Cytotoxicity and genotoxicity of Agaricus blazei methanolic extract fractions assessed using gene and chromosomal mutation assays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Allelic imbalance studies of chromosome 9 suggest major differences in chromosomal instability among nonmelanoma skin carcinomas

CONTEXTO: Vários estudos de perda de heterozigozidade na região 9p21-p22, que abriga os genes supressores tumorais CDKN2a/p16INK4a, p19ARF e p15INK4b, têm sido realizados em uma ampla série de tumores humanos, incluindo os melanomas familiares. Perdas e ganhos em outras regiões do cromossomo 9 também têm sido observados com freqüência e podem indicar mecanismos adicionais no processo de tumorigênese dos carcinomas basocelulares da pele. OBJETIVO: Investigar o equilíbrio alélico existente na região 9p21-p22 em carcinomas basocelulares. TIPO DE ESTUDO: Análise molecular de marcadores de microssatélites em tumores e controles. LOCAL: Dois serviços de dermatologia de atendimento terciário em universidades públicas de São Paulo e o Laboratório de Genética Molecular do Câncer da Universidade Estadual de Campinas (UNICAMP), Brasil. PARTICIPANTES: Examinamos 13 casos benignos, incluindo 4 queratoses solares, 3 queratoacantomas, 3 nevos melanocíticos, 2 doenças de Bowen e 1 neurofibroma cutâneo, além de 58 tumores malignos da pele: 14 de células escamosas, 40 carcinomas basocelulares e 4 melanomas; em pacientes consecutivamente encaminhados à clínica de Dermatologia da Unicamp e que concordaram em participar do estudo. VARIÁVEIS ESTUDADAS: O tumor principal e uma porção normal de pele não-adjacente foram removidos cirurgicamente de pacientes que consecutivamente procuraram os ambulatórios de dermatologia da Universidade Estadual de Campinas (UNICAMP) e da Universidade Estadual de São Paulo (Unesp), São Paulo, por causa de lesões cutâneas. Extraímos DNA tanto de tecido tumoral como do correspondente tecido normal de cada paciente. Para amplificar regiões de repetição polimórfica de microssatélites do cromossomo 9, foram utilizados quatro pares de primers, sendo dois deles destinados à região 9p21-p22. RESULTADOS: Identificamos oito casos (20%) de desequilíbrio alélico entre os carcinomas basocelulares, sendo dois casos de perda de heterozigozidade e seis casos de instabilidade de microssatélite na região 9p21-p22. Outros marcadores também mostravam anormalidades em três destes tumores, enquanto nenhuma alteração foi detectada entre os casos benignos e nos outros tumores malignos. CONCLUSÃO: Esta dependência fenotípica sugere que existem diferenças importantes no comportamento das formas mais comuns de tumores cutâneos não-melanocíticos em relação à sua tendência para instabilidade de microssatélite no cromossomo 9. Considerando-se que os genes CDKN2a/p16INK4a, p19ARF e p15INK4b não parecem responsáveis pelas anormalidades observadas, outros genes em 9p21-p22 podem estar envolvidos na etiopatogenia e na progressão dos carcinomas basocelulares.

Duffy blood group gene polymorphisms among malaria vivax patients in four areas of the Brazilian Amazon region

Background: Duffy blood group polymorphisms are important in areas where Plasmodium vivax predominates, because this molecule acts as a receptor for this protozoan. In the present study, Duffy blood group genotyping in P. vivax malaria patients from four different Brazilian endemic areas is reported, exploring significant associations between blood group variants and susceptibility or resistance to malaria.Methods: the P. vivax identification was determined by non-genotypic and genotypic screening tests. The Duffy blood group was genotyped by PCR/RFLP in 330 blood donors and 312 malaria patients from four Brazilian Amazon areas. In order to assess the variables significance and to obtain independence among the proportions, the Fisher's exact test was used.Results: the data show a high frequency of the FYA/FYB genotype, followed by FYB/FYB, FYA/FYA, FYA/FYB-33 and FYB/FYB-33. Low frequencies were detected for the FYA/FY(X), FYB/FY(X), FYX/FY(X) and FYB-33/FYB-33 genotypes. Negative Duffy genotype (FYB-33/FYB-33) was found in both groups: individuals infected and non-infected (blood donors). No individual carried the FY(X)/FYB-33 genotype. Some of the Duffy genotypes frequencies showed significant differences between donors and malaria patients.Conclusion: the obtained data suggest that individuals with the FYA/FYB genotype have higher susceptibility to malaria. The presence of the FYB-33 allele may be a selective advantage in the population, reducing the rate of infection by P. vivax in this region. Additional efforts may contribute to better elucidate the physiopathologic differences in this parasite/host relationship in regions endemic for P. vivax malaria, in particular the Brazilian Amazon region.

