Asunto(s)
Anticoagulantes/efectos adversos , Hidrocarburo de Aril Hidroxilasas/genética , Negro o Afroamericano/genética , Hemorragia Gastrointestinal/inducido químicamente , Polimorfismo de Nucleótido Simple , Warfarina/efectos adversos , Anticoagulantes/farmacocinética , Fibrilación Atrial/tratamiento farmacológico , Comorbilidad , Citocromo P-450 CYP2C9 , Análisis Mutacional de ADN , Hemorragia Gastrointestinal/genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ADN , Warfarina/farmacocinéticaRESUMEN
Most of the DNA damage produced by ionizing radiation is repaired by the base excision repair (BER) pathway. To determine whether the BER genes were up-regulated by low doses of ionizing radiation, we investigated their expression in TK6 human lymphoblastoid cells by measuring mRNA levels using real-time quantitative PCR. No induction at the transcriptional level of any of the base excision repair genes, NTH1 (NTHL1), OGG1, NEIL1, NEIL2, NEIL3, APE1, POLB, or accessory protein genes, LIG3, XRCC1 or XPG, was found at gamma-radiation doses ranging from 1 cGy to 2 Gy in a 24-h period. As has been measured in other cell lines, a dose-dependent induction of CDKN1A (WAF1) mRNA levels was observed in TK6 cells in the dose range of 0.5 to 2.0 Gy. We also examined BER enzyme activity on 8-oxoguanine-, dihydrouracil- and furan-containing oligonucleotide substrates and found no increase in extracts of TK6 cells after gamma-ray doses of 0.5-2.0 Gy. These data were corroborated by Western blot analysis of APE1 and NTH1, suggesting that the BER enzymes are also not up-regulated at the post-transcriptional level after ionizing radiation exposure.
Asunto(s)
Reparación del ADN , ADN/efectos de la radiación , Oxígeno/metabolismo , Radiación Ionizante , Western Blotting , Línea Celular Tumoral , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Humanos , Oligonucleótidos/química , ARN/química , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética , Regulación hacia ArribaRESUMEN
Cytochrome P450 (CYP) 2C9 is the principal enzyme responsible for the metabolism of numerous clinically important drugs. Two polymorphic alleles CYP2C9*2 and CYP2C9*3 have been documented which affect the metabolism and clinical toxicity of drugs such as phenytoin, warfarin, glipizide, and tolbutamide. The present study reports the first example of a null polymorphism in CYP2C9. This mutation dramatically affects the half-life and clinical toxicity of phenytoin. The study subject was a female African-American presented to the emergency department with phenytoin toxicity evidenced by mental confusion, slurred speech, memory loss and the inability to stand. She exhibited extremely poor clearance of phenytoin with an elimination half-life of approximately 13 days. Genotyping studies demonstrated that the patient did not possess any known variant CYP2C9 alleles. Phenytoin is metabolized to a minor extent by the polymorphic CYP2C19, but this individual did not possess any variant CYP2C19 alleles. Sequencing studies revealed that the individual was homozygous for a new CYP2C9 allele (CYP2C9*6) with the deletion of an adenine at base pair 818 of the cDNA. The clearance of phenytoin in this individual is estimated to be approximately 17% of that observed in normal patients. The frequency of this allele was 0.6% (95% confidence limits of 0.1 to 3.5%) in 79 African-Americans and 0% (95% confidence limits of 0 to 1.1%) in 172 Caucasians. The study also demonstrates the severe clinical consequences to patients with a null mutation in CYP2C9 after treatment with normal doses of phenytoin.
