RESUMEN

Cyclophilin A (CyPA, also known as PPIA) is an abundant and ubiquitously expressed protein belonging to the immunophilin family, which has intrinsic peptidyl-prolyl-(cis/trans)-isomerase enzymatic activity. CyPA mediates immunosuppressive action of the cyclic undecapeptide cyclosporine A and is also involved in multiple cellular processes, such as protein folding, intracellular trafficking, signal transduction and transcriptional regulation. CyPA is abundantly expressed in cancer cells, and, owing to its chaperone nature, its expression is induced upon the onset of stress. In this study, we demonstrated that a significant pool of this immunophilin is primarily an intramitochondrial factor that migrates to the nucleus when cells are stimulated with stressors. CyPA shows anti-apoptotic action per se and the capability of forming ternary complexes with cytochrome c and the small acidic co-chaperone p23, the latter interaction being independent of the usual association of p23 with the heat-shock protein of 90 kDa, Hsp90. These CyPA•p23 complexes enhance the anti-apoptotic response of the cell, suggesting that both proteins form a functional unit, the high level of expression of which plays a significant role in cell survival.

Asunto(s)

Apoptosis , Ciclofilina A , Ciclosporina , Células 3T3 , Animales , Proteínas Portadoras , Ciclofilina A/genética , Ciclofilina A/metabolismo , Células HeLa , Humanos , Ratones , Isomerasa de Peptidilprolil , Pliegue de Proteína , Ratas

RESUMEN

Great efforts have been made to identify promising antigens and vaccine formulations against schistosomiasis. Among the previously described Schistosoma vaccine candidates, cyclophilins comprise an interesting antigen that could be used for vaccine formulations. Cyclophilin A is the target for the cyclosporine A, a drug with schistosomicide activity, and its orthologue from Schistosoma japonicum induces a protective immune response in mice. Although Schistosoma mansoni cyclophilin A also represents a promising target for anti-schistosome vaccines, its potential to induce protection has not been evaluated. In this study, we characterized the cyclophilin A (SmCyp), initially described as Smp17.7, analyzed its allergenic potential using in vitro functional assays, and evaluated its ability to induce protection in mice when administered as an antigen using different vaccine formulations and strategies. Results indicated that SmCyp could be successfully expressed by mammalian cells and bacteria. The recombinant protein did not promote IgE-reporter system activation in vitro, demonstrating its probable safety for use in vaccine formulations. T and B-cell epitopes were predicted in the SmCyp sequence, with two of them located within the active isomerase site. The most immunogenic antigen, SmCyp (107-121), was then used for immunization protocols. Immunization with the SmCyp gene or protein failed to reduce parasite burden but induced an immune response that modulated the granuloma area. In contrast, immunization with the synthetic peptide SmCyp (107-121) significantly reduced worm burden (48-50%) in comparison to control group, but did not regulate liver pathology. Moreover, the protection observed in mice immunized with the synthetic peptide was associated with the significant production of antibodies against the SmCyp (107-121) epitope. Therefore, in this study, we identified an epitope within the SmCyp sequence that induces a protective immune response against the parasite, thus representing a promising antigen that could be used for vaccine formulation against schistosomiasis.

Asunto(s)

Ciclofilina A/inmunología , Epítopos/inmunología , Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Femenino , Proteínas del Helminto/inmunología , Inmunización/métodos , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/inmunología , Vacunación/métodos , Vacunas/inmunología

RESUMEN

The SELF PRUNING (SP) gene is a key regulator of growth habit in tomato (Solanum lycopersicum). It is an ortholog of TERMINAL FLOWER1, a phosphatidylethanolamine-binding protein with antiflorigenic activity in Arabidopsis (Arabidopsis thaliana). A spontaneous loss-of-function mutation (sp) has been bred into several industrial tomato cultivars, as it produces a suite of pleiotropic effects that are favorable for mechanical harvesting, including determinate growth habit, short plant stature, and simultaneous fruit ripening. However, the physiological basis for these phenotypic differences has not been thoroughly explained. Here, we show that the sp mutation alters polar auxin transport as well as auxin responses, such as gravitropic curvature and elongation of excised hypocotyl segments. We also demonstrate that free auxin levels and auxin-regulated gene expression patterns are altered in sp mutants. Furthermore, diageotropica, a mutation in a gene encoding a cyclophilin A protein, appears to confer epistatic effects with sp Our results indicate that SP affects the tomato growth habit at least in part by influencing auxin transport and responsiveness. These findings suggest potential novel targets that could be manipulated for controlling plant growth habit and improving productivity.

