RESUMEN
Antecedentes: El fracaso renal agudo (FRA) es una complicación frecuente tras la cirugía cardíaca y la angiografía coronaria que ensombrece el pronóstico de estos pacientes. El diagnóstico se basa en el ascenso de la creatinina sérica, que es tardío. Es necesaria la identificación y validación de nuevos biomarcadores precoces que permitan intervenciones más tempranas y eficaces. Objetivos: Evaluar la sensibilidad y especificidad de interleuquina-18 (IL-18) en orina, neutrophil gelatinase-associated lipocalin en orina (uNGAL) y cistatina C en suero para la detección precoz del FRA en una población de pacientes con síndrome coronario agudo o fallo cardíaco y sometidos a cirugía cardíaca o cateterismo. Métodos: Se incluyeron en el estudio 135 pacientes ingresados en una unidad de cuidados intensivos por síndrome coronario agudo o fallo cardíaco por patología coronaria o valvular y a los que se realizaron una angiografía cardíaca o una cirugía cardíaca de revascularización o recambio valvular. Se determinaron los biomarcadores a las 12 horas de la intervención y se monitorizó la creatinina sérica durante los siguientes seis días para el diagnóstico del FRA. Resultados: Para NGAL se obtuvo un área bajo la curva ROC (AUC) de 0,983 y para cistatina C e IL-18 de 0,869 y 0,727, respectivamente. Para un punto de corte de NGAL en orina de 31,9 ng/ml la sensibilidad fue del 100% y la especificidad del 91%. Conclusiones: uNGAL es un marcador precoz de FRA en pacientes con síndrome coronario o fallo cardíaco agudo y sometidos a cirugía cardíaca y angiografía cardíaca, con una capacidad predictiva superior a cistatina o a IL-18 (AU)
Background: Acute kidney injury (AKI) is a common complication in cardiac surgery and coronary angiography, which worsens patients' prognosis. The diagnosis is based on the increase in serum creatinine, which is delayed. It is necessary to identify and validate new biomarkers that allow for early and effective interventions. Aims: To assess the sensitivity and specificity of neutrophil gelatinase-associated lipocalin in urine (uNGAL), interleukin-18 (IL-18) in urine and cystatin C in serum for the early detection of AKI in patients with acute coronary syndrome or heart failure, and who underwent cardiac surgery or catheterization. Methods: The study included 135 patients admitted to the intensive care unit for acute coronary syndrome or heart failure due to coronary or valvular pathology and who underwent coronary angiography or cardiac bypass surgery or valvular replacement. The biomarkers were determined 12 hours after surgery and serum creatinine was monitored during the next six days for the diagnosis of AKI. Results: The area under the ROC curve (AUC) for NGAL was 0.983, and for cystatin C and IL-18 the AUCs were 0.869 and 0.727, respectively. At a cut-off of 31.9ng/ml for uNGAL the sensitivity was 100% and the specificity was 91%. Conclusions: uNGAL is an early marker of AKI in patients with acute coronary syndrome or heart failure and undergoing cardiac surgery and coronary angiography, with a higher predictive value than cystatin C or IL-18 (AU)
Asunto(s)
Humanos , Lesión Renal Aguda/fisiopatología , Angiografía Coronaria , Síndrome Coronario Agudo/fisiopatología , Insuficiencia Cardíaca/fisiopatología , Biomarcadores/análisis , Lipocalinas/orina , Cistatina C/sangre , Factores de RiesgoRESUMEN
BACKGROUND: In obesity, insulin resistance appears frequently after activation of proinflammatory molecules. Caspase-generated cytokeratin-18 (CK-18) fragments are produced during the apoptosis of hepatic cells. The main objective in the present study is to investigate the relationship between insulin resistance and caspase-generated CK-18 fragments in patients with severe obesity. METHODS: Sixty-two patients selected for bariatric surgery were clinically studied (sex, age, weight, waist diameter, body mass index, arterial pressure and type 2 diabetes mellitus) and analytic parameters were measured in blood (glucose concentration, cholesterol, triglycerides, insulin, glycosylated hemoglobin, aspartate aminotransferase, alanine aminotransferase, high-sensitivity C-reactive protein, adiponectin, interleukin 6, interleukin 18 and CK-18 fragments). Patient group division was based on 70th percentile of insulin resistance as measured by homeostasis model assessment (HOMA) and also according to liver histology. RESULTS: Patients with greater insulin resistance (percentile > 70th) showed higher values of CK-18 fragments, interleukin 6 and transaminases. A positive correlation between the HOMA score, value of CK-18 fragments and triglyceride level was found. A correlation between CK-18 fragments with interleukin 6, triglycerides and transaminases was also observed. HOMA score and value of CK-18 fragments correlated with the degree of liver fibrosis. CONCLUSIONS: Greater degree of insulin resistance induces apoptosis of hepatic cells as measured by the serum levels of CK-18 fragments.
