RESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) encodes four viral interferon regulatory factors (vIRF-1-4). We investigated the mechanism and consequences of vIRF-2-mediated inhibition of interferon-response element signalling following type I interferon (IFN) induction. Western blot and electrophoretic mobility-shift assays identified the interferon-stimulated gene factor-3 (ISGF-3) components STAT1 and IRF-9 as the proximal targets of vIRF-2 activity. The biological significance of vIRF-2 inhibition of ISGF-3 was demonstrated by vIRF-2-mediated rescue of the replication of the IFN-sensitive virus encephalomyocarditis virus. This study provides both a mechanism and evidence for KSHV vIRF-2-mediated suppression of the consequences of type 1 IFN-induced signalling.
Asunto(s)
Herpesvirus Humano 8/inmunología , Herpesvirus Humano 8/patogenicidad , Evasión Inmune , Factores Reguladores del Interferón/metabolismo , Interferón Tipo I/antagonistas & inhibidores , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/antagonistas & inhibidores , Factor de Transcripción STAT1/antagonistas & inhibidores , Proteínas Virales/metabolismo , Western Blotting , Ensayo de Cambio de Movilidad Electroforética , Virus de la Encefalomiocarditis/crecimiento & desarrollo , Virus de la Encefalomiocarditis/inmunología , Replicación Viral/inmunologíaRESUMEN
The notion of the necessary criticality (both with respect to path and to activity) of a network with imprecisely defined (by means of intervals or fuzzy intervals) activity duration times is introduced and analyzed. It is shown, in the interval case, that both the problem of asserting whether a given path is necessarily critical and the problem of determining an arbitrary necessarily critical path (more exactly, a subnetwork covering all the necessarily critical paths) are easy. The corresponding solution algorithms are proposed. However, the problem of evaluating whether a given activity is necessarily critical does not seem to be so easy. Certain conditions are formulated which, in some situations (but not in all possible situations), allow the necessary criticality of activities to be evaluated. The results obtained for networks with interval activity duration times are generalized to the case of networks with fuzzy activity duration times. Two effective algorithms for calculating the degree of necessary criticality of a fixed path, as well as an algorithm for determining the paths that are necessarily critical to the maximum degree, are proposed.
RESUMEN
Northern blotting has shown that mouse small intestine contains relatively large amounts of the nuclear factor-E2 p45-related factor (Nrf) 2 transcription factor but relatively little Nrf1. Regulation of intestinal antioxidant and detoxication enzymes by Nrf2 has been assessed using a mouse line bearing a targeted disruption of the gene encoding this factor. Both Nrf2-/- and Nrf2+/+ mice were fed a control diet or one supplemented with either synthetic cancer chemopreventive agents [butylated hydroxyanisole (BHA), ethoxyquin (EQ), or oltipraz] or phytochemicals [indole-3-carbinol, cafestol and kahweol palmitate, sulforaphane, coumarin (CMRN), or alpha-angelicalactone]. The constitutive level of NAD(P)H:quinone oxidoreductase (NQO) and glutathione S-transferase (GST) enzyme activities in cytosols from small intestine was typically found to be between 30% and 70% lower in samples prepared from Nrf2 mutant mice fed a control diet than in equivalent samples from Nrf2+/+ mice. Most of the chemopreventive agents included in this study induced NQO and GST enzyme activities in the small intestine of Nrf2+/+ mice. Increases of between 2.7- and 6.2-fold were observed in wild-type animals fed diets supplemented with BHA or EQ; increases of about 2-fold were observed with a mixture of cafestol and kahweol palmitate, CMRN, or alpha-angelicalactone; and increases of 1.5-fold were measured with sulforaphane. Immunoblotting confirmed that in the small intestine, the constitutive level of NQO1 is lower in the Nrf2-/- mouse, and it also showed that induction of the oxidoreductase was substantially diminished in the mutant mouse. Immunoblotting class-alpha and class-mu GST showed that constitutive expression of most transferase subunits is also reduced in the small intestine of Nrf2 mutant mice. Significantly, induction of class-alpha and class-mu GST by EQ, BHA, or CMRN is apparent in the gene knockout animal. No consistent change in the constitutive levels of the catalytic heavy subunit of gamma-glutamylcysteinyl synthetase (GCS(h)) was observed in the small intestine of Nrf2-/- mice. However, although the expression of GCS(h) was found to be increased dramatically in the small intestine of Nrf2+/+ mice by dietary BHA or EQ, this induction was essentially abolished in the knockout mice. It is apparent that Nrf2 influences both constitutive and inducible expression of intestinal antioxidant and detoxication proteins in a gene-specific fashion. Immunohistochemistry revealed that induction of NQO1, class-alpha GST, and GCS(h) occurs primarily in epithelial cells of the small intestine. This suggests that the variation in inducibility of NQO1, Gsta1/2, and GCS(h) in the mutant mouse is not attributable to the expression of the enzymes in distinct cell types but rather to differences in the dependency of these genes on Nrf2 for induction.