RESUMEN
In response to the endoplasmic reticulum (ER) stress induced by herpes simplex virus type 1 (HSV-1) infection, host cells activate the unfolded protein response (UPR) to reduce the protein-folding burden in the ER. The regulation of UPR upon HSV-1 infection is complex, and the downstream effectors can be detrimental to viral replication. Therefore, HSV-1 copes with the UPR to create a beneficial environment for its replication. UPR has three branches, including protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activated transcription factor 6 (ATF6). IRE1α is the most conserved branch of UPR which has both RNase and kinase activities. Previous studies have shown that IRE1α RNase activity was inactivated during HSV-1 infection. However, the effect of the two activities of IRE1α on HSV-1 replication remains unknown. Results in this study showed that IRE1α expression was up-regulated during HSV-1 infection. We found that in HEC-1-A cells, increasing RNase activity, or inhibiting kinase activity of IRE1α led to viral suppression, indicating that the kinase activity of IRE1α was beneficial, while the RNase activity was detrimental to viral replication. Further evidence showed that the kinase activity of IRE1α leads to the activation of the JNK (c-Jun N-terminal kinases) pathway, which enhances viral replication. Taken together, our evidence suggests that IRE1α is involved in HSV-1 replication, and its RNase and kinase activities play differential roles during viral infection.
Asunto(s)
Endorribonucleasas/metabolismo , Endorribonucleasas/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/farmacología , Ribonucleasas/metabolismo , Replicación Viral/efectos de los fármacos , Proteína 1 de Unión a la X-Box/farmacología , Animales , Antivirales/farmacología , Línea Celular , Chlorocebus aethiops , Replicación del ADN , Estrés del Retículo Endoplásmico , Endorribonucleasas/farmacocinética , Regulación de la Expresión Génica , Células HeLa , Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/patogenicidad , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/farmacocinética , Empalme del ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/análisis , Transducción de Señal/genética , Respuesta de Proteína Desplegada , Regulación hacia Arriba , Células Vero , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
Sequence-specificity of binding of ribonuclease binase to oligodeoxyribonucleotides immobilized in biochip gel pads was studied. Binding constants for the complexes between binase and selected oligonucleotides were measured. Oligodeoxyribonucleotides GAGAGAG and GAGAGAGAG were found to be high-specific in interaction with binase. These oligonucleotides were used as molecular probes for immobilization in a sorptive medium for subsequent isolation and concentrating of binase from diluted water solutions. Volume capacity of the developed sorptive mediums containing immobilized oligodeoxyribonucleotides GAGAGAG and GAGAGAGAG was found to be 2.6 and 2.3 mg of binase per 1 mL of sorbate correspondingly. After the procedure of affinity chromatography and elution ofbinase from sorptive medium the protein showed the same affinity of binding to oligonucleotides as the initial sample.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Endorribonucleasas/aislamiento & purificación , Análisis por Matrices de Proteínas/métodos , Adsorción , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Sitios de Unión/genética , Cromatografía de Afinidad , ADN/metabolismo , Proteínas de Unión al ADN/farmacocinética , Endorribonucleasas/farmacocinética , Humanos , Ácidos Nucleicos Inmovilizados/genética , Ácidos Nucleicos Inmovilizados/metabolismo , Sondas Moleculares/genética , Unión ProteicaRESUMEN
Microbial ribonucleases possess a broad spectra of biological activities, demonstrating stimulating properties at low concentrations and cytotoxicity and genotoxicity at high concentrations. The mechanisms of their penetration into the cells are not clear so far. This research is aimed to the study of Bacillus intermedius RNase (binase) penetration in alveolar lung epithelial cells--pneumocytes of type II. Using immunofluorescence we have shown for the first time have internalization of binase by primary non-differentiated pneumocytes ATII. The enzyme did not penetrate in pneumocytes MLE-12, which also derived from type II cells. However, binase was cytotoxic towards tumor MLE-12 cells, but not ATII cells. The obtained results testified the higher sensitivity of tumor cells towards binase compared with normal cells, and also showed that penetration of the enzyme into alveolar cells did not directly correlated with the cell death.
Asunto(s)
Endorribonucleasas/farmacocinética , Neoplasias Pulmonares/tratamiento farmacológico , Alveolos Pulmonares/citología , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/efectos de los fármacos , Animales , Antígenos Transformadores de Poliomavirus/genética , Muerte Celular/efectos de los fármacos , Diferenciación Celular , Endorribonucleasas/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Alveolos Pulmonares/efectos de los fármacosRESUMEN
The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca(2+) concentration in the endosome. In this paper, we monitored rotavirus entry by measuring alpha-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca(2+) concentration on virus entry was studied by inhibiting the endosomal H(+)-ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-alpha-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca(2+)-dependent endocytosis model.
Asunto(s)
Calcio/fisiología , Proteínas Fúngicas , Macrólidos , Rotavirus/fisiología , ATPasas de Translocación de Protón Vacuolares/fisiología , Animales , Antibacterianos/farmacología , Células Cultivadas , Ácido Egtácico/farmacología , Endocitosis , Endorribonucleasas/farmacocinética , Concentración de Iones de Hidrógeno , Metionina/metabolismo , Porcinos , Replicación ViralRESUMEN
BACKGROUND: Bovine seminal ribonuclease (BS RNase) was determined to have a specific suppressive effect on the proliferation of T lymphocytes in vitro. Its immunosuppressive effect was proven in skin grafting in mice as well. METHODS: The immunosuppressive effect of BS RNase was evaluated in tissue cultures and on a model of corneal transplantation in rabbits. The penetration of BS RNase into the anterior chamber was detected by immunoblotting of anterior chamber fluid obtained from animals treated either topically or subconjunctivally. RESULTS: In vitro blastic transformation of mouse T lymphocytes was significantly inhibited by BS RNase (concentrations 15-250 micrograms/ml). No such effect was observed on B lymphocytes. In the rabbit model of corneal graft rejection, BS RNase injected subconjunctivally prolonged mean graft survival time significantly (33.4 days) compared with placebo (salt solution; MST 17.7 days). No BS RNase was detected by immunoblotting in anterior chamber fluid after either topical or subconjunctival application. CONCLUSION: BS RNase showed significant immunosuppressive effect both in the blastic transformation test and in the rabbit high-risk model of corneal transplantation. Negative results of anterior chamber fluid immunoblotting indicate poor absorption of the drug.
Asunto(s)
Trasplante de Córnea , Endorribonucleasas/farmacología , Rechazo de Injerto/prevención & control , Terapia de Inmunosupresión/métodos , Linfocitos T/efectos de los fármacos , Administración Tópica , Animales , Cámara Anterior/metabolismo , Bovinos , División Celular , Células Cultivadas , Conjuntiva , Trasplante de Córnea/inmunología , Trasplante de Córnea/patología , Endorribonucleasas/farmacocinética , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Supervivencia de Injerto/efectos de los fármacos , Immunoblotting , Inyecciones , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Conejos , Linfocitos T/inmunologíaRESUMEN
"Binase" enzyme sample (a microbial ribonuclease) has been tested for mutagenicity in a set of tests. The set included Ames test Salmonella/microsome, Escherichia coli Rec-test, bacteriophage induction assay, DNA-repair synthesis in lymphoid cells. "Binase" is shown to possess a small genotoxic effect at high concentrations. Both animal and plant S-9 fractions eliminated the effect.