Asunto(s)
Apoptosis/fisiología , Hepatocitos/metabolismo , Inflamación/metabolismo , Resistencia a la Insulina/fisiología , Obesidad/metabolismo , Adulto , Glucemia , Presión Sanguínea , Índice de Masa Corporal , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibrosis/patología , Hepatocitos/patología , Humanos , Inflamación/patología , Insulina/sangre , Interleucina-18/sangre , Interleucina-6/sangre , Queratina-18/sangre , Lípidos/sangre , Hígado/patología , Masculino , Persona de Mediana Edad , Obesidad/patología , Oportunidad Relativa , Selección de Paciente , Estadísticas no ParamétricasRESUMEN
Mesophilic, hydrogenotrophic, sulfate-reducing bacteria were isolated from a deep-sea hydrothermal chimney sample collected at 13 degrees N on the East-Pacific Rise at a depth of 2,600 m. Two strains (BL5 and H9) were found to be phylogenetically similar to Desulfovibrio profundus (similarity >99%), whereas two other strains (H1 and AM13T) were found to be phylogenetically distinct (similarity 96.4%) from Desulfovibrio zosterae, their closest relative. Strain AM13T was characterized further. It was a barophilic, Gram-negative, non-sporulating, motile, vibrio-shaped or sigmoid bacterium possessing desulfoviridin. It grew at temperatures ranging from 20 to 40 degrees C, with an optimum at 35 degrees C in the presence of 2.5% NaCl. The pH range for growth was 6.7-8.2 with an optimum around 7.8. Strain AM13T utilized H2/CO2, lactate, formate, ethanol, choline and glycerol as electron donors. Electron acceptors were sulfate, sulfite and thiosulfate, but not elemental sulfur or nitrate. The G + C content of DNA was 47 mol%. Strain AM13T (= DSM 14728T = CIP107303T) differed from D. zosterae not only phylogenetically, but also genomically (DNA-DNA reassociation value between the two bacteria was 23.8%) and phenotypically. This isolate is therefore proposed as the type strain of a novel species of the genus Desulfovibrio, Desulfovibrio hydrothermalis sp. nov.
Asunto(s)
Desulfovibrio/clasificación , Desulfovibrio/aislamiento & purificación , ADN Bacteriano/genética , ADN Ribosómico/genética , Desulfovibrio/genética , Desulfovibrio/metabolismo , Calor , Presión Hidrostática , Datos de Secuencia Molecular , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Agua de Mar/microbiologíaRESUMEN
The mutagenicity of three nitric oxide (NO) donors, 3-morpholinosydnonimine (SIN-1), a compound generating the precursors of peroxynitrite NO and superoxide, diethylamine/NO (DEA/NO) and spermine/NO (SPER/NO), both releasing authentic NO was analyzed using Escherichia coli tester strains IC203, carrying a deletion of the oxyR gene, and its oxyR(+) parent IC188 (the alternative name of WP2 uvrA/pKM101). The OxyR protein is a redox-sensitive transcriptional activator of genes encoding antioxidant enzymes. Strains IC203 and IC188 contain error-prone DNA polymerases polV, encoded by the chromosomal umuDC genes, and polRI, encoded by mucAB genes carried by pKM101. SIN-1 was determined to be an oxidative mutagen giving a positive response only in IC203, whereas DEA/NO and SPER/NO induced similar positive responses in IC203 and IC188 and were considered as non-oxidative mutagens. The spectrum of ochre suppressors in Trp(+) revertants induced by SIN-1 in IC203 was characterized by a higher number of TA-->AT transversions and GC-->AT transitions, and a lower number of GC-->TA transversions, with respect to the untreated control. The mutagenicity of SIN-1 in IC203, probably induced by peroxynitrite through reactive derivatives, was enhanced in the presence of plumbagin (PLB), a superoxide generator. Superoxide generation by PLB, as well as formation of peroxynitrite in cells treated with SIN-1, evaluated by monitoring the oxidation, respectively, of dihydroethidium and dihydrorhodamine 123, were greater in IC203 than in IC188. Formation of peroxynitrite in IC203 treated with SIN-1 was stimulated by PLB. After treatment with DEA/NO and SPER/NO the number of revertants scored in IC188 was higher than in strains IC187, containing only polV, and IC204, deficient in both polV and polRI. For these compounds, induced suppressor revertants in IC187 and IC204 were almost exclusively GC-->AT transitions, whereas in IC188 significant levels of GC-->TA and TA-->AT transversions were also induced. Mutagenesis by both DEA/NO and SPER/NO was partially inhibited in the presence of PLB. The results show the usefulness of the new tester strain IC203 to differentiate NO-promoted mutagenic mechanisms that involve or do not involve oxygen radicals.