Asunto(s)
Alelos , Anticonvulsivantes/efectos adversos , Hidrocarburo de Aril Hidroxilasas , Población Negra/genética , Sistema Enzimático del Citocromo P-450/genética , Fenitoína/efectos adversos , Eliminación de Secuencia , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/genética , Administración Oral , Anticonvulsivantes/administración & dosificación , Anticonvulsivantes/sangre , Anticonvulsivantes/farmacocinética , Citocromo P-450 CYP2C9 , Femenino , Frecuencia de los Genes , Genotipo , Homocigoto , Humanos , Tasa de Depuración Metabólica/genética , Persona de Mediana Edad , Mutación , Fenitoína/administración & dosificación , Fenitoína/sangre , Fenitoína/farmacocinética , Polimorfismo Genético , Convulsiones/inducido químicamente , Convulsiones/etnología , Convulsiones/genética , Análisis de Secuencia de ADNRESUMEN
Cytochrome P450 (CYP) 2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel (Taxol). It is also the predominant P450 responsible for the metabolism of arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs) in human liver and kidney. In this study, we describe two new CYP2C8 alleles containing coding changes: CYP2C8*2 has an Ile269Phe substitution in exon 5 and CYP2C8*3 includes both Arg139Lys and Lys399Arg amino acid substitutions in exons 3 and 8. CYP2C8*2 was found only in African-Americans, while CYP2C8*3 occurred primarily in Caucasians. Neither occurred in Asians. The frequency of the CYP2C8*2 allele was 0.18 in African-Americans, and that of CYP2C8*3 was 0.13 in Caucasians. CYP2C8*1 (wild-type), CYP2C8*2 and CYP2C8*3 cDNAs were expressed in Escherichia coli, and the ability of these enzymes to metabolize both paclitaxel and arachidonic acid was assessed. Recombinant CYP2C8*3 was defective in the metabolism of both substrates. The turnover number of CYP2C8*3 for paclitaxel was 15% of CYP2C8*1. CYP2C8*2 had a two-fold higher Km and two-fold lower intrinsic clearance for paclitaxel than CYP2C8*1. CYP2C8*3 was also markedly defective in the metabolism of arachidonic acid to 11,12- and 14,15-EET (turnover numbers 35-40% that of CYP2C8*1). Thus, CYP2C8*3 is defective in the metabolism of two important CYP2C8 substrates: the anticancer drug paclitaxel and the physiologically important compound arachidonic acid. This polymorphism has important clinical and physiological implications in individuals homozygous for this allele.
Asunto(s)
Antineoplásicos Fitogénicos/farmacocinética , Ácido Araquidónico/farmacocinética , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Paclitaxel/farmacocinética , Polimorfismo Genético/genética , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/genética , Alelos , Línea Celular , Citocromo P-450 CYP2C8 , Genotipo , Humanos , Tasa de Depuración Metabólica , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Análisis de Secuencia de ADN/métodosRESUMEN
It has been postulated that ionizing radiation produces a unique form of cellular DNA damage called "clustered damages" or "multiply damaged sites". Here, we show that clustered DNA damages are indeed formed in Escherichia coli by ionizing radiation and are converted to lethal double-strand breaks during attempted base-excision repair. In wild-type cells possessing the oxidative DNA glycosylases that cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during postirradiation incubation and also in a dose-dependent fashion. E. coli mutants lacking these enzymes do not form double-strand breaks postirradiation and are substantially more radioresistant than wild-type cells. Furthermore, overproduction of one of the oxidative DNA glycosylases in mutant cells confers a radiosensitive phenotype and an increase in the number of double-strand breaks. Thus, the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.
Asunto(s)
Disparidad de Par Base , Daño del ADN , Reparación del ADN , ADN Bacteriano/efectos de la radiación , Escherichia coli/genética , ADN Glicosilasas , ADN Bacteriano/genética , Relación Dosis-Respuesta en la Radiación , Escherichia coli/efectos de la radiación , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Rayos XRESUMEN
Energy from low LET ionising radiation, such as X rays and gamma rays, is deposited in the water surrounding the DNA molecule such that between 2 to 5 radical pairs are generated within a radius of I to 4 nm. As a result, multiple single lesions, including oxidised purine or pyrimidine bases, sites of base loss, and single-strand breaks, can be formed in DNA from the same radiation energy deposition event. The single lesions in these so-called multiply damaged sites or clustered lesions are repaired by base excision repair. Here we show that clustered DNA damages are formed in bacterial cells by ionising radiation and are converted to lethal double-strand breaks during attempted repair. In wild type cells possessing the oxidative DNA glycosylases that recognise and cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during post-irradiation incubation and in a dose-dependent fashion. Mutant cells lacking these enzymes do not form double-strand breaks post-irradiation and are substantially more radioresistant than wild type cells. These radioresistant mutant cells can be made radiosensitive by overexpressing one of the oxidative DNA glycosylases. Thus the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.