Asunto(s)

Ciclofilina A/metabolismo , Frutas/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Transporte Biológico , Ciclofilina A/genética , Frutas/genética , Frutas/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Mutación , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/genética

RESUMEN

Yellow fever virus (YFV) replication is highly dependent on host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1-DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein that has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner. 59 proteins were found to be significantly increased when compared with a negative control, and CypA exhibited the greatest difference, with a 22-fold change. Fisher's exact test was significant for 58 proteins, and the p value of CypA was the most significant (0.000000019). The Ingenuity Systems software identified 16 pathways, and this analysis indicated sirolimus, an mTOR pathway inhibitor, as a potential inhibitor of CypA. Immunofluorescence and viral plaque assays showed a significant reduction in YFV replication using sirolimus and cyclosporine A (CsA) as inhibitors. Furthermore, YFV replication was strongly inhibited in cells treated with both inhibitors using reporter BHK-21-rep-YFV17D-LucNeoIres cells. Taken together, these data suggest that CypA-NS4B interaction regulates YFV replication. Finally, we present the first evidence that YFV inhibition may depend on NS4B-CypA interaction.

Asunto(s)

Ciclofilina A/metabolismo , Proteínas/genética , Replicación Viral/genética , Virus de la Fiebre Amarilla/genética , Ciclofilina A/genética , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacos , Sirolimus/administración & dosificación , Biología de Sistemas , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Virus de la Fiebre Amarilla/patogenicidad

RESUMEN

BACKGROUND AND OBJECTIVE: Periodontal disease is an infectious disease resulting from the immunoinflammatory response of the host to microorganisms present in the dental biofilm which causes tissue destruction. The objective of this study was to evaluate the immunohistochemical expression of matrix metalloproteinase 7 (MMP-7), extracellular matrix metalloproteinase inducer (EMMPRIN) and cyclophilin A (CypA) in periodontal disease. DESIGN: Gingival tissue samples were divided as follows: clinically healthy gingiva (n=32), biofilm-induced gingivitis (n=28), and chronic periodontitis (n=30). Histological sections of 3µm were submitted to immunoperoxidase method and undergone quantitative analysis. The results were analyzed statistically by the Mann-Whitney and Spearman correlation tests, with the level of significance set at 0.05 (α=0.05). RESULTS: Immunopositivity for MMP-7, EMMPRIN and CypA differed significantly between the three groups, with higher percentages of staining in chronic periodontitis specimens, followed by chronic gingivitis and healthy gingiva specimens (p<0.05). Immunoexpression of CypA and MMP-7 was higher in the intense inflammatory infiltrate observed mainly in cases of periodontitis (p<0.05). CypA expression was positively correlated with MMP-7 (r=0.831; p<0.001) and EMMPRIN (r=0.289; p=0.006). In addition, there was a significant positive correlation between probing depth and expression of MMP-7 (r=0.726; p<0.001), EMMPRIN (r=0.345; p=0.001), and CypA (r=0.803; p<0.001). CONCLUSION: These results suggest that MMP-7, EMMPRIN and CypA are associated with the pathogenesis and progression of periodontal disease.

Asunto(s)

Basigina/metabolismo , Periodontitis Crónica/metabolismo , Periodontitis Crónica/patología , Ciclofilina A/metabolismo , Metaloproteinasa 7 de la Matriz/metabolismo , Progresión de la Enfermedad , Femenino , Líquido del Surco Gingival/química , Humanos , Técnicas para Inmunoenzimas , Masculino

RESUMEN

OBJECTIVE: A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex). METHODS: Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder. RESULTS: Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively. CONCLUSION: Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene.

Asunto(s)

Actinas/genética , Encéfalo/fisiología , Ciclofilina A/genética , Ubiquitina C/genética , Actinas/análisis , Animales , Conducta Animal , Ciclofilina A/análisis , Femenino , Genes Esenciales/fisiología , Control Interno-Externo , ARN Mensajero/genética , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Valores de Referencia , Transcripción Reversa , Ubiquitina C/análisis

RESUMEN

Objective A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex). Methods Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder. Results Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively. Conclusion Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene. .

