RESUMEN
P58(IPK) is a tetratricopeptide repeat-containing cochaperone that is involved in stress-activated cellular pathways and that inhibits the activity of protein kinase PKR, a primary mediator of the antiviral and antiproliferative properties of interferon. To gain better insight into the molecular actions of P58(IPK), we generated NIH 3T3 cell lines expressing either wild-type P58(IPK) or a P58(IPK) deletion mutant, DeltaTPR6, that does not bind to or inhibit PKR. When treated with double-stranded RNA (dsRNA), DeltaTPR6-expressing cells exhibited a significant increase in eukaryotic initiation factor 2alpha phosphorylation and NF-kappaB activation, indicating a functional PKR. In contrast, both of these PKR-dependent events were blocked by the overexpression of wild-type P58(IPK). In addition, the P58(IPK) cell line, but not the DeltaTPR6 cell line, was resistant to dsRNA-induced apoptosis. Together, these findings demonstrate that P58(IPK) regulates dsRNA signaling pathways by inhibiting multiple PKR-dependent functions. In contrast, both the P58(IPK) and DeltaTPR6 cell lines were resistant to tumor necrosis factor alpha-induced apoptosis, suggesting that P58(IPK) may function as a more general suppressor of programmed cell death independently of its PKR-inhibitory properties. In accordance with this hypothesis, although PKR remained active in DeltaTPR6-expressing cells, the DeltaTPR6 cell line displayed a transformed phenotype and was tumorigenic in nude mice. Thus, the antiapoptotic function of P58(IPK) may be an important factor in its ability to malignantly transform cells.
Asunto(s)
Apoptosis , Chaperonas Moleculares/metabolismo , Inhibidores de Proteínas Quinasas , ARN Bicatenario/metabolismo , Proteínas Represoras/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , eIF-2 Quinasa/metabolismo , Células 3T3 , Animales , Factor 2 Eucariótico de Iniciación/metabolismo , Proteínas del Choque Térmico HSP40 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Chaperonas Moleculares/genética , Mutagénesis , FN-kappa B/metabolismo , Fenotipo , Fosforilación , Poli I-C/metabolismo , Poli I-C/farmacología , ARN Bicatenario/antagonistas & inhibidores , Conejos , Proteínas Represoras/genética , Transformación Genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
We present observations on the use of racial and cultural stereotypes in psychoanalytic psychotherapy with patients from the majority culture and with those from minority backgrounds. Earlier work has centered on black/white patient dyads and has not taken other possible combinations into account. "Race" and "culture" have sometimes been used synonymously. Our clinical experience indicates that there is some overlap in the themes of transferences to us as members of different racial minorities. We note, however, that for the African-American therapist, projections are more often based on racial stereotypes, whereas for the Chinese-American therapist, projections are based more on cultural assumptions. When careful attention is paid to the manifestations of racial and cultural stereotyping, much can be learned about the patient's inner life, to the benefit of the analytic work.
Asunto(s)
Cultura , Terapia Psicoanalítica , Grupos Raciales , Estereotipo , Transferencia Psicológica , Negro o Afroamericano/psicología , Actitud del Personal de Salud , Humanos , Grupos Minoritarios/psicología , Relaciones Médico-Paciente , ProyecciónRESUMEN
The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2alpha phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.
Asunto(s)
Hepacivirus/fisiología , Interferones/farmacología , Proteínas no Estructurales Virales/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Clonación Molecular , Cartilla de ADN , Dimerización , Escherichia coli , Regulación Enzimológica de la Expresión Génica , Hepacivirus/efectos de los fármacos , Hepacivirus/patogenicidad , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Reacción en Cadena de la Polimerasa , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transfección , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/química , Replicación Viral , eIF-2 Quinasa/químicaRESUMEN
The analgesic effects of the rat in response to electroacupuncture (EA) or low-dose morphine (3 mg/kg) show marked individual variations. In the midbrain periaqueductal gray (PAG) of the rat, the content of the neuropeptide cholecystokinin octapeptide (CCK-8) was found to be significantly higher in the low responder (LR) rats as compared to that in the high responders (HR). Since PAG has been shown to be a strategic site for CCK-8 to exert an anti-opioid action, a high CCK content in PAG may account for the low analgesic responsiveness to EA and morphine. In order to block the expression of the gene encoding preproCCK in the brain, antisense CCK expression vector pSV2-CCKAS was microinjected into the lateral cerebral ventricle of the rat, leading to a decrease of the CCK-mRNA as well as the CCK-8 content in rat brain. This effect started 4 days after the intracerebroventricular (i.c.v.) injection of the antisense expression vector, and lasted no more than 1 week. This procedure was shown to be very effective in converting LR rats into HR for EA analgesia and morphine analgesia, and also delayed the development of tolerance elicited by prolonged EA stimulation or repeated morphine administration. The time course of the augmentation of opioid analgesia (4-6 days after the i.c.v. injection of the expression vector) paralleled the decrease of the brain CCK-8 content. The results argue that blocking the CCK gene expression in the brain may tilt the balance between opioid and anti-opioid peptides in favor of the former, thus strengthening the EA analgesia and morphine analgesia, and delaying the development of opioid tolerance.