Asunto(s)
Reparación del ADN/fisiología , ADN/efectos de la radiación , Disparidad de Par Base , ADN/genética , Daño del ADN , ADN Glicosilasas , Relación Dosis-Respuesta en la Radiación , Humanos , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismoRESUMEN
This study tested the hypothesis that inhaled nitric oxide (NO) and combined NO and hyperoxia will result in less pulmonary dysfunction and delay onset of respiratory signs compared with hyperoxia-exposed newborn guinea pigs (GPs). GPs were exposed to room air (n = 14), 95% O(2) (n = 36), 20 parts per million (ppm) NO (n = 14), or combined 20 ppm NO and 95% O(2) (NO/O(2), n = 13) for up to 5 days. Data evaluated included latency interval for onset of respiratory distress, pressure volume curves, lung histology, and bronchoalveolar lavage (BAL) polymorphonuclear cells (PMNs), proteolytic activity, and total protein. NO-exposed GPs did not develop respiratory distress and had no evidence of pulmonary dysfunction. O(2)-exposed GPs developed respiratory distress after 1-5 days (median 4.0) vs. 3-5 days (median 5.0) for NO/O(2) exposure (P < 0.05). BAL from O(2)-exposed GPs showed increased PMNs compared with NO/O(2)-exposed GPs. O(2)- and NO/O(2)-exposed GPs had comparable reduced lung volumes, lung histology, and increased BAL proteinase activity and total protein. In summary 1) O(2) exposure resulted in multiple measures of pulmonary dysfunction in newborn GPs, 2) 5-day exposure to NO produced no noticeable respiratory effects and pulmonary dysfunction, and 3) short-term exposure (=5 days) to NO/O(2) delayed onset of respiratory distress and neither exacerbated nor attenuated pulmonary dysfunction compared with O(2) exposure alone.
Asunto(s)
Animales Recién Nacidos/fisiología , Hiperoxia/complicaciones , Enfermedades Pulmonares/prevención & control , Óxido Nítrico/administración & dosificación , Administración por Inhalación , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Quimotripsina/metabolismo , Cobayas , Recuento de Leucocitos , Pulmón/patología , Pulmón/fisiopatología , Enfermedades Pulmonares/etiología , Enfermedades Pulmonares/patología , Mediciones del Volumen Pulmonar , Neutrófilos , Oxígeno/administración & dosificación , Tripsina/metabolismoRESUMEN
Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), MutY DNA glycosylase, endonuclease VIII, and endonuclease III are oxidative base excision repair DNA glycosylases that remove oxidized bases from DNA, or an incorrect base paired with an oxidized base in the case of MutY. Since genes encoding other base excision repair proteins have been shown to be part of adaptive responses in E. coli, we wanted to determine whether the oxidative DNA glycosylase genes are induced in response to conditions that cause the type of damage their encoded proteins remove. The genes fpg, mutY, nei, and nth encode Fpg, MutY, endonuclease VIII, and endonuclease III, respectively. Multiprobe RNase protection assays were used to examine the transcript levels of these genes under conditions that induce the SoxRS, OxyR, and SOS regulons after a shift from anaerobic to aerobic growth and at different stages along the growth curve. Transcript levels for all four genes decreased as cells progressed from log-phase growth to stationary phase and increased after cells were shifted from anaerobic to aerobic growth. None of the genes were induced by hydrogen peroxide, paraquat, X rays, or conditions that induce the SOS response.
Asunto(s)
ADN Glicosilasas , Reparación del ADN , Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli , Escherichia coli/enzimología , N-Glicosil Hidrolasas/genética , Estrés Oxidativo , ARN Bacteriano , ARN Mensajero , Aerobiosis , Anaerobiosis , ADN-Formamidopirimidina Glicosilasa , Desoxirribonucleasa (Dímero de Pirimidina) , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Genes Bacterianos , Ribonucleasas , Transcripción GenéticaRESUMEN
In the bacterium Escherichia coli, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the nth and nei genes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C-->A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C-->T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in fpg mutY double mutants was significantly enhanced by the addition of the nei mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C-->T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.