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Objetivo Um número crescente de artigos publicados relaciona a expressão de genes específicos com diferentes padrões de comportamento em ratos. Os níveis de transcritos de ácido ribonucleico mensageiro são geralmente analisados por transcrição reversa, seguida de reação em cadeia da polimerase, e quantificados após a normalização com um controle interno ou gene de referência (gene housekeeping). No entanto, os genes housekeeping exibem expressão diferencial no sistema nervoso central, dependendo das condições fisiológicas e da área do cérebro a ser estudada. A escolha de um bom gene de controle interno é essencial para a obtenção de resultados confiáveis. Este estudo avaliou a expressão de três genes housekeeping (beta-actina, ciclofilina A e ubiquitina C) em diferentes áreas do sistema nervoso central de ratos (bulbo olfatório, hipocampo, estriado e córtex pré-frontal). Métodos Foram usadas ratas Wistar (fêmeas virgens, n=6) durante o período de diestro. O ácido ribonucleico total foi extraído a partir de cada região do cérebro; o ácido desoxirribonucleico complementar foi sintetizado por transcrição reversa e amplificado por reação em cadeia da polimerase quantitativo em tempo real utilizando SYBR® Green e primers específicos para cada um dos genes de referência. A estabilidade de expressão foi determinada utilizando NormFinder. Resultados A beta-actina foi o gene mais estável no hipocampo e estriado, enquanto a ciclofilina A e a ubiquitina C apresentaram maior estabilidade no córtex pré-frontal e no bulbo olfatório, respectivamente. Conclusão Com base em nosso trabalho, estudos posteriores de expressão gênica utilizando ratos como modelos animais devem levar ...

Asunto(s)

Animales , Femenino , Actinas/genética , Encéfalo/fisiología , Ciclofilina A/genética , Ubiquitina C/genética , Actinas/análisis , Conducta Animal , Ciclofilina A/análisis , Genes Esenciales/fisiología , Control Interno-Externo , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Valores de Referencia , Transcripción Reversa , ARN Mensajero/genética , Ubiquitina C/análisis

RESUMEN

A number of studies have demonstrated that simple elastic network models can reproduce experimental B-factors, providing insights into the structure-function properties of proteins. Here, we report a study on how to improve an elastic network model and explore its performance by predicting the experimental B-factors. Elastic network models are built on the experimental Cα coordinates, and they only take the pairs of Cα atoms within a given cutoff distance rc into account. These models describe the interactions by elastic springs with the same force constant. We have developed a method based on numerical simulations with a simple coarse-grained force field, to attribute weights to these spring constants. This method considers the time that two Cα atoms remain connected in the network during partial unfolding, establishing a means of measuring the strength of each link. We examined two different coarse-grained force fields and explored the computation of these weights by unfolding the native structures.

Asunto(s)

Algoritmos , Modelos Químicos , Simulación de Dinámica Molecular , Pliegue de Proteína , Proteínas/química , Azurina/química , Ciclofilina A/química , Citocromos c/química , Oligopéptidos/química , Proteínas de Plantas/química , Ribonucleasas/química , Temperatura , Ubiquitina/química

RESUMEN

Background: Cyclophilin A (CypA), a receptor for the immunosuppressive agent cyclosporin A (CsA), is a cis-trans peptidyl-prolyl isomerase (PPIase) which accelerates the cis-trans isomerization of prolyl-peptide bonds and interacts with a variety of proteins to regulate their activities. Results: The full-length cDNA of crab Eriocheir sinensis CypA (EsCypA) was cloned by EST and RACE technique. The complete sequence of EsCypA cDNA contained a 5' untranslated region (UTR) of 50 bp, a 3’ UTR of 233 bp with a polyA tail, and an open reading frame (ORF) of 495 bp encoding a polypeptide of 164 amino acids with the predicted molecular weight of 17.36 kDa. The deduced amino acid sequence of EsCypA contained two highly conserved signature sequences of peptidyl-prolyl cis-trans isomerase and a pro-isomerase domain. The mRNA transcripts of EsCypA were detectable in all the examined tissues, including haemocytes, gill, hepatopancreas, gonad, muscle and heart, with higher expression level in hepatopancreas and gonad. No significant difference in the relative mRNA expression level of EsCypA was observed during the whole course of bacteria challenge, whereas it was up-regulated during fungi challenge. The purified recombinant protein rEsCypA exhibited a significant PPIase activity and an antifungal activity. Conclusions: All these results indicated that it was a typical CypA member and potentially involved in the innate immune responses of crab.

