímica , Microscopía , Miosina Tipo II/biosíntesis , Taenia/anatomía & histologíaRESUMEN
Albendazole and mebendazole are widely used in the treatment of trichinellosis; however, chemotherapy failure has been reported. In an effort to develop new anthelminthic compounds, we examined a previously synthesized 2-(trifluoromethyl)-1H-benzimidazole derivative (1) that showed good in vitro activity against Trichinella spiralis muscle larvae but low in vivo efficacy. In order to improve the solubility of compound 1, an inclusion complex with 2-hydroxypropyl-ß-cyclodextrin (1/HP-ßCD) was prepared. When 1/HP-ßCD was tested in vivo, it significantly reduced the ML burden (84%). In addition, a proteomic analysis of T. spiralis ML treated with 1 revealed significant changes in the expression levels of proteins involved in energy metabolism and the cytoskeleton of the parasite. Compound (1) also induced extensive ultrastructural changes in the cuticle, hypodermis and midgut of the parasite.
Asunto(s)
Antihelmínticos/farmacología , Bencimidazoles/farmacología , Trichinella spiralis/efectos de los fármacos , Triquinelosis/parasitología , beta-Ciclodextrinas/farmacología , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Electroforesis en Gel Bidimensional , Regulación de la Expresión Génica/efectos de los fármacos , Larva , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Músculos/parasitología , Proteómica , Trichinella spiralis/ultraestructura , Triquinelosis/tratamiento farmacológicoRESUMEN
The trypanocidal effect of five benzimidazole derivatives (1-5) was determined in vitro and in vivo assays against two strains of Trypanosoma cruzi (NINOA and INC5). The in vitro trypanocidal activity was evaluated by measuring the percentage of lysis of bloodstream trypomastigotes of T. cruzi. Results point to 5-chloro-1H-benzimidazole-2-thiol (1) as the best activity profile compound with a 50% lytic concentration (LC(50)) of 0.014 mM (NINOA strain) and 0.32 mM (INC5 strain). Reference drugs were nifurtimox (Nfx) and benznidazole (Bnz), which on NINOA strain displayed a LC(50)=0.60 mM and LC(50)=0.78 mM, respectively; while on INC5 strain they exhibited LC(50) values of 0.31 mM and 0.69 mM, respectively. The in vivo trypanocidal activity of 1-5 on parasitemia in a murine model acute Chagas' disease indicated that 1 and Nfx showed similar activity on INC5 strain, while 5-chloro-1-methyl-1H-benzimidazole-2-thiol (2) and its regioisomer, 6-chloro-1-methyl-1H-benzimidazole-2-thiol (3), displayed better activity than Nfx and Bnz on NINOA strain. All compounds showed low cytotoxicity against Vero cells, with selective index 38-3000 times higher to the parasite.
Asunto(s)
Antiprotozoarios/administración & dosificación , Antiprotozoarios/farmacología , Bencimidazoles/administración & dosificación , Bencimidazoles/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Trypanosoma cruzi/efectos de los fármacos , Animales , Antiprotozoarios/química , Antiprotozoarios/toxicidad , Bencimidazoles/química , Bencimidazoles/toxicidad , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Parasitemia/tratamiento farmacológico , Pruebas de Sensibilidad Parasitaria , Resultado del Tratamiento , Células VeroRESUMEN
No existen, hasta el momento, imágenes que muestren la disposición de la citoarquitectura de parásitos adultos de Taenia solium, parásitos los cuales se encuentran en el intestino de portadores humanos asintomáticos. Las causas de ello podrían tener como base el que cuando se recuperan los parásitos, ellos han sufrido alteraciones debidas a la respuesta inmune de sus hospederos o bien, por el efecto que han producido en los parásitos los fármacos antihelmínticos que hayan sido usados en el tratamiento de los pacientes. Una de las alternativas que se han encontrado para la obtención de parásitos adultos, es la obtención de tenias a partir del modelo de teniosis experimental en hámsteres dorados e inmunosuprimidos y que gracias a este modelo se han podido efectuar diferentes tipos de estudios de los parásitos de esta fase infectiva. El propósito de este reporte es presentar imágenes de ultraestructura, obtenidas mediante Microscopía Electrónica de Barrido, de un corte transversal obtenido de un proglótido de una tenia recuperada de una infección experimental. Las imágenes se obtuvieron a diferentes aumentos y muestran aspectos relacionados con la superficie tegumentaria, el tegumento sincicial continuo, la capa germinal que incluye el soma de algunas células subtegumentarias y los ductos del sistema protonefridial tanto vacíos como llenos con corpúsculos calcáreos. Las imágenes ultraestructurales obtenidas muestran una forma de observación de la anatomía microscopica de los parásitos en estudio y ello contribuye a ampliar el conocimiento de los mismos en relación a aspectos de su biología celular y su fisiología.
