RESUMEN
OBJECTIVE: Oxidative stress plays an important role in the development of diabetic neuropathy (DN). Antioxidant enzymes reduce enhanced oxidative stress in the peripheral nerve. Genetic variations within the antioxidant genes therefore could be implicated in the pathogenesis of DN. METHODS: Using a PCR-RFLP assay, a total of 216 Russian type 1 diabetic (T1D) patients with DN and 250 T1D individuals without DN have been tested to verify whether the -262T > C and 1167C > T polymorphisms of the catalase (CAT), 197Pro > Leu amino acid substitution of the glutathione peroxidase 1 (GPX1) and +/null polymorphism of the glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genes contribute to susceptibility to DN. RESULTS: Association between the -262T > C polymorphism of the CAT gene and DN was shown. The -262TT genotype of the CAT gene was significantly associated with higher erythrocyte catalase activity in blood of DN patients compared to the -262CC genotype (17.8 +/- 2.7 x 104 IU/g Hb vs. 13.5 +/- 3.2 x 104 IU/g Hb, P = 0.0022). CONCLUSIONS: These data suggest a protective role of the -262T allele of the CAT gene against the rapid development of DN in T1D (Odds Ratio = 0.7 [95% confidence interval 0.54-0.9], P = 0.002).
Asunto(s)
Catalasa/genética , Diabetes Mellitus Tipo 1/genética , Neuropatías Diabéticas/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Adulto , Citosina , ADN/sangre , ADN/genética , ADN/aislamiento & purificación , Diabetes Mellitus Tipo 1/enzimología , Neuropatías Diabéticas/enzimología , Femenino , Genotipo , Humanos , Masculino , Valores de Referencia , Federación de Rusia , TiminaRESUMEN
Alleles and genotypes of polymorphic markers of paraoxonase 1 and paraoxonase 2 genes (PON1 and PON2) encoding enzymes of the body antioxidative defense were compared in type 1 diabetes mellitus patients with or without diabetic nephropathy. The patients with nonoverlapping ("polar") phenotypes constituted different groups. The first group contained patients with diabetic nephropathy (DN+, n = 62), clinical proteinuria (albuminuria above 300 mg per day), and at least 15-year disease duration. In control group, the patients had no diabetic nephropathy (DN-, n = 68), their albuminuria was below 200 mg per day, and disease duration was at least 20 years. Comparative analysis with exact Fisher's test revealed no significant differences in frequencies of alleles and genotypes of the PON1 gene polymorphic marker Gln192Arg and of PON2 gene polymorphic markers Ala148Gly and Cys311Ser. Our results suggest that the polymorphic markers studied are not associated with diabetic nephropathy among Russian patients in Moscow.
Asunto(s)
Arildialquilfosfatasa/genética , Diabetes Mellitus Tipo 1/genética , Nefropatías Diabéticas/genética , Polimorfismo Genético , Biomarcadores , Estudios de Casos y Controles , Ligamiento Genético , Humanos , Moscú , Valor Predictivo de las PruebasRESUMEN
The allele and genotype frequencies of polymorphic markers of NOS1, NOS2 and NOS3 genes, encoding three types of NO synthases, were compared in type 1 diabetes patients with and without diabetic polyneuropathty. 180 type 1 diabetes patients (T1DM) of Russian or Eastern Slavonic origin, living in Moscow city, were divided into two groups using non-overlapping (polar) phenotypes. 86 patients had overt DPN and T1DM duration in this group was less than 5 years (DPN+ group) and 94 patients had no clinical DPN and T1DM duration was more than 10 years (DPN- group). We have not found the significant differences of allele and genotype frequencies of polymorphic markers (CA)n of NOS1 gene, (CCTTT)n of NOS2 gene, ecNOS4a/4b and Glu298Asp of NOS3 gene that indicates that all these markers are not associated with diabetic polyneuropathty. Only in the case of (CCTTT)n marker of NOS2 gene we have found a tendency for the association of 14 allele with DPN development. The carriers of this allele have the lower risk of DPN in T1DM.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Neuropatías Diabéticas/genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Óxido Nítrico Sintasa/genética , Polimorfismo Genético , HumanosRESUMEN
The allele and genotype frequency distributions of polymorphic markers of genes coding for antioxidant enzymes were compared for type 1 diabetes mellitus patients with or without diabetic polyneuropathy (DPN). The groups (total 180 patients) had nonoverlapping (polar) phenotypes. Group DPN+ included 86 patients with DPN and diabetic record no more than 5 years. Control group DPN- included patients without DPN and diabetic record of at least 10 years. Comparative analysis with Fisher's exact test revealed a significant difference in allele and genotype frequency distributions of the T(-262)C polymorphic marker of the CAT gene. Polymorphic markers C1167T of the CAT gene, Pro/Leu of the GPX1 gene, 0/+ of the GSTT1 gene, and 0/+ of the GSTM1 gene showed no significant difference in allele or genotype frequency distribution. On this evidence, these markers were not associated with DPN in the sample examined.