Asunto(s)

Animales , Braquiuros/genética , Braquiuros/inmunología , Ciclofilina A , Antifúngicos , Filogenia , Alineación de Secuencia , Análisis de Secuencia , Inmunidad

RESUMEN

The aim of this study was to assess the effect of leucine supplementation on elements of the ubiquitin-proteasome system (UPS) in rat skeletal muscle during immobilization. This effect was evaluated by submitting the animals to a leucine supplementation protocol during hindlimb immobilization, after which different parameters were determined, including: muscle mass; cross-sectional area (CSA); gene expression of E3 ligases/deubiquitinating enzymes; content of ubiquitinated proteins; and rate of protein synthesis. Our results show that leucine supplementation attenuates soleus muscle mass loss driven by immobilization. In addition, the marked decrease in the CSA in soleus muscle type I fibers, but not type II fibers, induced by immobilization was minimized by leucine feeding. Interestingly, leucine supplementation severely minimized the early transient increase in E3 ligase [muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1] gene expression observed during immobilization. The reduced peak of E3 ligase gene expression was paralleled by a decreased content of ubiquitinated proteins during leucine feeding. The protein synthesis rate decreased by immobilization and was not affected by leucine supplementation. Our results strongly suggest that leucine supplementation attenuates muscle wasting induced by immobilization via minimizing gene expression of E3 ligases, which consequently could downregulate UPS-driven protein degradation. It is notable that leucine supplementation does not restore decreased protein synthesis driven by immobilization.

Asunto(s)

Leucina/uso terapéutico , Músculo Esquelético/efectos de los fármacos , Atrofia Muscular/patología , Atrofia Muscular/prevención & control , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Administración Oral , Animales , Ciclofilina A/genética , Suplementos Dietéticos , Regulación Enzimológica de la Expresión Génica , Suspensión Trasera , Histocitoquímica , Insulina/sangre , Leucina/administración & dosificación , Leucina/farmacología , Masculino , Músculo Esquelético/anatomía & histología , Músculo Esquelético/patología , Atrofia Muscular/sangre , ARN/genética , ARN/aislamiento & purificación , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina-Proteína Ligasas/genética

RESUMEN

The suppressive subtractive hybridization technique was previously used by the authors to identify candidate genes for meat quality in pig. A set of ESTs homologous (>95%) to genes involved in muscle metabolism is reported in the present paper. Four ESTs homologous to MYH1, KALRN, MLC2V, and SNX13 genes plus two genes (AK1, PPIA) used as housekeeping for muscle tissue were assigned to porcine chromosomes using the INRA-Minnesota 7000 rads radiation hybrid panel (IMpRH). Our data confirm and refine the cytogenetic position of the KALRN, AK1, PPIA genes, improve the existing physical map of MYH1 and assign two new genes (MLC2V and SNX13) to swine chromosomes.

Asunto(s)

Adenilato Quinasa/genética , Ciclofilina A/genética , Proteínas Activadoras de GTPasa/genética , Isoenzimas/genética , Cadenas Pesadas de Miosina/genética , Cadenas Ligeras de Miosina/genética , Proteínas Serina-Treonina Quinasas/genética , Porcinos/genética , Animales , Mapeo Cromosómico , Cromosomas/genética , Etiquetas de Secuencia Expresada , Ligamiento Genético , Genoma , Mapeo de Híbrido por Radiación/métodos

RESUMEN

The first proteomic analysis of Trypanosoma cruzi resistance to Benznidazole (BZ) is presented. The differential proteome of T. cruzi with selected in vivo resistance to Benznidazole (BZR and Clone27R), its susceptible pairs (BZS and Clone9S), and a pair from a population with Benznidazole- in vitro-induced resistance (17LER) and the susceptible pair 17WTS were analyzed by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) for protein identification. Out of 137 spots analyzed through MS, 110 were identified as 56 distinct proteins. Out of the 56 distinct proteins, 36 were present in resistant, 9 in susceptible, and 11 in both phenotypes. Among the proteins identified in resistant samples, 5 were found in Cl 27R and in BZR (calpain-like cysteine peptidase, hypothetical protein conserved 26 kDa, putative peptidase, peroxiredoxin and tyrosine amino transferase) and 4 in Cl 27R and 17LER (cyclophilin A, glutamate dehydrogenase, iron superoxide dismutase and nucleoside diphosphate kinase). As for the proteins identified in Benznidazole-susceptible samples, PGF-2a was found in BZS and 17WTS. A functional category analysis showed that the proteins involved with transcription and protein destination were overexpressed for the Benznidazole-resistant phenotype. Thus, the present study provides large-scale, protein-related information for investigation of the mechanism of T. cruzi resistance to Benznidazole.

