Thymidylate synthase and methylenetetrahy-drofolate reductase gene polymorphisms and gastric cancer susceptibility in a population of Northern Brazil.

The folate metabolic pathway, which is involved in DNA synthesis and methylation, is associated with individual susceptibility to several diseases, including gastric tumors. In this study, we investigated four polymorphisms [thymidylate synthase enhancer region, single nucleotide polymorphism thymidylate synthase 5' (TS5'), TS3' untranslated region, and methylenetetrahydrofolate reductase (MTHFR) 677C> T] in 2 genes related to the folate pathway, TS and MTHFR, and their possible association with the risk gastric cancer development in a population from Pará state, Brazil. For the TS enhancer region, TS3' untranslated region, and single nucleotide polymorphism TS5' polymorphisms, no significant results were obtained. For the MTHFR 677C>T polymorphism, TT genotype carriers had a higher risk of developing tumors in the antrum (P = 0.19 vs CC and P = 0.02 vs CT) and intestine (odds ratio = 4.18, 95% confidence interval = 0.66-26.41; P = 0.252 vs CC and odds ratio = 2.25, 95% confidence interval = 0.32-15.75; P = 0.725 vs CT). Those carrying at least 1 T allele had an increased risk of lymph node metastasis (odds ratio = 3.00, 95% confidence interval = 0.88-10.12; P = 0.133). Our results suggest that polymorphisms in MTHFR affect the susceptibility to gastric tumors in the Brazilian population and may be a factor causing poor prognosis in such patients.

Comparative phylogenetic study of Stichotrichia (Alveolata: Ciliophora: Spirotrichea) based on 18S-rDNA sequences.

Since molecular phylogenies of stichotrich ciliates started to be published, some remarkable contradictions to morphology-based classifications have been reported, such as the Convergent Evolution of Urostylids and Uroleptids (CEUU) hypothesis, the Halteria paradox, the polyphyly of Oxytricha and of Stichotrichia. We hypothesized the internal phylogeny of 18S-rDNA from 53 morphological species of stichotrichs and their relationships with Hypotrichia and Oligotrichia using parsimony and neighbor-joining methods, including new data from Pseudouroleptus caudatus and Strongylidium pseudocrassum. Competing phylogenetic scenarios were compared using statistical tests, and the results suggest the reconsideration of both CEUU and the position of Halteria among flexible-body oxytrichids. The polyphyly of Oxytricha was not rejected and the monophyly of Stichotrichia was accepted based on parsimony analysis if Pseudoamphisiella is considered an external (discocephalid related) taxon.

Evolution of the SEC1 gene in New World monkey lineages (Primates, Platyrrhini).

The structure and evolution of the SEC1 gene were examined for the first time in New World primates of the genera Alouatta, Aotus, Ateles, Brachyteles, Callicebus, Callithrix, Cebus, Chiropotes, Lagothrix, Leontopithecus, Pithecia, Saguinus, and Saimiri. This gene has a high CG content (63.8%) and an estimated heterogeneous size ranging from 795 (Callithrix) to 1041 bp (Pithecia), due to numerous indel events. Similar to other fucosyltransferases, three conserved regions are shared by these primates, except for the callitrichines, Aotus and Pithecia, in which indel events resulted in premature stop codons that are related to the production of a supposedly non-functional protein. Phylogenetic analysis of the SEC1 gene, transition/transversion rates, and nucleotide sequence alignment support the hypothesis that primate SEC1 evolved by divergent evolution, and that the lack of activity in some lineages occurred independently at least twice in New World primates, once in the Aotus-Cebus-Callitrichinae group and again in Pithecia. Likelihood-based inference of ancestral states for the activity of SEC1 leads us to suppose that inactivation of SEC1 in the Callitrichinae was a result of a more complex series of events than in Pithecia.

Polymorphisms of the TP53 codon 72 and WRN codon 1367 in individuals from Northern Brazil with gastric adenocarcinoma.

