Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects: I - gene expression profile of highly expressed phospholipases A(2)

Snake venom glands are a rich source of bioactive molecules such as peptides, proteins and enzymes that show important pharmacological activity leading to in local and systemic effects as pain, edema, bleeding and muscle necrosis. Most studies on pharmacologically active peptides and proteins from snake venoms have been concerned with isolation and structure elucidation through methods of classical biochemistry. As an attempt to examine the transcripts expressed in the venom gland of Bothrops jararacussu and to unveil the toxicological and pharmacological potential of its products at the molecular level, we generated 549 expressed sequence tags (ESTs) from a directional cDNA library. Sequences obtained from single-pass sequencing of randomly selected cDNA clones could be identified by similarities searches on existing databases, resulting in 197 sequences with significant similarity to phospholipase A(2) (PLA(2)), of which 83.2% were Lys49-PLA(2) homologs (BOJU-1), 0.1% were basic Asp49-PLA(2)s (BOJU-II) and 0.6% were acidic Asp49-PLA(2)s (BOJU-III). Adjoining this very abundant class of proteins we found 88 transcripts codifying for putative sequences of metalloproteases, which after clustering and assembling resulted in three full-length sequences: BOJUMET-I, BOJUMET-II and BOJUMET-III; as well as 25 transcripts related to C-type lectin like protein including a full-length cDNA of a putative galactose binding C-type lectin and a cluster of eight serine-proteases transcripts including a full-length cDNA of a putative serine protease. Among the full-length sequenced clones we identified a nerve growth factor (Bj-NGF) with 92% identity with a human NGF (NGHUBM) and an acidic phospholipase A2 (BthA-I-PLA(2)) displaying 85-93% identity with other snake venom toxins. Genetic distance among PLA(2)s from Bothrops species were evaluated by phylogenetic analysis. Furthermore, analysis of full-length putative Lys49-PLA(2) through molecular modeling showed conserved structural domains, allowing the characterization of those proteins as group II PLA(2)s. The constructed cDNA library provides molecular clones harboring sequences that can be used to probe directly the genetic material from gland venom of other snake species. Expression of complete cDNAs or their modified derivatives will be useful for elucidation of the structure-function relationships of these toxins and peptides of biotechnological interest. (C) 2004 Elsevier SAS. All rights reserved.

Markers of tumors susceptibility in head and neck cancer

Genetic polymorphisms are associated with a number of enzymes involved in the induction of head and neck carcinomas. It has been suggested that such polymorphisms may be linked to cancer susceptibility. Using a control-case study molecular genetic approach, we have investigated the association between polymorphisms genes (CYPs, GSTs and NAT2 genes) and susceptibility in head and neck cancer.

Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genomics and proteomics approaches to the study of cancer-stroma interactions

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

IL-1ra anti-inflammatory cytokine polymorphism is associated with risk of gastric cancer and chronic gastritis in a Brazilian population, but the TNF-beta pro-inflammatory cytokine is not

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

Haplotype effects on matrix metalloproteinase-1 gene promoter activity in cancer cells

Increased expression of matrix metalloproteinase-1 (MMP1) is associated with poor prognosis in cancers. Several single nucleotide polymorphisms (-1607GG > G, -839G > A, -755G > T, -519A > G, -422T > A, -340C > T, and 320C > T) in the MMP1 gene promoter have recently been identified. In this study, we assessed the functional effects of these polymorphisms on MMP1 gene promoter activity in cell lines of melanoma (A2058 and A375), breast cancer (MCF7 and MDA-MB-231), lung cancer (A549 and H69), and colorectal cancer (HT-29, SW-620) by comparing the promoter strengths of 10 most common haplotypes deriving from these polymorphisms. In A2058 cells, the GG-G-G-A-T-T-T and GG-G-G-A-C-T haplotypes had 2-fold higher promoter activity than the GG-G-T-A-T-T-C, GG-G-G-A-A-T-T, GG-G-G-A-T-T-C, and GG-G-G-A-A-C-T haplotypes, which in turn, had 3-fold higher promoter activity than the G-G-T-A-A-C-T, G-A-T-G-T-T-T, G-A-T-G-A-C-T, and G-A-T-G-A-T-G haplotypes. In A375 and MDA-MB-231 cells, high expression haplotypes include not only the -1607GG-bearing haplotypes but also the G-A-T-G-A-T-T haplotype containing the -1607G allele. A similar trend was detected in A549 cells. In addition, in A549 cells, the GG-G-G-A-T-T-T haplotype had > 2-fold higher promoter activity than several other 1607GG-bearing haplotypes. In MCF7 cells, the GG-G-G-A-T-T-T and G-G-T-A-A-C-T haplotypes had 1.5- to 4-fold higher promoter activity than the other haplotypes. These results suggest that the polymorphisms exert haplotype effects on the transcriptional regulation of the MMP1 gene in cancer cells, and indicate a need to examine haplotypes rather than any single polymorphism in genetic epidemiologic studies of the MMP1 gene in cancers.