Asunto(s)
Analgesia por Acupuntura , Analgésicos Opioides/farmacología , Colecistoquinina/fisiología , Electroacupuntura , Morfina/farmacología , ARN sin Sentido/farmacología , Animales , Química Encefálica/efectos de los fármacos , Química Encefálica/fisiología , Colecistoquinina/biosíntesis , Colecistoquinina/genética , Femenino , Expresión Génica/efectos de los fármacos , Inyecciones Intraventriculares , Sustancia Gris Periacueductal/metabolismo , Plásmidos , Reacción en Cadena de la Polimerasa , ARN sin Sentido/administración & dosificación , Radioinmunoensayo , Ratas , Ratas WistarRESUMEN
Hepatitis C virus (HCV) is the major cause of non-A non-B hepatitis and a leading cause of liver dysfunction worldwide. While the current therapy for chronic HCV infection is parenteral administration of type 1 interferon (IFN), only a fraction of HCV-infected individuals completely respond to treatment. Previous studies have correlated the IFN sensitivity of strain HCV-1b with mutations within a discrete region of the viral nonstructural 5A protein (NS5A), termed the interferon sensitivity determining region (ISDR), suggesting that NS5A may contribute to the IFN-resistant phenotype of HCV. To determine the importance of HCV NS5A and the NS5A ISDR in mediating HCV IFN resistance, we tested whether the NS5A protein could regulate the IFN-induced protein kinase, PKR, a mediator of IFN-induced antiviral resistance and a target of viral and cellular inhibitors. Using multiple approaches, including biochemical, transfection, and yeast genetics analyses, we can now report that NS5A represses PKR through a direct interaction with the protein kinase catalytic domain and that both PKR repression and interaction requires the ISDR. Thus, inactivation of PKR may be one mechanism by which HCV avoids the antiviral effects of IFN. Finally the inhibition of the PKR protein kinase, by NS5A is the first described function for this HCV protein.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Interferón Tipo I/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas no Estructurales Virales/fisiología , Sitios de Unión , Catálisis , Farmacorresistencia Microbiana , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas no Estructurales Virales/genética , eIF-2 QuinasaRESUMEN
Double stranded RNA-dependent protein kinase (PKR) is a double stranded RNA-activated, interferon-induced serine-threonine kinase that participates in both the antiviral and antiproliferative properties of interferon. We previously found that influenza virus inhibited PKR function by recruiting or activating a cellular inhibitor termed P58(IPK). The present study was undertaken to complement our earlier analyses, which demonstrated that P58(IPK) efficiently inhibited PKR autophosphorylation and activity in vitro. We now report that P58(IPK) down-regulates PKR and, in turn, stimulates protein synthetic rates inside the cell. Using transfection analysis, we show that P58(IPK) stimulates translation of secreted embryonic alkaline phosphatase reporter gene mRNA. Furthermore, we found that at least two regions of the P58(IPK) molecule were required for PKR inhibitory activity in COS-1 cells: (i) the DnaJ similarity region at the carboxyl terminus (amino acids 391-504); and (ii) the tetratricopeptide repeat 6 (TPR6) domain (amino acids 222-255) located in the middle of the P58(IPK) protein and within the eukaryotic protein synthesis initiation factor 2alpha homology region. P58(IPK) variants lacking either one of these regions were unable to stimulate secreted embryonic alkaline phosphatase protein synthetic rates. Consistent with this data is the observation that the DeltaTPR6 mutant (the P58(IPK) variant lacking the TPR6 motif) failed to block PKR activity in vitro. Based on these data and our earlier in vitro functional and PKR-P58(IPK) binding analyses, a revised model of PKR regulation by P58(IPK) is presented.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas de Choque Térmico/metabolismo , Biosíntesis de Proteínas , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Represoras/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Células COS , Bovinos , Proteínas del Choque Térmico HSP40 , Modelos Biológicos , eIF-2 QuinasaRESUMEN
In this chapter we consider the cross-cultural relevance of the Oedipus complex by investigating three "oedipal" myths from three disparate cultures: Chinese, Indian, and European. Although all three myths concern the same mother-father-son triad, they stress different aspects and proscriptions. These are interpreted as reflecting the different values that are central to the respective cultures. The relationship between these values and child-rearing practices is discussed. We conclude that fundamental psychoanalytic concepts are relevant cross-culturally, although the content of the Oedipus complex may vary from society to society.