Intraspecific evolution of Rhizoctonia solani AG-1 IA associated with soybean and rice in Brazil based on polymorphisms at the ITS-5.8S rDNA operon

Rhizoctonia solani AG-1 IA causes leaf blight on soybean and rice. Despite the fact that R. solani AG-1 IA is a major pathogen affecting soybean and rice in Brazil and elsewhere in the world, little information is available on its genetic diversity and evolution. This study was an attempt to reveal the origin, and the patterns of movement and amplification of epidemiologically significant genotypes of R. solani AG-1 IA from soybean and rice in Brazil. For inferring intraspecific evolution of R. solani AG-1 IA sampled from soybean and rice, networks of ITS-5.8S rDNA sequencing haplotypes were built using the statistical parsimony algorithm from Clement et al. (2000) Molecular Ecology 9: 1657-1660. Higher haplotype diversity (Nei M 1987, Molecular Evolutionary Genetics Columbia University Press, New york: 512p.) was observed for the Brazilian soybean sample of R. solani AG-1 IA (0.827) in comparison with the rest of the world sample (0.431). Within the south-central American clade (3-2), four haplotypes of R. solani AG-1 IA from Mato Grosso, one from Tocantins, one from Maranhao, and one from Cuba occupied the tips of the network, indicating recent origin. The putative ancestral haplotypes had probably originated either from Mato Grosso or Maranhao States. While 16 distinct haplotypes were found in a sample of 32 soybean isolates of the pathogen, the entire rice sample (n=20) was represented by a single haplotype (haplotype 5), with a worldwide distribution. The results from nested-cladistic analysis indicated restricted gene flow with isolation by distance (or restricted dispersal by distance in nonsexual species) for the south-central American clade (3-2), mainly composed by soybean haplotypes.

Evolutionary chromosomal differentiation among four species of Conoderus Eschscholtz, 1829 (Coleoptera, Elateridae, Agrypninae, Conoderini) detected by standard staining, C-banding, silver nitrate impregnation, and CMA(3)/DA/DAPI staining

The speciose Brazilian Elateridae fauna is characterized by high karyotypic diversity, including one species (Chalcolepidius zonatus Eschscholtz, 1829) with the lowest diploid number within any Coleoptera order. Cytogenetic analysis of Conoderus dimidiatus Germar, 1839, C. scalaris (Germar, 1824,) C. ternarius Germar, 1839, and C. stigmosus Germar, 1839 by standard and differential staining was performed with the aim of establishing mechanisms of karyotypic differentiation in these species. Conoderus dimidiatus, C. scalaris, and C. ternarius have diploid numbers of 2n(male) = 17 and 2n(female) = 18, and a X0/XX sex determination system, similar to that encountered in the majority of Conoderini species. The karyotype of C. stigmosus was characterized by a diploid number of 2n=16 and a neoXY/neoXX sex determination system that was highly differentiated from other species of the genus. Some features of the mitotic and meiotic chromosomes suggest an autosome/ancestral X chromosome fusion as the cause of the neoXY system origin in C. stigmosus. C-banding and silver impregnation techniques showed that the four Conoderus species possess similar chromosomal characteristics to those registered in most Polyphaga species, including pericentromeric C band and autosomal NORs. Triple staining techniques including CMA(3)/DA/DAPI also provided useful information for differentiating these Conoderus species. These techniques revealed unique GC-rich heterochromatin associated with NORs in C. scalaris and C. stigmosus and CMA(3)-heteromorphism in C. scalaris and C. ternarius.

Effects of genetic polymorphisms CYP1A1, GSTM1, GSTT1 and GSTP1 on urinary 1-hydroxypyrene levels in sugarcane workers

Sugarcane workers in Brazil are exposed to various genotoxic compounds, including polycyclic aromatic hydrocarbons (PAHs), derived from an incomplete combustion process of burnt sugarcane fields. The effects of the occupational exposure to sugarcane fields burning were measured in urine samples of sugarcane workers from the northwest of the State of São Paulo when exposed (harvesting) and when non-exposed (non-harvesting). The urinary levels of 1-hydroxypyrene (1-OHP) and the influence of the genetic polymorphisms CYP1A1, GSTM1, GSTT1 and GSTP1 were evaluated. Our results showed that the 1-OHP levels were significantly higher (P < 0.0000) in the exposed sugarcane workers (0.318 mu mol mol(-1) creatinine) than in the non-exposed workers (0.035 mu mol mol(-1) creatinine). In an unvaried analysis, no influence regarding the polymorphisms was observed. However, multivariate regression analysis showed that the CYP1A1*4 polymorphism in the exposed group, and age and the GSTP1 polymorphism in the non-exposed group significantly influenced urinary 1-OHP excretion levels (P < 0.10). The same group of sugarcane workers was significantly more exposed to PAHs during the harvesting period than during the non-harvesting period. (c) 2006 Elsevier B.V. All rights reserved.