Asunto(s)
Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Molsidomina/análogos & derivados , Mutágenos/toxicidad , Donantes de Óxido Nítrico/toxicidad , Superóxidos/metabolismo , Daño del ADN , Dietilaminas/toxicidad , Escherichia coli/metabolismo , Genes Bacterianos/efectos de los fármacos , Genes Supresores/efectos de los fármacos , Modelos Genéticos , Molsidomina/toxicidad , Pruebas de Mutagenicidad , Naftoquinonas/toxicidad , Espermina/toxicidadRESUMEN
Strain IC203, deficient in OxyR, and its oxyR(+) parent WP2 uvrA/pKM101 (denoted IC188) are the basis of a new bacterial reversion assay, the WP2 Mutoxitest, which has been used in the evaluation of 80 chemicals for oxidative mutagenicity. The following 31 oxidative mutagens were recognized by their greater mutagenic response in IC203 than in IC188: (1) peroxides: hydrogen peroxide (HP), t-butyl hydroperoxide (BOOH) and cumene hydroperoxide (COOH); (2) benzoquinones (BQ): 2-methyl-1,4-BQ, 2,6-dimethyl-1,4-BQ and 2,3, 5,6-tetramethyl-1,4-BQ; (3) naphthoquinones (NQ): 1,4-NQ, 2-methyl-1, 4-NQ and 2-hydroxy-1,4-NQ; (4) phenol derivatives: catechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, t-butylhydroquinone, gallic acid and 4-aminophenol; (5) catecholamines: DL- and L-dopa, DL- and L-epinephrine, dopamine and L-norepinephrine; (6) thiols: L-cysteine methyl ester, L-cysteine ethyl ester, L-penicillamine and dithiothreitol; (7) diverse: 3,4-dihydroxyphenylacetic acid, hypoxanthine and xanthine, both in the presence of xanthine oxidase, L-ascorbic acid plus copper (II) and phenazine methosulfate. Among these oxidative mutagens, 25 were found to be uniquely positive in IC203. With the exception of BOOH and COOH, mutagenesis by all oxidative mutagens was inhibited by catalase present in rat liver S9, indicating that it is mediated by HP generation, probably in autoxidation reactions. These catalase-sensitive oxidative mutagens were poor inducers of mutations derived from 8-oxoguanine lesions, whereas such mutations were efficiently induced by organic hydroperoxides. The results support the usefulness of incorporating IC203 in the bacterial battery for testing of chemicals. The well-characterized oxidative mutagens available with the use of the WP2 Mutoxitest may serve as a reference in studies on the genotoxicity of oxidative stress.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN , Escherichia coli/genética , Pruebas de Mutagenicidad/métodos , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Proteínas Bacterianas/metabolismo , Catalasa/farmacología , Catecolaminas/farmacología , Proteínas de Escherichia coli , Hígado/metabolismo , Mutación , Estrés Oxidativo , Peróxidos/farmacología , Fenoles/farmacología , Quinonas/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Low doses of L-cysteine (CYS), cysteinyl-glycine (CYSGLY) and reduced glutathione (GSH) activated by gamma-glutamyl transpeptidase (GGT) were mutagenic in strain IC203 (oxyR), whereas higher doses were required to observe a weak mutagenicity in the oxyR+ strain WP2 uvrA/pKM101 (denoted IC188). This indicates that thiol mutagenesis is suppressed by OxyR-regulated antioxidant defenses and confirms its oxidative character. The mutagenesis by low doses of CYS, CYSGLY and GSH + GGT detected in IC203 was abolished by rat liver S9, through the activity of catalase, as well as by the metal chelator diethyldithiocarbamate (DETC), supporting the dependence of this mutagenesis on H2O2 production, probably in thiol autoxidation reactions in which transition metals are involved. Surprisingly, low DETC concentrations greatly potentiate the mutagenicity of low CYS doses. Mutagenesis by high doses of CYS and CYSGLY occurred in both IC203 and IC188 in the presence of liver S9, and was resistant to inhibition by catalase, although it was prevented by DETC. Mutagenesis by GSH activated by rat kidney S9, rich in GGT, was detected in IC203 and IC188 only at high doses since catalase and glutathione peroxidase, both present in kidney S9, might inhibit its induction by low GSH doses. In the presence of liver S9, almost deficient in GGT, GSH was not mutagenic. The mutagenicity of a high GSH dose occurring in the presence either of GGT plus liver S9 or of kidney S9 was weakly prevented by DETC.