Gastric cancer is the second most frequent type of neoplasia and also the second most common cause of death in the world. TP53 codon 72, which produces variant proteins with an arginine (Arg) or proline (Pro), has been reported to be associated with cancers of the lung, oesophagus, stomach and cervix. Werner's syndrome (WS) is a premature ageing disease caused by a mutation in the WRN gene. The WRN protein acts as a DNA helicase and as an exonuclease. WRN codon 1367 produces variant proteins with an Arg or cysteine (Cys). This polymorphism has been studied, in order to understand the clinical impact of the molecular variants in WS and in age-related disorders. In the present study, the TP53 codon 72 and the WRN codon 1367 polymorphisms were investigated in 54 gastric adenocarcinoma patients (31 diffuse-type and 25 intestinal-type) and 54 controls. DNA samples were extracted, and PCR-RFLP was utilised for genotyping TP53 codon 72 and WRN codon 1367. The allele frequencies of the TP53 polymorphism were: Arg=0.74 and Pro=0.26. The allele frequencies of the WRN polymorphism were: Cys=0.73 and Arg=0.27. The crude genotypic frequencies in gastric cancer patients were similar to those of the controls, but in the WRN codon 1367 polymorphisms the mean age tended to be higher in the Arg/Arg genotypes. There also was an association, although not statistically significant, between the presence of Helicobacter pylori and the genotypes Cys/Cys and Cys/Arg and a higher percentage of cardia cancer among the Arg/Arg genotypes, and of non-cardia cancer among genotypes Cys/Cys and Cys/Arg. These findings may be a reflection of differences in the interaction between WRN codon 1367 polymorphisms and local factors in the stomach. To our knowledge, this is the first study to examine a genetic polymorphism of the WRN gene in cancer. The precise mechanisms of action of the TP53 and WRN polymorphisms involved in the aetiopathogeny of this disease need further investigation.

Molecular study of the tumour suppressor gene PTEN in gastric adenocarcinoma in Brazil.

Among all tumours diagnosed worldwide, gastric adenocarcinoma is the second most frequent type of malignancy. In Brazil, it is estimated to be the fifth most frequent type of neoplasia. According to the classification of Laurén, these tumours are divided into well differentiated and ill differentiated gastric adenocarcinomas. There are studies suggesting that the first type develops through remodulation of genes involved in the suppressor pathway and the second through remodulation of genes belonging to the mutational pathway. The gene PTEN is located in region 10q23 and is altered in several human tumours. In gastric cancer, this gene is thought to take part in the suppressor pathway. In our study, DNA was obtained from 48 gastric adenocarcinoma samples, amplified, screened for all exons of the PTEN gene by PCR-SSCP and then confirmed by sequencing. There was only one sample that presented an alteration and that was a transversion. Our results corroborate the hypothesis that somatic alterations in the PTEN gene are rare events in gastric cancer.

Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor

Gastric cancer is the second most frequent type of neoplasia and also the second most important cause of death in the world. Virtually all the established cell lines of gastric neoplasia were developed in Asian countries, and western countries have contributed very little to this area. In the present study we describe the establishment of the cell line ACP01 and characterize it cytogenetically by means of in vitro immortalization. Cells were transformed from an intestinal-type gastric adenocarcinoma (T4N2M0) originating from a 48-year-old male patient. This is the first gastric adenocarcinoma cell line established in Brazil. The most powerful application of the cell line ACP01 is in the assessment of cytotoxicity. Solid tumor cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The ACP01 cell line is triploid, grows as a single, non-organized layer, similar to fibroblasts, with focus formation, heterogeneous division, and a cell cycle of approximately 40 h. Chromosome 8 trisomy, present in 60 percent of the cells, was the most frequent cytogenetic alteration. These data lead us to propose a multifactorial triggering of gastric cancer which evolves over multiple stages involving progressive genetic changes and clonal expansion.

Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor.

Gastric cancer is the second most frequent type of neoplasia and also the second most important cause of death in the world. Virtually all the established cell lines of gastric neoplasia were developed in Asian countries, and western countries have contributed very little to this area. In the present study we describe the establishment of the cell line ACP01 and characterize it cytogenetically by means of in vitro immortalization. Cells were transformed from an intestinal-type gastric adenocarcinoma (T4N2M0) originating from a 48-year-old male patient. This is the first gastric adenocarcinoma cell line established in Brazil. The most powerful application of the cell line ACP01 is in the assessment of cytotoxicity. Solid tumor cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The ACP01 cell line is triploid, grows as a single, non-organized layer, similar to fibroblasts, with focus formation, heterogeneous division, and a cell cycle of approximately 40 h. Chromosome 8 trisomy, present in 60% of the cells, was the most frequent cytogenetic alteration. These data lead us to propose a multifactorial triggering of gastric cancer which evolves over multiple stages involving progressive genetic changes and clonal expansion.