Epstein-barr virus infection and single nucleotide Polymorphisms in the promoter region of interleukin 10 gene in patients with Hodgkin lymphoma

Context. - Hodgkin lymphoma is a neoplastic disease in which the immune system plays a major role in its pathogenesis. Interleukin 10 ( IL-10), an immunosuppressive cytokine actively produced in patients with Hodgkin lymphomas, favors the survival of the Hodgkin/Reed-Sternberg cells. Individual variations in IL-10 levels may be due, in part, to the presence of single nucleotide polymorphisms in the IL10 gene promoter.Objective. - To evaluate whether particular single nucleotide polymorphisms in the IL10 gene are found more frequently in Hodgkin lymphoma cases associated with Epstein-Barr virus infection.Design. - the identification of single nucleotide polymorphisms at positions -1082 and -819/-592 in the IL10 gene was performed by polymerase chain reaction and restriction length fragment polymorphisms analysis in 65 cases of Hodgkin lymphoma and 50 cases of reactive benign follicular lymphoid hyperplasia ( non-Hodgkin lymphoma control group).Results. - the frequency of the genotype GG at position -1082 was found to be significantly higher in patients with Epstein-Barr virus-positive Hodgkin lymphoma compared with Epstein-Barr virus-negative cases.Conclusions. - the results suggest that the presence of specific single nucleotide polymorphisms in the IL10 gene, notably those associated with high IL-10 production, may play a role in the susceptibility to Epstein-Barr virus -positive Hodgkin lymphoma development.

Analysis of CDKN1A polymorphisms: markers of cancer susceptibility?

The CDKN1A (TP21)(2) gene encodes a 21-kD protein that is a critical downstream mediator of wild-type TP53 and an important regulator of the cell cycle. Failure in the function of this gene would be expected to result in abnormal cell proliferation and transformation. Tumor-associated mutations of the coding region of the TP21 are rare. on the other hand, some TP21 polymorphisms have been identified and characterized by single base substitutions. In the present study, we investigated the potential role of TP21 gene polymorphisms in skin, head, and neck tumorigenesis. A total of 261 samples were examined by polymerase chain reaction single-strand conformational analysis, and one mutation at codon 31 and four polymorphisms in exons 2 (codon 55) and 3 [nucleotide (nt)590] and in promoter region (nt2298) were identified. In conclusion, this investigation confirmed the rarity of mutations in this gene, arguing against a role for TP21 mutations in skin, head, and neck cancers. Also, our results show significant differences in nt2298 allele frequencies between normal individuals and skin malignant tumors (P < 0.05). The results suggest that this polymorphism affects TP21 transactivator binding and may be important during the pathogenesis of skin cancer. (C) 2003 Elsevier B.V. All rights reserved.

Identification and analysis of single nucleotide polymorphisms (SNPs) in citrus

Most of the cultivated species of citrus have narrow genetic basis. Relationships among species and cultivars are obscured by sexual compatibility, polyembryony, apomixis and a high incidence of somatic mutations. DNA analysis is crucial in genetic studies not only for citrus breeding programs but also for characterization of hybrids and species. In this paper, single nucleotide polymorphisms ( SNPs) were investigated in 58 accessions of Citrus, hybrids and related genera. Genomic sequences of 'Pera IAC' sweet orange ( Citrus sinensis L. Osbeck) were used for primer design and selection of sequence tagged sites (STSs) for identification of SNPs. Analysis of 36 STSs showed identical sequences among 40 of the 41 sweet orange accessions studied. However, these accessions were heterozygous for many SNPs. Ten selected STSs were analyzed in 17 additional accessions from 13 species and hybrids. Comparing to the 'Pera IAC' sweet orange accession, a total of 150 polymorphic nucleotides were identified and most of the alterations were transitions ( 52.7%). The greatest number of SNPs was observed in Poncirus trifoliata ( L.) Raf. and the smallest in 'Ponkan' mandarin ( Citrus reticulata Blanco). At the intra-specific level, 'Bafa Gigante' ( Citrus sinensis L. Osbeck) was the only sweet orange accession with a divergent SNPs genotype, which corroborates the hypothesis of a hybrid origin for this accession. Although the STSs analyzed represent randomly sampled genomic sequences, they provided consistent information about the level of polymorphism and showed the potential of SNPs markers for characterization and phylogenetic studies.