Asunto(s)
Proteínas de Unión al ADN , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Pruebas de Mutagenicidad/métodos , Proteínas Represoras/metabolismo , Compuestos de Sulfhidrilo/toxicidad , Factores de Transcripción/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cisteína/toxicidad , Dipéptidos/toxicidad , Ditiocarba/farmacología , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Glutatión/metabolismo , Glutatión/toxicidad , Peróxido de Hidrógeno/toxicidad , Riñón/química , Riñón/metabolismo , Hígado/química , Hígado/metabolismo , Mutagénesis , Oxidantes/toxicidad , Oxidación-Reducción , Ratas , Proteínas Represoras/genética , Extractos de Tejidos/metabolismo , Extractos de Tejidos/farmacología , Factores de Transcripción/genética , gamma-Glutamiltransferasa/metabolismo , terc-Butilhidroperóxido/toxicidadRESUMEN
Strain IC203, deficient in the OxyR function, was sensitive to both cytotoxic and mutagenic effects of isoniazid (INH) whereas its parent, WP2 uvrA/pKM101, was resistant to these effects. Four other hydrazine compounds, hydrazine hydrate (HZH), phenylhydrazine (PHZ), hydralazine (HLZ) and nialamide (NLD), were mutagenic in WP2 uvrA/pKM101. Increases in mutagenicity were observed in IC203 for HZH and PHZ but not for HLZ and NLD. Growth inhibition zones by HZH, PHZ and NLD were larger in IC203 than in WP2 uvrA/pKM101. The enhancements in the effects of INH, HZH and PHZ in IC203 with respect to its oxyR+ parent are considered to be caused by the production of reactive oxygen species. This is consistent with its inhibition in IC203 by S9 from liver of uninduced rats, probably through the action of catalase. Mutagenicities of INH, PHZ and HLZ were low in strains IC204, a derivative of WP2 uvrA carrying a deletion of the umuDC genes, and IC206, a derivative of IC204 deficient in the MutY glycosylase. In these strains, HZH and NLD induced a high level of revertants which carry suppressor mutations resulting exclusively from G:C-A:T transitions, thus suggesting a direct reaction of the two hydrazines with cytosine.
Asunto(s)
Proteínas de Unión al ADN , Escherichia coli/efectos de los fármacos , Isoniazida/toxicidad , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Animales , Escherichia coli/clasificación , Proteínas de Escherichia coli , Isoniazida/análogos & derivados , Hígado/fisiología , Mutagénesis , Oxidación-Reducción , Ratas , Especies Reactivas de Oxígeno/fisiología , Especificidad de la EspecieRESUMEN
New Escherichia coli strains have been added to the WP2 mutagenicity test for the specific detection of oxidative mutagens. Strain IC203 derives from WP2 uvrA/pKM101 and is highly sensitive to oxidative stress due to a deficiency in the OxyR function. Following exposure to t-butyl hydroperoxide (BuOOH) or menadione (MD), but not to 4-nitroquinoline 1-oxide (4NQO), strain IC203 (oxyR) shows increased mutability with respect to the oxyR+ parent. The advantage that the OxyR deficiency confers on IC203 strain in detecting oxidative mutagens is not obtained with strains deficient in either katG or ahpCF, two OxyR-regulated genes. Strain IC206, a derivative of WP2 uvrA carrying a deletion of the umuDC genes and deficient in the MutY glycosylase, has also been added to the WP2 test for the detection of SOS-independent mutations promoted by 8-oxoguanine lesions. Induction of these mutations was observed after treatment with BuOOH, but not after MD or 4NQO exposure. The two new strains, IC203 and IC206, can be useful for the screening of mutations resulting from oxidative stress as well as in studies on antioxidants preventing mutagenesis.