Asunto(s)
ADN Glicosilasas , Reparación del ADN , Desoxirribonucleasa (Dímero de Pirimidina) , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Guanosina/análogos & derivados , Mutagénesis , Citosina/química , ADN-Formamidopirimidina Glicosilasa , Escherichia coli/enzimología , Guanosina/metabolismo , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Oxidación-ReducciónRESUMEN
Extrahepatic tissue distribution of the mRNAs for the four human CYP2Cs (2C8, 2C9, 2C18, and 2C19) was examined in kidney, testes, adrenal gland, prostate, brain, uterus, mammary gland, ovary, lung, and duodenum. CYP2C mRNAs were detected by RT-PCR using specific primers for each individual CYP2C. CYP2C8 mRNA was detected in the kidney, adrenal gland, brain, uterus, mammary gland, ovary, and duodenum. CYP2C9 mRNA was detected in the kidney, testes, adrenal gland, prostate, ovary, and duodenum. CYP2C18 mRNA was found only in the brain, uterus, mammary gland, kidney, and duodenum and CYP2C19 mRNA was found only in the duodenum. Immunoblot analysis of small intestinal microsomes detected both 2C9 and 2C19 proteins. In addition, genomic clones for CYP2C8 were sequenced, and long-distance PCR was performed to determine the complete gene structure. CYP2C8 spanned a 31 kb region. Comparative analysis of the 2.4 kb upstream region of CYP2C8 with CYP2C9 revealed two previously unidentified transcription factors sites, C/EBP and HPF-1, and the latter might be involved in hepatic expression. Although CYP2C8 has been shown to be phenobarbital inducible, neither a barbiturate-responsive regulatory sequence (a Barbie box) nor a phenobarbital-responsive enhancer module (PBREM) was found within the upstream region analyzed.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Genes/genética , Hígado/metabolismo , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/genética , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Sistema Enzimático del Citocromo P-450/metabolismo , ADN/química , ADN/genética , Exones , Femenino , Expresión Génica , Humanos , Intrones , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Distribución TisularRESUMEN
Cytochrome P-450 (CYP) 2C19 is responsible for the metabolism of a number of therapeutic agents such as S-mephenytoin, omeprazole, proguanil, certain barbiturates, diazepam, propranolol, citalopram and imipramine. Genetic polymorphisms in this enzyme are responsible for the poor metabolizers (PM) of mephenytoin, which represent approximately 13-23% of Asians and 3-5% of Caucasians. Several polymorphisms contribute to this phenotype. We have isolated two new allelic variants that contribute to the PM phenotype in Caucasians. CYP2C19*7 contained a single T --> A nucleotide transversion in the invariant GT at the 5' donor splice site of intron 5. The second PM allele, CYP2C19*8, consisted of a T358C nucleotide transition in exon 3 that results in a Trp120Arg substitution. In a bacterial expression system, CYP2C198 protein exhibited a dramatic (approximately 90% and 70%) reduction in the metabolism of S-mephenytoin and tolbutamide, respectively, when compared with the wild-type CYP2C191B protein. Restriction fragment length polymerase chain reaction tests were developed to identify the new allelic variants.
Asunto(s)
Anticonvulsivantes/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Mefenitoína/metabolismo , Oxigenasas de Función Mixta/genética , Alelos , Citocromo P-450 CYP2C19 , Inhibidores Enzimáticos del Citocromo P-450 , Escherichia coli/genética , Escherichia coli/metabolismo , Exones , Francia , Genotipo , Humanos , Intrones , Pulmón/enzimología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Fenotipo , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Población BlancaRESUMEN
