RESUMEN
We have examined MC1R variant allele frequencies in the general population of South East Queensland and in a collection of adolescent dizygotic and monozygotic twins and family members to define statistical associations with hair and skin color, freckling, and mole count. Results of these studies are consistent with a linear recessive allelic model with multiplicative penetrance in the inheritance of red hair. Four alleles, D84E, R151C, R160W, and D294H, are strongly associated with red hair and fair skin with multinomial regression analysis showing odds ratios of 63, 118, 50, and 94, respectively. An additional three low-penetrance alleles V60L, V92M, and R163Q have odds ratios 6, 5, and 2 relative to the wild-type allele. To address the cellular effects of MC1R variant alleles in signal transduction, we expressed these receptors in permanently transfected HEK293 cells. Measurement of receptor activity via induction of a cAMP-responsive luciferase reporter gene found that the R151C and R160W receptors were active in the presence of NDP-MSH ligand, but at much reduced levels compared with that seen with the wild-type receptor. The ability to stimulate phosphorylation of the cAMP response element binding protein (CREB) transcription factor was also apparent in all stimulated MC1R variant allele-expressing HEK293 cell extracts as assessed by immunoblotting. In contrast, human melanoma cell lines showed wide variation in the their ability to undergo cAMP-mediated CREB phosphorylation. Culture of human melanocytes of known MC1R genotype may provide the best experimental approach to examine the functional consequences for each MC1R variant allele. With this objective, we have established more than 300 melanocyte cell strains of defined MC1R genotype.
Asunto(s)
Alelos , Variación Genética , Pigmentación/genética , Receptores de Corticotropina/genética , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Genotipo , Humanos , Melanocitos/citología , Melanocitos/metabolismo , Fenotipo , Pigmentación/fisiología , Receptores de Corticotropina/metabolismo , Receptores de Melanocortina , alfa-MSH/metabolismoRESUMEN
Migraine is a debilitating disorder affecting approximately 12% of Caucasian populations. The disease has a large genetic component, although at present the type and number of genes involved is unclear. Candidate gene studies may be useful strategies for identifying genes involved in complex diseases such as migraine, especially if the gene being examined contributes only a minor effect to the overall phenotype. Nitric oxide (NO) is emerging as a key molecule affecting the pain associated with migraine. Since NO synthase (NOS) enzymes catalyze the synthesis of NO, the genes that code for these enzymes are good candidates for migraine molecular genetic analysis. This study investigated the role of a functionally relevant bi-allelic tetranucleotide polymorphism located in the promoter region of the human inducible nitric oxide synthase (iNOS) gene in migraine etiology. A large group of migraine affected individuals (n = 261) were genotyped and compared with an age- and sex-matched group of unaffected controls (n = 252). Results of a chi-squared analysis indicated that allele distributions for both migraine cases and controls were not significantly different (chi2 = 1.93, P = 0.16). These findings offer no evidence for an allelic association of the tested iNOS polymorphism with the common forms of the disease and therefore do not support a role for this gene in migraine pathogenesis.
Asunto(s)
Predisposición Genética a la Enfermedad , Trastornos Migrañosos/genética , Óxido Nítrico Sintasa/genética , Adulto , Alelos , Frecuencia de los Genes , Pruebas Genéticas , Genotipo , Humanos , Análisis por Apareamiento , Repeticiones de Microsatélite , Trastornos Migrañosos/clasificación , Óxido Nítrico Sintasa de Tipo II , Reacción en Cadena de la Polimerasa , Polimorfismo GenéticoRESUMEN
Certain dietary constituents can protect against chemically induced carcinogenesis in rodents. A principal mechanism by which these chemopreventive compounds exert their protective effects is likely to be via induction of carcinogen detoxification. This can be mediated by conjugation with glutathione, which is synthesized by the sequential actions of glutamate-cysteine ligase (GLCL) and glutathione synthetase. We have demonstrated that dietary administration of the naturally occurring chemopreventive agents, ellagic acid, coumarin or alpha-angelicalactone caused an increase in GLCL activity of between approximately 3- and 5-fold in rat liver. Treatment with the synthetic antioxidant ethoxyquin or the classic inducer phenobarbital caused < 2-fold induction of GLCL activity in rat liver, which was not found to be significant. The increases in GLCL activity were accompanied by increases (between 2- and 4-fold) in levels of both the catalytic heavy subunit (GLCLC) and regulatory light subunit (GLCLR). No substantial induction of GLCL was observed in rat kidney. The glutathione S-transferase (GST) subunits A1, A3, A4, A5, P1 and M1 were all found to be inducible in rat liver by most of the agents. The greatest levels of induction were observed for GST P1, following treatment with coumarin (20-fold), alpha-angelicalactone (10-fold) or ellagic acid (6-fold), and GST A5, following treatment with coumarin (7-fold), alpha-angelicalactone (6-fold) and ethoxyquin (6-fold). Glutathione synthetase was induced approximately 1.5-fold by coumarin, alpha-angelicalactone, ellagic acid and ethoxyquin. The expression of glutathione-related enzymes was also examined in preneoplastic lesions induced in rat liver by aflatoxin B(1). The majority of gamma-glutamyltranspeptidase (GGT)-positive preneoplastic foci contained increased levels of GLCLC relative to the surrounding tissue. This was usually found to be accompanied by an increase in GLCLR. Cells in the inner cortex of rat kidney were found to contain the highest levels of both GLCLC and GLCLR. The same cells showed the strongest staining for GGT activity.