Asunto(s)
Proteínas de Unión al ADN , Escherichia coli/efectos de los fármacos , Mutágenos/toxicidad , Escherichia coli/genética , Proteínas de Escherichia coli , Guanina/análogos & derivados , Guanina/toxicidad , Oxidación-Reducción , Especies Reactivas de Oxígeno , Proteínas Represoras/fisiología , Respuesta SOS en Genética , Factores de Transcripción/fisiologíaRESUMEN
The induction of SOS-independent mutations by exposure to benzo[a]pyrene (BaP) was screened in Escherichia coli strains lacking SOS mutagenesis proteins and deficient in MutY or MutM glycosylases, which prevent mutations by 8-hydroxyguanine (GO lesion). Mutagenicity assays, performed in the presence of S9 mix, indicated a great increase in the reversion of the trpE65 ochre mutation in both mutY and mutY mutM strains, whereas a lower increase was observed in a mutM strain. This mutability by BaP was observed in either uvr- or uvr+ strains. Moreover, it was increased when strains carried a deletion of the oxyR gene that abolished the OxyR response to oxidative stress, and reduced in the presence of the oxyR2 allele that rendered constitutive such response. It is suggested that SOS-independent mutations in cells treated with BaP arise from adenine/GO mispairs. The interaction of radical scavengers with BaP ultimate metabolites could cause oxidative stress capable of producing GO lesions. Strains lacking mutagenesis proteins and deficient in base excision repair systems, such as those dependent on MutY and MutM glycosylases, could be useful for screening the induction of SOS-independent mutations.
Asunto(s)
Proteínas Bacterianas/genética , Benzo(a)pireno/toxicidad , ADN Glicosilasas , Proteínas de Escherichia coli , Escherichia coli/enzimología , Mutágenos/toxicidad , N-Glicosil Hidrolasas/metabolismo , Respuesta SOS en Genética , Adenosina Trifosfatasas/genética , Alelos , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN , ADN-Formamidopirimidina Glicosilasa , Escherichia coli/genética , Pruebas de Mutagenicidad , Estrés Oxidativo/genética , Peróxidos/farmacología , Proteínas Represoras/genética , Factores de Transcripción/genética , terc-ButilhidroperóxidoRESUMEN
Mutations induced by oxidative DNA damage appear to occur by two pathways, differing in their dependence on SOS mutagenesis. We have analysed the specificity of mutations produced by each pathway. Base substitutions generating extragenic suppressors were characterized in Trp+ revertants of Escherichia coli strains carrying the trpE65 ochre mutation, which were hypersensitive to oxidative mutagenesis due to a deletion of the oxyR gene. In strain IC3821, containing MucA/B proteins and therefore proficient for SOS mutagenesis, the more frequently scored base substitutions, either spontaneous or induced by t-butyl hydroperoxide (BuOOH), were T:A-A:T transversions, followed by G:C-A:T transitions, while the frequency of G:C-T:A transversions was lower. This SOS-dependent mutability could be promoted by abasic sites. In strains IC3894 (mutY) and IC3981 (mutY mutM), lacking mutagenesis proteins, SOS-independent revertants arose almost exclusively via G:C-T:A transversions probably derived from oxidatively damaged 8-oxoguanine/adenine mispairs. Formation of these mispairs in IC3894 and IC3981 would be enhanced by BuOOH treatment since it caused a significant increase in the revertant number. Strains IC3894 and IC3981 could have a complementary role to that of IC3821 to analyse the mutagenicity and the mutational specificity of oxidants.