Inheritance of resistance to powdery mildew in pea and pathogenesis-related aspects

A herança da resistência ao oídio na cultivar de ervilha MK-10 e alguns aspectos histológicos da infecção foram estudados. Para o estudo da herança, as gerações F1, F2, retrocuzamentos e geração F3 de MK-10 com duas populações suscetíveis foram avaliadas. Nas avaliações histológicas observou-se a porcentagem de conídios germinados, porcentagem de conídios que formaram apressório, porcentagem de conídios que estabeleceram colônia e número de haustórios por colônia. Para comparar as razões de segregação obtidas no estudo da herança da resistência, adotou-se o teste do Qui-quadrado (X²) e para os dados das análises histológicas, utilizou-se o teste Tukey a 5% de probabilidade. Concluiu-se que a resistência de MK-10 ao oídio é devida a um par de alelos recessivos e que a resistência é expressa na fase de pré-penetração, completada por uma morte celular localizada pós-penetração, característica da presença do par de alelos recessivos er1er1.

Implications of genetic variability of Trypanosoma cruzi for the pathogenesis of Chagas disease

O Trypanosoma cruzi, agente etiológico da doença de Chagas, apresenta elevado grau de variabilidade genética intra-específica, com possíveis implicações na forma clínica da doença, como o desenvolvimento de cardiopatia, do megaesôfago e do megacólon de forma isolada ou em associação. Este tropismo tecidual envolvido na patogênese da doença não está totalmente esclarecido. Assim, nesta revisão são abordados alguns aspectos referentes à diversidade genética dos parasitas isolados, às formas clínicas da doença de Chagas, ao processo de infecção do parasita na célula hospedeira e resposta imune. Outros aspectos também são enfocados, como os fatores imunossupressivos liberados pelo parasita que atuam na regulação das respostas imunes, a inibição da apoptose da célula hospedeira, assim como da patogênese do megaesôfago chagásico que pode estar relacionada à interação hospedeiro- parasita e sua associação com risco aumentado para o desenvolvimento do carcinoma epidermóide do esôfago. Porém, apesar dos avanços no entendimento desta doença, ainda não é possível estabelecer o verdadeiro perfil da variabilidade genética do parasita com a forma clínica da doença de Chagas.

Genetic variability in natural populations of Zeyheria montana mart. from the Brazilian Cerrado

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

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Aneuploidies, deletion, and overexpression of TP53 gene in intestinal metaplasia of patients without gastric cancer

Gastric carcinogenesis is attributable to interacting environmental and genetic factors, through a sequence of events including intestinal metaplasia. Using a fluorescence in situ hybridization technique, we investigated the occurrence of ancuploidies of chromosomes 3, 7, 8, 9, and 17, TP53 gene deletion, and expression of p53 in 21 intestinal metaplasia (IM) samples from cancer-free patients and in 20 gastric adenocarcinoma samples. Aneuploidies were found in 71% (15/21) of the IM samples. Trisomy of chromosomes 7 and 9 occurred mainly in complete-type IM; in the incomplete type, trisomy of chromosomes 7 and 8 were more commonly found. The TP53 gene deletion was observed in 60% (3/5) of the IM cases, and immunohistochemistry revealed p53 overexpression in 12% (2/17) of the analyzed IM cases. All gastric adenocarcinoma cases presented higher frequencies of trisomy or tetrasomy of chromosomes 3, 7, 8, 9, and 17. The TP53 deletion was found in all three of the gastric adenocarcinoma analyzed for it, and immunohistochemistry detected overexpression of protein p53 in 80% (12/15) of the analyzed cases. Our study revealed for the first time the presence of aneuploidies of chromosomes 7, 8, 9, and 17 and of TP53 gene deletion and overexpression in IM samples from cancer-free patients. These results suggest that IM and gastric adenocarcinoma may share the same genetic alterations. (C) 2004 Elsevier B.V. All rights reserved.

Predisposing genes and increased chromosome aberrations in lung cancer cigarette smokers

Genotoxic effects linking cigarette smoking with lung cancer have not been consistently demonstrated, therefore claims for the cause-effect relationships are vigorously contested. Using matched populations of 22 lung cancer patients who have been cigarette smokers (LCP), 22 non-cancerous cigarette smokers (SC) and 13 non-smokers (NSC), we have applied the fluorescence in situ hybridization (FISH) tandem probe assay to elucidate the frequency of chromosome breakage among the participants. Two probes were used, a classical satellite probe which hybridizes to the large heterochromatin region of chromosome 1, and an alpha-satellite probe which targets a small region adjacent to the heterochromatin probe. The highest frequency of structural aberrations was observed in LCP (1.4 +/- 0.1) followed by SC (1.25 +/- 0.1) and NSC (0.4 +/- 0.1). Aberration frequencies were not significantly different between LCP and SC (p > 0.05), however, a statistically significant difference was detected between the smoker populations combined (LCP and SC) and the NSC (p < 0.001). The breakage frequencies showed a positive correlation with duration of smoking for LCP (r = 0.5; p < 0.01), but not for SC (P > 0.05). In addition, the aberration frequencies were influenced by the inheritance of polymorphic glutathione S-transferase (GST) genes. LCPs missing one or the other GST (GSTM1 or GSTT1) genes were found to have significantly higher chromosome breaks compared to LCPs with both genes present (p < 0.05), Our data indicate that genetic predisposition and chromosome aberrations may be mechanistically related to the initiation of lung carcinogenesis; therefore, they may be useful biomarkers for lung cancer among cigarette smokers. (C) 1997 Elsevier B.V. B.V.