Asunto(s)
4-Butirolactona/análogos & derivados , Aflatoxina B1/toxicidad , Anticarcinógenos/farmacología , Carcinógenos/toxicidad , Glutamato-Cisteína Ligasa/metabolismo , Neoplasias Hepáticas Experimentales/enzimología , Lesiones Precancerosas/enzimología , 4-Butirolactona/farmacología , Aflatoxina B1/antagonistas & inhibidores , Animales , Antioxidantes/farmacología , Carcinógenos/antagonistas & inhibidores , Dominio Catalítico/genética , Cumarinas/farmacología , Dieta , Ácido Elágico/farmacología , Inducción Enzimática/efectos de los fármacos , Etoxiquina/farmacología , Glutamato-Cisteína Ligasa/biosíntesis , Glutamato-Cisteína Ligasa/genética , Glutatión/metabolismo , Inactivación Metabólica , Hígado/efectos de los fármacos , Hígado/enzimología , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/prevención & control , Masculino , Fenobarbital/farmacología , Lesiones Precancerosas/inducido químicamente , Ratas , Ratas Endogámicas F344 , Ratas WistarRESUMEN
gamma-Glutamylcysteine synthetase (GCS) is reported to catalyse the rate-limiting step in glutathione biosynthesis, and is a heterodimer composed of a catalytic subunit [heavy subunit (GCSh) of Mr 73000] and a regulatory subunit [light subunit (GCSl) of Mr 31000]. In the present study, we have demonstrated for the first time a potential role for GCSl in resistance towards doxorubicin and cadmium chloride. Addition of recombinant GCSl to HeLa cell extracts in vitro was found to result in an increase in GCS activity of between 2- and 3-fold. Transient transfections of COS-1 cells with the GCSl cDNA cause an increase in GCS activity of approx. 2-fold, and a small but significant (P=0.008) increase in glutathione levels from 126.9+/-4. 2 nmol/mg protein to 178.8+/-19.1 nmol/mg protein. We proceeded to make a HeLa cell line (LN73), which stably overexpresses GCSl. These cells overexpress GCSl approx. 20-fold above basal levels. LN73 was found to have a 2-fold increase in GCS activity (437.3+/-85.2 pmol/min per mg) relative to the control cell line, HL9 (213.4+/-71. 8 pmol/min per mg). In contrast with the transient transfections in COS-1 cells, stable overexpression of GCSl was found not to be associated with an increase in glutathione content. However, when the LN73 and HL9 cells were treated with the glutathione-depleting agent, diethylmaleate, the LN73 cells were found to have an enhanced ability to regenerate glutathione, compared with HL9 cells. The cell lines were treated with various anti-cancer drugs, and their cytotoxicity was examined. No obvious differences in toxicity were observed between the different cell lines following treatment with cisplatin and melphalan. The redox-cycling agent doxorubicin, however, was found to be more toxic (approx. 2-fold) to the HL9 cells than the LN73 cells. When the cells were treated with the carcinogenic transition-metal compound, cadmium chloride, LN73 cells were found to be approx. 3-fold more resistant than HL9 cells.
Asunto(s)
Glutamato-Cisteína Ligasa/metabolismo , Animales , Antineoplásicos/farmacología , Células COS , Cloruro de Cadmio/toxicidad , Carcinógenos/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN Complementario/genética , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Glutamato-Cisteína Ligasa/biosíntesis , Glutamato-Cisteína Ligasa/genética , Glutatión/biosíntesis , Células HeLa , Humanos , Maleatos/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
We have shown that in HepG2 cells treatment with 75 microM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of gamma-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.
Asunto(s)
Carcinoma Hepatocelular/enzimología , Glutamato-Cisteína Ligasa/biosíntesis , Neoplasias Hepáticas/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Carcinoma Hepatocelular/genética , Catálisis , Clonación Molecular , Inducción Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/química , Glutamato-Cisteína Ligasa/genética , Glutamato-Cisteína Ligasa/aislamiento & purificación , Humanos , Hidroquinonas/farmacología , Neoplasias Hepáticas/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/fisiología , Análisis de Secuencia de ADN , Células Tumorales CultivadasRESUMEN
Vaccines made by inactivating pathogenic microorganisms have been dramatically successful in controlling diseases in humans and animals. Despite their successes, they have a major disadvantage in that several inoculations are required for them to be effective. To overcome this problem, a commercial inactivated vaccine preparation against tickborne encephalitis was combined with human growth hormone (HGH). This formulation produced complete protection in a murine model with only one dose of vaccine, apparently by binding hormone and antigen to an insoluble matrix containing aluminium hydroxide. Thus it is postulated that when virus-specific lymphocytes are attracted to the site of injection, the hormone is at a high local concentration and stimulates the clonal expansion of antigen-specific T cells. The development of genetically engineered HGH now gives unlimited supplies of hormone, potentially resulting in an increase in efficacy of a wide variety of vaccines, especially those needing prolonged immunization schedules such as those being developed to combat human immunodeficiency virus infection.