Asunto(s)
ADN Glicosilasas , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Escherichia coli/genética , Mutación , N-Glicosil Hidrolasas/fisiología , Peróxidos/toxicidad , Proteínas Represoras/fisiología , Respuesta SOS en Genética , Factores de Transcripción/fisiología , ADN-Formamidopirimidina Glicosilasa , Escherichia coli/efectos de los fármacos , N-Glicosil Hidrolasas/genética , Especies Reactivas de Oxígeno/toxicidad , terc-ButilhidroperóxidoRESUMEN
The Escherichia coli strain IC3821, a delta oxyR derivative of WP2 uvrA trpE65, was more sensitive to mutagenicity promoted by t-butyl hydroperoxide and cumene hydroperoxide than the isogenic oxyR+ control. Mutagenicity of menadione, a redox cycling quinone, was clearly detected in the delta oxyR strain, whereas only a slight mutagenic response was observed in the oxyR+ strain. Plumbagin, another quinone structurally similar to menadione, was not mutagenic to any of the strains. These mutagenic responses appeared to involve the SOS processing of oxidative DNA lesions and were mediated by MucA/B proteins more efficiently than by UmuD/C. In cells lacking mutagenesis proteins, induction of SOS-independent mutations by the two alkyl hydroperoxides required a deficiency in the MutY DNA glycosylase and was increased by the presence of the delta oxyR mutation. In contrast, the two quinones assayed were unable to induce SOS-independent mutations in the MutY-deficient strains.
Asunto(s)
Derivados del Benceno/toxicidad , ADN Glicosilasas , Proteínas de Unión al ADN , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Mutágenos/toxicidad , N-Glicosil Hidrolasas/genética , Peróxidos/toxicidad , Proteínas Represoras/genética , Respuesta SOS en Genética/efectos de los fármacos , Factores de Transcripción/genética , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Eliminación de Gen , Mutación/efectos de los fármacos , Oxidación-Reducción , terc-ButilhidroperóxidoRESUMEN
Escherichia coli and Salmonella typhimurium strains deficient in the OxyR-regulated adaptive response to oxidative stress were used to study the mode in which spontaneous SOS-dependent mutations are generated in a distressed bacterial population. When assayed on supplemented selective medium, the E. coli strain IC3821 (trpE65), carrying the delta oxyR30 mutation and containing the plasmid pRW144 (mucA/B), showed a frequency of spontaneous Trp+ revertants similar to that of the oxyR+ control. Instead, the IC3821 strain exhibited an enhancement in the clonal occurrence of spontaneous revertants arising at random during growth on a nonselective medium. A similar enhancement was observed for the S. typhimurium strain TA4125 (hisG428 delta oxyR2). The mutator effect observed in oxyR- cells would be induced by an increased background of reactive oxygen species; it provides a model for studying the mutability of a cell population constantly exposed to mutation-inducing agents. In the IC3821 strain, revertants were induced by t-butyl hydroperoxide with higher efficiency than in oxyR+. We suggest that strain IC3821 could be useful for the detection of SOS-dependent mutagenesis induced by chemical oxidants.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN , Escherichia coli/genética , Mutagénesis/genética , Estrés Oxidativo/genética , Proteínas Represoras , Salmonella typhimurium/genética , Factores de Transcripción , Adaptación Biológica , Relación Dosis-Respuesta a Droga , Escherichia coli/efectos de los fármacos , Proteínas de Escherichia coli , Eliminación de Gen , Modelos Genéticos , Peróxidos/farmacología , Especies Reactivas de Oxígeno/farmacología , Respuesta SOS en Genética/genética , Salmonella typhimurium/efectos de los fármacos , Selección Genética , terc-ButilhidroperóxidoRESUMEN
We have studied the influence of the processing of MucA protein on the occurrence of base substitution mutations. Escherichia coli strains carrying the trpA23 missense mutation and having a full deletion of the chromosomal umuD/C operon were transformed with plasmids encoding the MucB protein together with either wild-type MucA or the nonprocessable MucA202 protein. The efficient reversion of the trpA23 allele by G.C-T.A transversions in benzo[a]pyrene (B[a]P)-treated cells required the function of a matured MucA protein. This processed protein was also necessary for the occurrence of G.C-T.A transversions targeted at spontaneous DNA lesions and for the SOS mutator effect dependent on the constitutive coprotease activity of the RecA730 protein. In contrast, G.C-T.A transversions reverting trpA23 were spontaneously generated by an SOS-independent mechanism in cells deficient in the MutY DNA glycosylase.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Glicosilasas , Mutágenos/metabolismo , N-Glicosil Hidrolasas/metabolismo , Mutación Puntual , Supresión Genética/genética , Benzo(a)pireno/toxicidad , Biotransformación , Reparación del ADN , Escherichia coli/genética , Péptido Hidrolasas/metabolismo , Procesamiento Proteico-Postraduccional , Rec A Recombinasas/metabolismo , Respuesta SOS en Genética , Triptófano Sintasa/genéticaRESUMEN