Genetic polymorphisms in the cytochrome P450 (CYP) family are widely known to contribute to interindividual differences in the pharmacokinetics of many drugs. Several alleles for the CYP2C9 gene have been reported. Individuals homozygous for the Leu359 variant (CYP2C9*3) have been shown to have significantly lower drug clearances compared with Ile359 (CYP2C9*1) homozygous individuals. A male Caucasian who participated in six bioavailability studies in our laboratory over a period of several years showed extremely low clearance of two drugs: phenytoin and glipizide (both substrates of CYP2C9), but not for nifedipine (a CYP3A4 substrate) and chlorpheniramine (a CYP2D6 substrate). His oral clearance of phenytoin was 21% of the mean of the other 11 individuals participating in the study, and his oral clearance of glipizide, a second generation sulfonylurea structurally similar to tolbutamide, was only 188% of the mean of the other 10 individuals. However, his oral clearance of nifedipine and chlorpheniramine did not differ from individuals in other studies performed at our laboratories. An additional blood sample was obtained from this individual to determine if he possessed any of the known CYP2C9 or CYP2C19 allelic variants that would account for his poor clearance of the CYP2C9 substrates (phenytoin and glipizide) compared with the CYP3A4 (nifedipine) and CYP2D6 (chlorpheniramine) substrates. The results of the genotype testing showed that this individual was homozygous for the CYP2C9*3 allele and did not possess any of the known defective CYP2C19 alleles. This study establishes that the Leu359 mutation is responsible for the phenytoin and glipizide/tolbutamide poor metabolizer phenotype.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Clorfeniramina/farmacocinética , Sistema Enzimático del Citocromo P-450/genética , Glipizida/farmacocinética , Nifedipino/farmacocinética , Fenitoína/farmacocinética , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/genética , Adulto , Alelos , Clorfeniramina/sangre , Citocromo P-450 CYP2C9 , Genotipo , Glipizida/sangre , Homocigoto , Humanos , Masculino , Nifedipino/sangre , Fenotipo , Fenitoína/sangreRESUMEN
Cardiac troponin I (cTnl) is highly specific for cardiac muscle. In this study, we compared the utility of CK and CK-MB index versus cTnl in the assessment of myocardial infarction in 155 patients being evaluated for myocardial damage. As a cardiac marker for MI, Troponin I seems to be superior to CK-MB. In the subset of patients with renal disease, cTnl has definite advantages over CK-MB. In addition, the use of cTnl has the potential to replace the measurement of lactate dehydrogenase isoenzymes.
Asunto(s)
Creatina Quinasa/sangre , Isquemia Miocárdica/diagnóstico , Troponina I/sangre , Biomarcadores/sangre , Humanos , Isoenzimas , Fallo Renal Crónico/sangre , L-Lactato Deshidrogenasa/sangre , Isquemia Miocárdica/sangre , Curva ROC , Estudios RetrospectivosRESUMEN
A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large interracial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. In the present study, we identified two new mutations in CYP2C19 in a single Swiss Caucasian PM outlier (JOB 1) whose apparent genotype (CYP2C19*1/CYP2C19*2) did not agree with his PM phenotype. These mutations consisted of a single base pair mutation (G395A) in exon 3 resulting in an Arg132-->Gln coding change and a (G276C) mutation in exon 2 resulting in a coding change Glu92-->Asp. However, the G276C mutation and the G395A mutation resided on separate alleles. Genotyping tests of a family study of JOB1 showed that the exon 2 change occurred on the CYP2C19*2 allele, which also contained the known splice mutation in exon 5 (this variant is termed CYP2C19*2B to distinguish it from the original splice variant now termed CYP2C19*2A). The exon 3 mutation resided on a separate allele (termed CYP2C19*6). In all other respects this allele was identical to one of two wild-type alleles, CYP2C19*1B. The incidence of CYP2C19*6 in a European Caucasian population phenotyped for mephenytoin metabolism was 0/344 (99% confidence limits of 0 to 0.9%). Seven of 46 Caucasian CYP2C19*2 alleles were CYP2C19*2B(15%) and 85% were CYP2C19*2A. The Arg132Gln mutation was produced by site-directed mutatgenesis and the recombinant protein expressed in a bacterial cDNA expression system. Recombinant CYP2C19 6 had negligible catalytic activity toward S-mephenytoin compared with CYP2C19 1B, which is consistent with the conclusion that CYP2C19*6 represents a PM allele. Thus, the new CYP2C19*6 allele contributes to the PM phenotype in Caucasians.