Asunto(s)
Comparación Transcultural , Complejo de Edipo , Adulto , Niño , Crianza del Niño , Preescolar , China , Europa (Continente) , Femenino , Identidad de Género , Humanos , India , Masculino , Mitología , Relaciones Padres-Hijo , Valores SocialesRESUMEN
BACKGROUND: The frequency with which stools contain Shiga-like toxin producing Escherichia coli not belonging to serotype O157:H7 is unknown in the United States. The aim of this study was to determine the frequency with which these E. coli are present in stools from children from Seattle submitted for bacteriologic analysis. METHODS: 2225 coliform colonies from 445 stools submitted for bacterial culture from Seattle children were probed with the structural genes of Shiga-like toxins I and II in a 1-year prospective study. The adherence and actin aggregating characteristics of these E. coli were subsequently determined. RESULTS: Five (1.1%) patients had non-O157:H7 Shiga-like toxin producing E. coli, a rate of isolation higher than Shigella or Yersinia (0.2% each) but lower than Campylobacter (2.5%), E. coli O157:H7 (2.9%), or Salmonella (3.4%). Only one of the five patients had bloody diarrhea. None developed hemolytic uremic syndrome. All strains adhered in a localized pattern to, and induced actin aggregation in, HeLa cells, and produced a toxin that was lethal to Vero cells. CONCLUSIONS: Non-O157:H7 Shiga-like toxin producing E. coli are present in stools submitted for bacterial culture in a North American childhood population. Their role in childhood diarrhea warrants better definition.
Asunto(s)
Toxinas Bacterianas/análisis , Escherichia coli/patogenicidad , Heces/microbiología , Adolescente , Adhesión Bacteriana , Toxinas Bacterianas/genética , Niño , Preescolar , Diarrea/etiología , Escherichia coli/aislamiento & purificación , Humanos , Lactante , Hibridación de Ácido Nucleico , Proyectos Piloto , Estudios Prospectivos , Toxina Shiga I , Toxina Shiga IIRESUMEN
An examination of Chinese philosophy serves to highlight the differences between Chinese and Western Europeans with regard to beliefs about the nature of man, the ideal man, and general world view. These guide child rearing practices which in turn encourage specific personality traits and values. This paper deals with some implications that these differences have for psychoanalytic theory.
Asunto(s)
Crianza del Niño , Comparación Transcultural , Filosofía , Teoría Psicoanalítica , Niño , Preescolar , China , Humanos , Lactante , Religión y Psicología , SocializaciónRESUMEN
We wanted to establish an in vitro human model for AIDS-associated dementia and pursue the hypothesis that this disease process may be a result of soluble factors produced by HIV-infected macrophages. Human brain aggregates were prepared from nine different brain specimens, and were treated with supernatants from in vitro HIV-infected macrophages (SI), uninfected macrophages (SU), infected T cells, or macrophage-conditioned media from four AIDS patients. Seven of nine treated brains exposed to SI showed peripheral rarefaction after 1 wk of incubation that by ultrastructural analysis showed cytoplasmic vacuolation. Aggregates from two of three brain cultures treated with SI for 3 wk became smaller, an approximately 50% decrease in size. The degree of apparent toxicity in brains exposed to patient-derived macrophage supernatants paralleled the proportion of macrophages found to be expressing HIV p24. Ultrastructural abnormalities were not observed in brains treated with supernatants from HIV-infected T cells, uninfected macrophages, or LPS-activated macrophages. Levels of five neurotransmitter amino acids were decreased in comparison to the structural amino acid leucine. These findings suggest that HIV-infected macrophages, infected both in vitro as well as derived from AIDS patients' peripheral blood, produce factors that cause reproducible histochemical, ultrastructural, and functional abnormalities in human brain aggregates.