We have studied the mutability of Salmonella typhimurium tester strains carrying plasmids in which either the umuDC or the umuD'C operon of Escherichia coli have been cloned. Reversion of the hisD3052 frameshift mutation by benzo[a]pyrene (B[a]P), aflatoxin B1 (AFB1) and 1-nitropyrene (1-NP), was very efficiently promoted by UmuD' (the activated form of UmuD) and UmuC proteins. In contrast, UmuD'C proteins promoted a moderate reversion of the missense hisG46 allele by B[a]P, and were not effective in mediating this reversion by AFB1. The Salmonella tester strain carrying the hisD3052 allele and containing the E. coli UmuD'C proteins has a sensitivity toward frameshift mutagens similar to that of the MucAB containing strain TA98, and may be useful for obtaining a high level of mutants generated by the SOS mutagenic mechanism in the absence of MucAB proteins.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Mutación del Sistema de Lectura , Mutagénesis Insercional , Respuesta SOS en Genética , Salmonella typhimurium/genética , Aflatoxina B1/toxicidad , Proteínas Bacterianas/metabolismo , Benzo(a)pireno/toxicidad , Biotransformación , ADN Polimerasa Dirigida por ADN , Escherichia coli/genética , Genes Bacterianos , Microsomas Hepáticos/enzimología , Pruebas de Mutagenicidad , Mutágenos/toxicidad , Operón , Plásmidos , Pirenos/toxicidad , Salmonella typhimurium/efectos de los fármacos , Especificidad de la Especie , Supresión GenéticaRESUMEN
The correlation between the proficiency at promoting mutagenesis of MucA/B proteins and MucA processing has been considered to be very high (Hauser et al., 1992) on the basis of the results of UV mutagenicity (Shiba et al., 1990). Here we show that this correlation is only partial. We have assayed the mutagenicity of benzo[a]pyrene (B[a]P) and aflatoxin B1 (AFB1) in Salmonella typhimurium tester strains containing plasmids which encode MucA proteins with an altered cleavage site. Reversion of the frameshift hisD3052 mutation by B[a]P or AFB1 was observed in the presence of non-cleavable MucA protein although at a lower level than that found in cells containing wild-type MucA protein. Reversion of the base substitution hisG46 mutation by AFB1 requires a significant processing of MucA, while lower levels of this processing would be enough for the hisG46 reversion by B[a]P. These results suggest that the specificity of mutations induced by mutagens forming DNA adducts is influenced by the activity of MucA protein. They also show the relevance of mutagenicity assays in the mechanistic studies of mutagenesis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mutación del Sistema de Lectura , Mutágenos/toxicidad , Procesamiento Proteico-Postraduccional , Aflatoxina B1/toxicidad , Proteínas Bacterianas/genética , Benzo(a)pireno/toxicidad , Histidina/genética , Salmonella typhimurium/efectos de los fármacosRESUMEN
Escherichia coli B, unlike both E. coli K12 and Salmonella typhimurium, is sensitive to the rough-specific phage C21. This sensitivity is probably due to the incomplete lipopolysaccharide core of the E. coli B cells, which confers on them a partial permeability to large molecules. Derivatives of WP2 uvrA, a tryptophan-requiring E. coli B strain, were rendered still more permeable by selecting for C21-resistant clones. The new permeable strains, when tested for mutagenesis induced by polycyclic hydrocarbons, showed a mutagenic response higher than that of the parental strains.
Asunto(s)
Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Pruebas de Mutagenicidad , Mutágenos , Compuestos Policíclicos/toxicidad , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Benzopirenos/toxicidad , Permeabilidad de la Membrana Celular , Colifagos , Hidroxiacetilamino Fluoreno/toxicidad , Lipopolisacáridos/química , Metilcolantreno/toxicidad , Pruebas de Sensibilidad Microbiana , Especificidad de la Especie , Triptófano/metabolismoRESUMEN
We have isolated spontaneous mutant strains of Escherichia coli KL16 showing different levels of nalidixic acid (NAL) resistance. From 40 independent mutants, 36 had gyrA and four had gyrB mutations. Most of the gyrA mutations (30/36) conferred high level NAL resistance. In contrast, the only gyrB mutation that conferred a relatively high level of NAL resistance also determined enhanced susceptibility to quinolones with a piperazinyl substituent at C7 position of the quinolone ring (amphoteric quinolones). This gyrB mutation (denoted gyrB1604), jointly with a gyrA mutation (denoted gyrA972) which confers a high level of quinolone resistance, were used to construct strain IC2476, carrying the two gyr mutant alleles. The susceptibility of this strain to amphoteric quinolones (pipemidic acid, norfloxacin and ciprofloxacin) was similar to that of the gyrA972 single mutant. This result indicates that the change in GyrA subunit which determines a high level of quinolone-resistance has the capacity to mask the hypersusceptibility to amphoteric quinolones promoted by the GyrB1604 mutant subunit. This capacity was further confirmed by studying the effects of ciprofloxacin (CFX) on gyrase inhibition in the gyrA972 gyrB1604 strain.