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Glutatión Transferasa/biosíntesis , Intestino Delgado/enzimología , Leucina Zippers/fisiología , NAD(P)H Deshidrogenasa (Quinona)/biosíntesis , Transactivadores/fisiología , Factores de Transcripción/fisiología , Animales , Anticarcinógenos/farmacología , Antioxidantes/farmacología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Dieta , Inducción Enzimática/efectos de los fármacos , Factores de Unión al ADN Específico de las Células Eritroides , Expresión Génica , Glutamato-Cisteína Ligasa/biosíntesis , Glutatión Transferasa/metabolismo , Inactivación Metabólica , Intestino Delgado/efectos de los fármacos , Leucina Zippers/genética , Masculino , Ratones , Ratones Noqueados , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor de Transcripción NF-E2 , Subunidad p45 del Factor de Transcripción NF-E2 , Factor 2 Relacionado con NF-E2 , Factor Nuclear 1 de Respiración , Factores Nucleares de Respiración , Transactivadores/biosíntesis , Transactivadores/genética , Factores de Transcripción/genéticaRESUMEN
An overview is provided of the cancer chemoprevention actions of phenolic antioxidants and 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin). These agents principally appear to exert their beneficial effects through induction of phase II drug-metabolizing enzymes such as glutathione S-transferase (GST). The requirement for oxidative metabolism of the synthetic antioxidants to carbonyl-containing compounds, including quinones, in order that they can induce gene expression is discussed. Previous work has shown that the basic leucine zipper transcription factor Nrf2 is involved in induction of GST by the phenolic antioxidant butylated hydroxyanisole (BHA). Evidence is provided from a mouse possessing a targeted disruption of the Nrf2 gene that, in murine liver, the transcription factor regulates basal expression of several class Alpha and class Mu GST subunits, but not class Pi GST. In the Nrf2 knock-out mouse, hepatic induction of class Alpha and class Mu GST by BHA and the synthetic antioxidant ethoxyquin is similarly impaired, suggesting that these agents affect gene activation by a related mechanism. Significantly, residual induction of GST by antioxidants is apparent in the Nrf2 mutant mouse, indicating the existence of an alternative mechanism of gene activation.
Asunto(s)
Antioxidantes/farmacología , Hidroxianisol Butilado/farmacología , Proteínas de Unión al ADN/metabolismo , Etoxiquina/farmacología , Glutatión Transferasa/biosíntesis , Hígado/metabolismo , Transactivadores/metabolismo , Animales , Western Blotting , Proteínas de Unión al ADN/genética , Inducción Enzimática , Masculino , Ratones , Ratones Mutantes , Factor 2 Relacionado con NF-E2 , Transactivadores/genética , Factores de Transcripción/metabolismoRESUMEN
Structurally diverse compounds can confer resistance to aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Treatment with either phytochemicals [benzyl isothiocyanate, coumarin (CMRN), or indole-3-carbinol] or synthetic antioxidants and other drugs (butylated hydroxyanisole, diethyl maleate, ethoxyquin, beta-naphthoflavone, oltipraz, phenobarbital, or trans-stilbene oxide) has been found to increase hepatic aldo-keto reductase activity toward AFB1-dialdehyde and glutathione S-transferase (GST) activity toward AFB1-8,9-epoxide in both male and female rats. Under the conditions used, the natural benzopyrone CMRN was a major inducer of the AFB1 aldehyde reductase (AFAR) and the aflatoxin-conjugating class-alpha GST A5 subunit in rat liver, causing elevations of between 25- and 35-fold in hepatic levels of these proteins. Induction was not limited to AFAR and GSTA5: treatment with CMRN caused similar increases in the amount of the class-pi GST P1 subunit and NAD(P)H: quinone oxidoreductase in rat liver. Immunohistochemistry demonstrated that the overexpression of AFAR, GSTA5, GSTP1, and NAD(P)H:quinone oxidoreductase affected by CMRN is restricted to the centrilobular (periacinar) zone of the lobule, sometimes extending almost as far as the portal tract. This pattern of induction was also observed with ethoxyquin, oltipraz, and trans-stilbene oxide. By contrast, induction of these proteins by beta-naphthoflavone and diethyl maleate was predominantly periportal. Northern blotting showed that induction of these phase II drug-metabolizing enzymes by CMRN was accompanied by similar increases in the levels of their mRNAs. To assess the biological significance of enzyme induction by dietary CMRN, two intervention studies were performed in which the ability of the benzopyrone to inhibit either AFB1-initiated preneoplastic nodules (at 13 weeks) or AFB1-initiated liver tumors (at 50 weeks) was investigated. Animals pretreated with CMRN for 2 weeks prior to administration of AFB1, and with continued treatment during exposure to the carcinogen for a further 11 weeks, were protected completely from development of hepatic preneoplastic lesions by 13 weeks. In the longer-term dietary intervention, treatment with CMRN before and during exposure to AFB1 for a total of 24 weeks was found to significantly inhibit the number and size of tumors that subsequently developed by 50 weeks. These data suggest that consumption of a CMRN-containing diet provides substantial protection against the initiation of AFB1 hepatocarcinogenesis in the rat.