There are no clear morphological evidences of the cytoarchitecture of intestinal adult tapeworms of Taenia solium recovered from infected humans. Parasites could be altered because of the host´s immunological response or by the direct action of drugs used for antihelminthic treatment. Experimental taeniosis in immunosuppressed golden hamsters is a useful way for recovering and studying adult parasites. The purpose of this report is to show images, taken at the ultrastructural level by Scanning Electron Microscopy (SEM), of a cross-sectioned strobilar chain from an adult tapeworm. The parasite was recovered from an experimental infection. Images were taken at several magnifications; they show the brush border tegumental surface, the syncytial tegument, the germinal layer, some cell bodies and the protonephridial system ducts: empty or filled with calcareous corpuscles. Ultrastructural images taken using SEM of T. solium adult parasites, recovered from experimental infections, could be a new way for observing the microscopic anatomy of these parasites and for increasing the knowledge of aspects related to their cellular biology and physiology.
Asunto(s)
Animales , Taenia solium/anatomía & histología , Taenia solium/citología , Taenia solium/microbiología , Taenia solium/parasitología , Taenia solium/ultraestructura , Microscopía Electrónica de Rastreo/métodosRESUMEN
The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T.cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids.
Asunto(s)
Actinas/metabolismo , Trypanosoma cruzi/metabolismo , Células 3T3 , Actinas/análisis , Actinas/genética , Actinas/inmunología , Animales , Anticuerpos Antiprotozoarios/inmunología , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica , Ratones , Microscopía Confocal , Filogenia , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/inmunologíaRESUMEN
Using a murine model of cysticercosis caused by the Taenia crassiceps ORF strain, we developed a fluorescent quantitative evaluation of the action of two well known anti-helminthic drugs: albendazole sulfoxide and praziquantel. The fluorescence emitted by a biotransformed CellTracker Probe known as CellTracker Green CMFDA in the vesicular fluids of cysticerci was estimated, and the results were compared with macroscopic observations of the parasites. The pharmacological EC(50) value of each drug and changes in the level of biotransformation of the fluorescent tracker caused by the drugs could be easily calculated. These drug-induced changes in biotransformation could be related to changes in the GSH/GSSG ratio of parasites. Both the cysticercosis murine model and the CMFDA biotransformation assay could be used as an in vitro screening method to evaluate potential or well known cysticidal drugs.
Asunto(s)
Albendazol/análogos & derivados , Antihelmínticos/farmacología , Cysticercus/efectos de los fármacos , Fluoresceínas , Colorantes Fluorescentes , Praziquantel/farmacología , Albendazol/farmacología , Animales , Biotransformación , Cysticercus/metabolismo , Femenino , Fluoresceínas/farmacocinética , Colorantes Fluorescentes/farmacocinética , Ratones , Ratones Endogámicos BALB C , Microscopía FluorescenteRESUMEN
Type II myosin, the primary component of the thick filament of muscle fibers, is organized as a dimeric high molecular weight protein, and is composed of a pair of heavy chains (MHC) and two pairs of light chains. Myosin II transforms ATP energy into mechanical force. All type II myosins are conserved proteins but they have two variable regions that are located in different places of the molecule. Myosin molecules are encoded by a multigene family and many isoforms are generated. The expression of myosins depends on the developmental stage and on the type and degree of contractile activity and tissue, therefore several myosin isoforms are found in the same organism. Here we describe the use of different techniques that allowed demonstrating the presence of isoforms of the heavy chain type II myosin of Taenia solium cysticerci (larvae) and tapeworms (adults), a cestode parasite of importance in public health in many developing countries. Myosin was purified and used in comparative proteolytic fragmentation, ATPase activity, detection of antigenic differences and electrophoretic separation. The results obtained showed biochemical and immunochemical differences among cysticerci and tapeworms, and demonstrate the presence of myosin isoforms in T. solium that are probably associated to physiological requirements of each developmental stage.
Asunto(s)
Fibras Musculares Esqueléticas/química , Músculos/química , Cadenas Pesadas de Miosina/química , Miosina Tipo II/química , Taenia solium/química , Taenia solium/crecimiento & desarrollo , Adenosina Trifosfatasas/metabolismo , Animales , Antígenos/inmunología , Epítopos/inmunología , Larva/química , Larva/crecimiento & desarrollo , Larva/inmunología , Fibras Musculares Esqueléticas/inmunología , Músculos/inmunología , Cadenas Pesadas de Miosina/inmunología , Cadenas Pesadas de Miosina/aislamiento & purificación , Miosina Tipo II/inmunología , Miosina Tipo II/aislamiento & purificación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Péptido Hidrolasas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Porcinos , Taenia solium/inmunologíaRESUMEN
Identification, localization and partial biochemical characterization of actins expressed in the larval stage of the cestode parasite Taenia solium has been carried out. Frozen tissue sections of cysticerci, the larval stage of this parasite, were reacted with rhodamine-phalloidin, parasite actin was purified by polymerization in the presence of K(+), Mg(++) and ATP actin was analyzed by SDS-PAGE and two-dimensional gel electrophoresis, and immunoblotting of actin was performed in PVDF membranes and with commercial anti-actin monoclonal antibodies. Parasitic tissues showed different fibrous actin fluorescence patterns, which correlated with the expression of isoactins. Purified globular actin had a similar molecular mass to rabbit commercial actin (approximately 44 kDa). Actin was resolved into seven isoforms, indicating a family of actin genes.