Asunto(s)
Antiinfecciosos/farmacología , ADN-Topoisomerasas de Tipo II/genética , Escherichia coli/efectos de los fármacos , Mutación , Girasa de ADN , ADN-Topoisomerasas de Tipo II/fisiología , Farmacorresistencia Microbiana , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Ácido Nalidíxico/farmacología , Relación Estructura-ActividadRESUMEN
We examined, in Escherichia coli, the influence of recA mutant alleles on the level of quinolone resistance promoted by mutations in the gyrA gene. We found that the recA142 mutation, abolishing all the activities of RecA protein, greatly reduced the level of resistance to the quinolone ciprofloxacin, whereas the recA430 allele affecting the SOS inducing ability of RecA, reduced ciprofloxacin resistance to a lesser extent. The recA142 mutation did not cause enhancement of ciprofloxacin induced DNA breakage in gyrA mutants, indicating that the stabilization of DNA-gyrase complexes by the quinolone is not influenced by a RecA mutant protein. We suggest that RecA protein plays a role in the repair of quinolone damage, principally through a recombinational mechanism and, to a lesser degree, through the induction of the SOS response.
Asunto(s)
ADN-Topoisomerasas de Tipo II/genética , Escherichia coli/genética , Genes Bacterianos/efectos de los fármacos , Mutación , Quinolonas/farmacología , Rec A Recombinasas/genética , Reparación del ADN , Farmacorresistencia Microbiana , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Respuesta SOS en GenéticaRESUMEN
Changes in plasmid DNA supercoiling were measured following treatment of Escherichia coli cells, carrying topoisomerase mutations, with the quinolone ciprofloxacin. In quinolone-susceptible cells (top+ gyr+) as well as in topA mutants and in gyrB mutants, plasmid DNA was relaxed after the addition of ciprofloxacin. In cells partially resistant to quinolones, low ciprofloxacin levels led to an increase in negative superhelicity of plasmid DNA, whereas at higher ciprofloxacin concentrations, DNA became relaxed. Cells exhibiting partial resistance to quinolones carried either a gyrA mutation alone or a combination of gyrA and gyrB mutations. Moreover, they showed a reduction in gyrase activity, indicated by the supercoiling of a reporter plasmid. Therefore, we conclude that a low level of quinolone action and a DNA with a lower-than-normal level of superhelicity are the two essential conditions for obtaining a ciprofloxacin-promoted increase in plasmid DNA supercoiling. In contrast, deficiency in topoisomerase I is not required for this effect.
Asunto(s)
Ciprofloxacina/farmacología , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal/efectos de los fármacos , Escherichia coli/enzimología , Conformación de Ácido Nucleico/efectos de los fármacos , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo II/genética , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Mutación , Plásmidos , Quinolinas/farmacologíaRESUMEN
We studied the influence of DNA topological changes on Escherichia coli recA gene expression. This was monitored by measuring beta-galactosidase activity in cells containing a recA-lacZ fusion. To modulate DNA supercoiling we used mutations in the genes encoding for topoisomerase I and DNA gyrase. After either UV irradiation or treatment with the gyrase inhibitor ciprofloxacin, induction of the recA gene was reduced in topA10 mutants, this reduction being alleviated when gyrA or gyrB mutations causing DNA relaxation were present. A reduced induction of recA was also observed after incubation of cells carrying the recA441 mutation at 42 degrees C in the presence of adenine. Using bacteria deficient in the LexA repressor, we have demonstrated that the topA10 mutation reduces the constitutive expression of the recA gene. We suggest that the increase in negative supercoiling resulting from topoisomerase I deficiency interferes with transcription from the recA promoter. The reduction in the expression of the recA gene in topA10 bacteria could determine their increased UV sensitivity as well as their partial defectiveness in SOS mutability.