Asunto(s)
Alelos , Anticonvulsivantes/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/genética , Mefenitoína/metabolismo , Oxigenasas de Función Mixta/genética , Población Blanca/genética , Citocromo P-450 CYP2C19 , Humanos , Mutagénesis Sitio-DirigidaRESUMEN
Five murine cytochrome P450 (CYP) 2C cDNAs were cloned and characterized, including four new members of this subfamily: CYP2C37, CYP2C38, CYP2C39, and CYP2C40. The cDNAs ranged from 1716 to 1812 bp in length and encoded polypeptides of 490 amino acid residues except for CYP2C40, which contained an additional glutamic acid residue at the carboxyl terminus. The amino acid identity of the murine CYP2Cs ranged from 69 to 92%, while the overall amino acid identity was 60%; however, within the six putative substrate recognition sites the identity was only 25 to 41%, suggesting possible differences in substrate specificity and product profiles. The CYP2C cDNAs were expressed in Escherichia coli following modification of the N-terminus. All five recombinant CYP2Cs metabolized arachidonic acid, but with different metabolic profiles and catalytic rates. Based on coelution with authentic standards on reverse-phase HPLC, themajor metabolites were tentatively identified asfollows: CYP2C29 and CYP2C39 produced 14, 15-cis-epoxyeicosatrienoic acid (EET); CYP2C37 produced 12-hydroxyeicosatetraenoic acid (HETE); CYP2C38 produced 11,12-EET; and CYP2C40 produced an unidentified metabolite that coeluted with 16-,17-, and 18-HETEs. The turnover numbers for CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 were 0.34, 1.12, 5.15, 0.51, and 0.15 nmol/nmol/min, respectively. Reverse transcriptase-polymerase chain reaction demonstrated the presence of CYP2C29 mRNA in liver as well as in extrahepatic tissues including brain, kidney, lung, heart, and intestine. CYP2C38 and CYP2C40 were found in liver, brain, kidney, and intestine, with trace amounts in lung and heart, while CYP2C37 and CYP2C39 appeared to be liver specific.
Asunto(s)
Ácido Araquidónico/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Isoenzimas/biosíntesis , Isoenzimas/genética , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/biosíntesis , Esteroide Hidroxilasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Clonación Molecular , Sistema Enzimático del Citocromo P-450/metabolismo , Activación Enzimática , Femenino , Humanos , Isoenzimas/metabolismo , Masculino , Ratones , Microsomas Hepáticos/enzimología , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Esteroide Hidroxilasas/metabolismoRESUMEN
Two members of the canine cytochrome P4502C subfamily [CYP2C21 and CYP2C41 (sequence has been submitted to Genbank with accession number AF016248)] were cloned from three beagle liver cDNA libraries. The two canine CYP2C cDNAs exhibited 70% nucleotide and amino acid identity as well as 74-83% nucleotide and 67-76% amino acid identity with the human CYP2Cs. Canine CYP2C41 is more homologous to the human CYP2Cs than CYP2C21. The two canine CYP2C cDNAs exhibited a slightly lower nucleotide and amino acid identity (66-77%) with the rat P450CYPs, 2C11 and 2C12. Reverse transcription-polymerase chain reaction-based restriction enzyme tests for CYP2C21 and 2C41 mRNAs as well as polymerase chain reaction-based tests for genomic DNA were developed. CYP2C21 cDNA was present in the livers of all dogs tested (N = 9), but CYP2C41 was present in only 1 of the 9 (11%). Genomic tests found that the gene coding for CYP2C21 was also present in all dogs tested (N = 25), of which 15 were beagles and 10 mixed breeds. In contrast, the gene coding for CYP2C41 was present in only 16% (4 out of 25) of the dogs. An even distribution of the CYP2C41 gene was found between the sexes and between beagles and mixed breeds. This unique polymorphism in the canine CYP2C subfamily may be a source of variability in the metabolic clearance in dogs of xenobiotics that are metabolized by the cytochrome P450 2C subfamily of enzymes.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/química , Regulación de la Expresión Génica/genética , Hígado/enzimología , Esteroide 16-alfa-Hidroxilasa , Esteroide Hidroxilasas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Perros , Femenino , Genotipo , Isoenzimas/química , Masculino , Datos de Secuencia Molecular , Polimorfismo Genético/genética , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity. The mechanisms of action of the dominant negative cdc42(D118A) mutant and the lethal, gain of function cdc42(G12V) mutant were examined. Cdc42(D118A,C188S)p and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the two-hybrid system, which correlated with the temperature dependence of the cdc42(D118A) phenotype and supported a Cdc24p sequestration model for the mechanism of cdc42(D118A) action. Five