Partial genetic characterization of Stearoyl Coa-Desaturase's structural region in Bubalus bubalis

Conjugated Linoleic Acids (CLAs) comprise a family of positional and geometric isomers of linoleic acid. The main form of CLA, cis-9, trans-11-C18:2 show positive effects in cancer prevention and treatment. The major dietary sources of these fatty acids are derived from ruminant animals, in particular dairy products. In these animals, the endogenous synthesis mainly occurs in mammary gland by the action of enzyme Stearoyl CoA Desaturase (SCD). Different levels of expression and activity of SCD in mammary gland can explain partially the variation of CLA levels in fat milk. Considering a great fat concentration in bubaline milk and the benefit of a high and positive correlation between fat milk and CLA production, this study was carried on with the intention of sequencing and characterizing part of the gene that codifies SCD in buffaloes. Genomic DNA was extracted from blood samples of lactating bubaline which begins to the breed Murrah. After the (acho que nao precisa desse the) extractions, PCR (Polymerase Chain Reaction) reactions were made by using primers Z (sic) (sic) D1 and E1 (sic) (sic) F1. The fragments obtained in PCR were cloned into T vectors and transformed in competent cells DH10B line. After this, three samples of each fragment were sequenced from 5' and 3' extremities using a BigDye kit in an automatic sequencer. Sequences were edited in a consensus of each fragment and were submitted to BLAST-n / NCBI for similarity comparisions among other species. The sequence obtained with Z (sic) (sic) D1 primers shows 938 bp enclosing exons 1 and 2 and intron 1. The primers E1 (sic) (sic) F1 show 70 bp corresponding to exon 3 of bubaline SCD gene. Similarities were obtained between 85% and 97% among bubaline sequences and sequences of SCD gene described in human, mouse, rat, swine, bovine, caprine and ovine species. This study has permitted the identification and partial characterization of SCD codifing region in Bubalus bubalis specie.

The generation and utilization of a cancer-oriented representation of the human transcriptome by using expressed sequence tags

Whereas genome sequencing defines the genetic potential of an organism, transcript sequencing defines the utilization of this potential and links the genome with most areas of biology. To exploit the information within the human genome in the fight against cancer, we have deposited some two million expressed sequence tags (ESTs) from human tumors and their corresponding normal tissues in the public databases. The data currently define approximate to23,500 genes, of which only approximate to1,250 are still represented only by ESTs. Examination of the EST coverage of known cancer-related (CR) genes reveals that <1% do not have corresponding ESTs, indicating that the representation of genes associated with commonly studied tumors is high. The careful recording of the origin of all ESTs we have produced has enabled detailed definition of where the genes they represent are expressed in the human body. More than 100,000 ESTs are available for seven tissues, indicating a surprising variability of gene usage that has led to the discovery of a significant number of genes with restricted expression, and that may thus be therapeutically useful. The ESTs also reveal novel nonsynonymous germline variants (although the one-pass nature of the data necessitates careful validation) and many alternatively spliced transcripts. Although widely exploited by the scientific community, vindicating our totally open source policy, the EST data generated still provide extensive information that remains to be systematically explored, and that may further facilitate progress toward both the understanding and treatment of human cancers.

Heritability Estimate and Genetic Correlations of Reproductive Features in Nellore Bulls, Offspring of Super Precocious, Precocious and Normal Cows Under Extensive Farming Conditions

The present work aimed to estimate heritability and genetic correlations of reproductive features of Nellore bulls, offspring of mothers classified as superprecocious (M1), precocious (M2) and normal (M3). Twenty one thousand hundred and eighty-six animals with average age of 21.29 months were used, evaluated through the breeding soundness evaluation from 1999 to 2008. The breeding soundness features included physical semen evaluation (progressive sperm motility and sperm vigour), semen morphology (major, minor and total sperm defects), scrotal circumference (SC), testicular volume (TV) and SC at 18 months of age (SC18). The components of variance, heritability and genetic correlations for and between the features were estimated simultaneously by restricted maximum likelihood, with the use of the vce software system vs 6. The heritability estimates were high for SC18, SC and TV (0.43, 0.63 and 0.54; 0.45, 0.45 and 0.44; 0.42, 0.45 and 0.41, respectively for the categories of mothers M1, M2 and M3) and low for physical and morphological semen aspects. The genetic correlations between SC18 and SC were high, as well as between these variables with TV. High and positive genetic correlations were recorded among SC18, SC and TV with the physical aspects of the semen, although no favourable association was verified with the morphological aspects, for the three categories of mothers. It can be concluded that the mothers sexual precocity did not affect the heritability of their offspring reproduction features.