Asunto(s)

Resistencia a Medicamentos , Nitroimidazoles/farmacología , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Animales , Ciclofilina A/análisis , Ciclofilina A/metabolismo , Cisteína Endopeptidasas/análisis , Cisteína Endopeptidasas/metabolismo , Electroforesis en Gel Bidimensional , Glutamato Deshidrogenasa/análisis , Glutamato Deshidrogenasa/metabolismo , Hidroxiprostaglandina Deshidrogenasas/análisis , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Nucleósido-Difosfato Quinasa/análisis , Nucleósido-Difosfato Quinasa/metabolismo , Péptido Hidrolasas/análisis , Péptido Hidrolasas/metabolismo , Peroxirredoxinas/análisis , Peroxirredoxinas/metabolismo , Proteoma/análisis , Proteínas Protozoarias/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Superóxido Dismutasa/análisis , Superóxido Dismutasa/metabolismo , Espectrometría de Masas en Tándem , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Tirosina Transaminasa/análisis , Tirosina Transaminasa/metabolismo

RESUMEN

Host cell factors modulate retroviral infections. Among those, cyclophilin A (CypA) promotes virus infectivity by facilitating virus uncoating or capsid unfolding or by preventing retroviral capsid interaction with cellular restriction factors. In Aotus species, a retrotransposed copy of CypA inserted into the tripartite motif 5 (TRIM5) gene encodes a fusion protein which may block human immunodeficiency virus type 1 by targeting the incoming virus to ubiquitin-ligated degradation or by interfering with normal uncoating of the incoming particle, rendering those monkeys resistant to infection. In this study, we have extensively analyzed representative specimens from all New World primate genera and shown that the retrotransposed CypA copy is only present in Aotus. We have shown that this inserted copy diverged from its original counterpart and that this occurred prior to Aotus radiation, although no positive selection was observed. Finally, our data underscores the need for a precise taxonomic identification of primate species used as models for retroviral infections and novel antiviral approaches.

Asunto(s)

Ciclofilina A/genética , Infecciones por VIH/genética , Primates/fisiología , Proteínas/genética , Retroelementos/genética , Animales , Ciclofilina A/metabolismo , Evolución Molecular , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Proteínas/metabolismo , Ubiquitina-Proteína Ligasas

RESUMEN

Cyclophilins are peptidyl-prolyl cis-trans isomerases involved in catalyzing conformational changes and accelerating the rate of protein folding and refolding in several cellular systems. In the present study, we analyzed the expression pattern and intracellular distribution of the cellular isomerase cyclophilin A (CypA) during vaccinia virus (VV) infection. An impressive increase in CypA stability was observed, leading to a practically unchanged accumulation of CypA during infection, although its synthesis was completely inhibited at late times. By confocal microscopy, we observed that CypA went through an intense reorganization in the cell cytoplasm and colocalized with the virosomes late in infection. CypA relocation to viral factories required the synthesis of viral postreplicative proteins, and treatment of infected cells with cyclosporine (CsA) prevented CypA relocation, clearly excluding the virosomes from CypA staining. Immunoelectron microscopy of VV-infected cells showed that CypA was incorporated into VV particles during morphogenesis. Biochemical and electron microscopic assays with purified virions confirmed that CypA was encapsidated within the virus particle and localized specifically in the core. This work suggests that CypA may develop an important role in VV replication.

Asunto(s)

Ciclofilina A/metabolismo , Virus Vaccinia/fisiología , Vaccinia/metabolismo , Virión/fisiología , Western Blotting , Línea Celular , Cromatografía de Afinidad , Técnica del Anticuerpo Fluorescente , Expresión Génica , Fracciones Subcelulares/metabolismo , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Virión/genética , Virión/metabolismo

RESUMEN

myo-Inositol hexakisphosphate (IP(6)) is an abundant intracellular component of animal cells. In this study we describe the presence of extracellular IP(6) in the hydatid cyst wall (HCW) of the larval stage of the cestode parasite Echinococcus granulosus. The HCW comprises an inner cellular layer and an outer, acellular (laminated) layer up to 2 mm in thickness that protects the parasite from host immune cells. A compound, subsequently identified as IP(6), was detected in and purified from an HCW extract on the basis of its capacity to inhibit complement activation. The identification of the isolated compound was carried out by a combination of NMR, MS and TLC. The majority of IP(6) in the HCW was found in the acellular layer, with only a small fraction of the compound being extracted from cells. In the laminated layer, IP(6) was present in association with calcium, and accounted for up to 15% of the total dry mass of the HCW. IP(6) was not detected in any other structures or stages of the parasite. Our results imply that IP(6) is secreted by the larval stage of the parasite in a polarized fashion towards the interface with the host. This is the first report of the secretion of IP(6), and the possible implications beyond the biology of E. granulosus are discussed.

Asunto(s)

Echinococcus/química , Ácido Fítico/análisis , Animales , Antígenos Helmínticos/análisis , Calcio/análisis , Pared Celular/química , Cromatografía en Capa Delgada , Ciclofilina A/análisis , Echinococcus/crecimiento & desarrollo , Espacio Extracelular/química , Proteínas del Helminto/análisis , Magnesio/análisis , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ácido Fítico/aislamiento & purificación