Asunto(s)
Catalasa/genética , Diabetes Mellitus Tipo 1/genética , Nefropatías Diabéticas/genética , Marcadores Genéticos , Glutatión Peroxidasa/genética , Polimorfismo Genético , Superóxido Dismutasa/genética , Adolescente , Adulto , Secuencia de Bases , Niño , Cartilla de ADN , Diabetes Mellitus Tipo 1/enzimología , Nefropatías Diabéticas/enzimología , Femenino , Humanos , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
Single-nucleotide polymorphisms of the genes for mitochondrial (SOD2) and extracellular (SOD3) superoxide dismutases were tested for association with diabetic polyneuropathy (DPN) in diabetes mellitus (DM) type 1. Patients (n = 180) were divided into two groups with nonoverlapping (polar) phenotypes. Group DPN+ included 86 individuals with DPN and DM type 1 record of no more than 5 years. Group DPN-included 94 patients with DM type 1 record of more than 10 years but without clinical signs of DPN. Fisher's exact test revealed significant differences in allele and genotype frequencies for the two groups. Higher frequencies of SOD2 allele Val and genotype Val/Val and of SOD3 allele Arg and genotype Arg/Arg were established for group DPN+. On this evidence, SOD2 and SOD3 were associated with DPN in DM type 1.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Neuropatías Diabéticas/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Superóxido Dismutasa/genética , Adolescente , Adulto , Alanina/genética , Arginina/genética , Diabetes Mellitus Tipo 1/complicaciones , Neuropatías Diabéticas/etiología , Femenino , Frecuencia de los Genes , Glicina/genética , Humanos , Masculino , Valina/genéticaRESUMEN
Oxidative stress plays a key role in the development of microvascular complications of diabetes mellitus (DM). Antioxidant enzymes protect against the rapid onset of diabetic polyneuropathy (DPN) by reducing oxidative stress. Genetic variations that affect activity or expression levels of the antioxidant enzymes may therefore be associated with susceptibility to DPN. We examined polymorphic markers Ala(-9)Val in SOD2 gene and Arg213Gly in SOD3 gene for possible relation to DPN in Russian type 1 diabetic patients. Four hundred Russian white patients with type 1 diabetes were studied using neurological examination according to recommendations of the San Antonio Conference on Diabetic Neuropathy. Two groups were formed from the general sample. Definition of frequency distribution of the polymorphic markers was performed in these groups using the polymerase chain reaction. Genes encoding the enzymes Mn-SOD and extracellular superoxide dismutase (EC-SOD) were found to be associated with the pathogenesis of DPN.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Neuropatías Diabéticas/genética , Predisposición Genética a la Enfermedad/genética , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Neuropatías Diabéticas/epidemiología , Genotipo , Hemoglobina Glucada/análisis , Humanos , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Federación de Rusia , Fumar , Población BlancaRESUMEN
BACKGROUND: Oxidative stress, resulting in a marked increase in the level of oxygen free radicals (OFR), has been implicated in the etiology of diabetic neuropathy (DN). Antioxidant enzymes may protect against the rapid onset and progression of DN, by reducing the excess of OFR and peroxide. Mutations and polymorphisms in the genes encoding such enzymes may therefore result in predisposition to DN. We investigated the role of genes encoding two antioxidant enzymes, mitochondrial (Mn-SOD) and extracellular (EC-SOD) superoxide dismutase, in DN pathogenesis in a Russian population. We studied Ala(-9)Val and Ile58Thr polymorphisms of the Mn-SOD gene and Arg213Gly dimorphism of the EC-SOD gene