cdc42 mutations were isolated that led to decreased interactions with Cdc24p. The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42(D118A, C188S)p-Cdc24p interactions and could suppress the cdc42(D118A) mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. The cdc42(G12V) mutant phenotypes were suppressed by the intragenic T35A and K183-187Q mutations and in skm1Delta and cla4Delta cells but not ste20Delta cells, suggesting that the mechanism of cdc42(G12V) action is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors of cdc42(G12V) were also identified that displayed a dominant negative phenotype at 16 degrees C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Unión al GTP/genética , Genes Dominantes , Genes Letales , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Mutación , Fenotipo , Saccharomyces cerevisiae/enzimología , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Temperatura , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiaeRESUMEN
The 4'-hydroxylation of the S-enantiomer of the anticonvulsant drug mephenytoin exhibits a genetic polymorphism in humans. This polymorphism shows marked interracial heterogeneity, with the poor metabolizer (PM) phenotype representing 2 to 5% of Caucasian and 13 to 23% of Asian populations. Two defective CYP2C19 alleles, CYP2C19*2 and CYP2C19*3, have been described which account for approximately 87% of Caucasian and > 99% of Oriental PM alleles. The present study identifies a new allele (CYP2C19*4) in Caucasian PMs which contains an A-->G mutation in the initiation codon. A new polymerase chain reaction-restriction fragment length polymorphism genotyping test was developed, and the incidence of this allele was examined in a European Caucasian population which had been phenotyped for mephenytoin metabolism. One of nine putative PMs was heterozygous for CYP2C19*2/CYP2C19*4, which suggests that CYP2C19*4 represents a defective allele. Six of the seven remaining putative PMs available for genotyping were explained by CYP2C19*2. The frequency of the CYP2C19*4 allele in Caucasians was 0.6%. An additional Caucasian PM from a separate study was also heterozygous for CYP2C19*2 and CYP2C19*4. To verify that CYP2C19*4 represented a defective CYP2C19 allele, the initiation codon of the normal CYP2C19*1 cDNA was mutated to a GTG, and both cDNAs were expressed in yeast. Recombinant CYP2C19 protein was detected by Western blot analysis of colonies transformed with CYP2C19*1 cDNA, but not in those transformed with CYP2C19*4 cDNA. The two cDNAs were also used in an in vitro coupled transcription/translation assay. CYP2C19 protein was translated only from the CYP2C19*1 allele. These data indicate that CYP2C19*4 represents a new PM allele.
Asunto(s)
Anticonvulsivantes/metabolismo , Hidrocarburo de Aril Hidroxilasas , Codón , Sistema Enzimático del Citocromo P-450/genética , Mefenitoína/metabolismo , Oxigenasas de Función Mixta/genética , Mutación , Alelos , Citocromo P-450 CYP2C19 , Femenino , Humanos , MasculinoRESUMEN
The metabolism of the anticonvulsant drug mephenytoin exhibits a genetic polymorphism in humans. This polymorphism exhibits marked racial heterogeneity, with the poor metabolizer PM phenotype representing 13-23% of oriental populations, but only 2-5% of Caucasian populations. Two defective CYP2C19 alleles (CYP2C19*2 and CYP2C19*3) have been described, which account for more than 99% of Oriental poor metabolizer alleles but only approximately 87% of Caucasian poor metabolizer alleles. Therefore, additional defects presumably contribute to the poor metabolizer in Caucasians. Recent studies have found a third mutation CYP2C19*4, which accounts for approximately 3% of Caucasian poor metabolizer alleles. A fourth rare mutation (CYP2C19*5A) (C99,A991,Ile331;C1297T,Arg433-->Trp) resulting in an Arg433 to Trp substitution in the heme-binding region has been reported in a single Chinese poor metaboliser outlier belonging to the Bai ethnic group. The present study identifies a second variant allele CYP2C19*5B (C99-->T; A991-->G, Ile331-->Val; C1297-T, Arg433-->Trp in one of 37 Caucasian poor metabolizers. The frequency of the CYP2C19*5 alleles is low in Chinese (approximately 0.25% in the Bai ethnic group) and Caucasians (< 0.9%). However, these alleles contribute to the poor metabolizer phenotype in both ethnic groups and increases the sensitivity of the genetic tests for identifying defective alleles to approximately 100% in Chinese poor metabolizers and 92% in Caucasian poor metabolizers genotyped in our laboratory. The Arg433 to Trp mutation in the heme-binding region essentially abolishes activity of recombinant CYP2C19*5A toward S-mephenytoin and tolbutamide, which is consistent with the conclusion that CYP2C19*5 represents poor metabolizer alleles.