in type 1 diabetic patients with (n = 82) and without DN (n = 84). RESULTS: We developed and used a new polymerase chain reaction (PCR) assays for rapid detection of polymorphisms. These assays involved the use of mismatch PCR primers to create restriction sites in the amplified product only in presence of the polymorphic base. The PCR product was than digested with BshTI, Eco32I or Eco52I to detect Ala(-9)Val, Ile58Thr or Arg213Gly polymorphic site respectively. The frequencies of the Ala allele (50.6% vs. 68.5%, p < 0.002) and the Ala/Ala genotype (17.1% vs. 39.3%, p < 0.005) of the Mn-SOD gene were significantly lower in DN patients than in diabetic subjects without DN. In contrast, the Val allele (49.4% vs. 31.5%, p < 0.002) and the Val/Val genotype (15.9% vs. 2.4%, p < 0.01) were significantly more frequent in the DN patients than in the control group. CONCLUSIONS: Ala(-9)Val substitution in the Mn-SOD gene was associated with DN in a Russian population
RESUMEN
Polyclonal rabbit antibodies against a conjugate synthesized through condensing BSA and disubstituted roridin A hemisuccinate allowed roridin A to be determined in solutions at a sensitivity of 0.2 ng/ml. The cross-reactivity of structural analogues--roridin A, verrucarin, and verrucarol--amounted to 100, 2.5, and 0.03%, respectively. The data showed that these antibodies determine roridin A in an indirect heterogeneous enzyme immunoassay in cereal straw samples at a sensitivity of 20 micrograms/kg.
Asunto(s)
Anticuerpos/inmunología , Formación de Anticuerpos , Micotoxinas/inmunología , Animales , Reacciones Cruzadas , Micotoxinas/química , Conejos , Tricotecenos/química , Tricotecenos/inmunologíaRESUMEN
Zearalenone-6'-carboxymethyloxime was synthesized, and its conjugates with albumins and gelatin were prepared. Polyclonal rabbit antibodies against the conjugate with bovine serum albumin were shown to be highly specific to zearalenone and to have a lower cross-reactivity toward its structural analogues (alpha-zearalenol--28%, beta-zearalenol--6%, zearalanone--12%, and alpha-zearalanol--5%). The sensitivity of enzyme immunoassay using gelatin-based immobilized conjugates for determination of zearalenone in solutions was 1 ng/ml, and this allowed us to determine this substance in feed at a threshold concentration of 200 micrograms/kg.
Asunto(s)
Antígenos/inmunología , Oximas/inmunología , Zearalenona/análogos & derivados , Albúminas/química , Alimentación Animal/análisis , Animales , Bovinos , Reacciones Cruzadas , Gelatina/química , Técnicas para Inmunoenzimas , Oximas/química , Conejos , Sensibilidad y Especificidad , Zearalenona/química , Zearalenona/inmunologíaRESUMEN
Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9-291.7 micrograms/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 micrograms/kg.
Asunto(s)
Grano Comestible/química , Micotoxinas/análisis , Ocratoxinas/análisis , Grano Comestible/microbiología , Técnicas para InmunoenzimasRESUMEN
Indirect enzyme immunoassay based on immobilized conjugate of aflatoxin B1 carboxymethyloxime with bovine serum albumin and polyclonal rabbit antibodies allows determining aflatoxin B1 with a low relative cross-reactivity against aflatoxin B2, G1, G2, M1, B2a and G2a and sterigmatocystin (15.5, 15.5, 1.7, 1.0, 0.03, 0.03 and 0.01%, respectively) with a sensitivity of 0.04 ng per well or 4.0 ng per ml organic solvent.
Asunto(s)
Aflatoxina B1/análisis , Técnicas para Inmunoenzimas/métodosRESUMEN
Based on indirect solid-phase competitive enzyme immunoassay, a method for determination of T-2 toxin in grain was designed. Determination errors were measured on samples of contaminated grain. The method makes allows determination of the toxin levels ranging from 30 to 1000 ng/g.