ABO blood groups in the primate species of Cebidae from the Amazon region.

The ABO blood groups were determined in blood and saliva collected from 40 Aotus infulatus, 74 Saimiri sciureus, and 96 Cebus apella from the Amazonian region along the Tocantins river. Saliva samples were tested for human ABH antigens by a standard hemagglutination inhibition test. Aotus infulatus showed monomorphism, exhibiting only the B blood group. Saimiri sciureus exhibited the A (67) and AB (7) phenotypes. All four phenotypes have been found in C. apella: O (8), A (52), B (19) and AB (17). The observed distribution was as expected assuming Hardy-Weinberg equilibrium. The titers of ABH substances varied among the species and phenotypes. The B-like agglutinogen, common to all New World monkey species tested, was detected in the red blood cells. Sera were used to detect naturally occurring antibodies and the results showed discrepancies between serum and saliva phenotypes in all species studied.

Molecular phylogeny of the New World monkeys (Platyrrhini, primates) based on two unlinked nuclear genes: IRBP intron 1 and epsilon-globin sequences.

Nuclear sequences of the 1.8 kilobase (kb) long intron 1 of the interstitial retinol-binding protein gene (IRBP), previously determined for 11 of the 16 extant genera of New World monkeys (superfamily Ceboidea, infraorder Platyrrhini), have now been determined for the remaining 5 genera. The maximum parsimony trees found, first with IRBP sequences alone and then with tandemly combined IRBP and epsilon-globin gene sequences from the same species, supported a provisional cladistic classification with the following clusters. Subtribes Callitrichina (Callithrix, Cebuella), Callimiconina (Callimico), Leontopithecina (Leontopithecus) and Saguina (Saguinus) constitute subfamily Callitrichinae, and subfamilies Callitrichinae, Aotinae (Aotus), and Cebinae (Cebus, Saimiri) constitute family Cebidae. Subtribes Chiropotina (Chiropotes, Cacajao) and Pitheciina (Pithecia) constitute tribe Pitheciini; and tribes Pitheciini and Callicebini (Callicebus) constitute subfamily Pitheciinae. Subtribes Brachytelina (Brachyteles, Lagothrix) and Atelina (Ateles) constitute tribe Atelini, and tribes Atelini and Alouattini (Alouatta) constitute subfamily Atelinae. The parsimony results were equivocal as to whether Pitheciinae should be grouped with Atelinae in family Atelidae or have its own family Pitheciidae. The cladistic groupings of extant ceboids were also examined by different stochastic evolutionary models that employed the same stochastic process of nucleotide substitutions but alternative putative phylogenetic trees on which the nucleotide substitutions occurred. Each model, i.e., each different tree, predicted a different multinomial distribution of nucleotide character patterns for the contemporary sequences. The predicted distributions that were closest to the actual observed distributions identified the best fitting trees. The cladistic relationships depicted in these best fitting trees agreed in almost all cases with those depicted in the maximum parsimony trees.

DNA evidence on the phylogenetic systematics of New World monkeys: support for the sister-grouping of Cebus and Saimiri from two unlinked nuclear genes.

Previous inferences from epsilon-globin gene sequences on cladistic relationships among the 16 extant genera of Ceboidea (the New World monkeys) were tested by strength of grouping and bootstrap values for the clades in the most parsimonious trees found: for this epsilon data set enlarged with additional Cebus and Saimiri orthologues; for another nuclear DNA sequence data set consisting of IRBP (interstitial retinol-binding protein gene) intron 1 orthologues; and for tandemly combined epsilon and IRBP sequences. Different ceboid species of the same genus always grouped strongly together as demonstrated by results on Cebus (capuchin monkeys), Saimiri (squirrel monkeys), Callicebus (titi monkeys), Aotus (night monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys). Other strong groupings that could be represented as monophyletic taxa in a cladistic classification were: Cebuella (pygmy marmoset) and Callithrix (marmoset) into subtribe Callitrichina; Callitrichina, Callimico (Goeldi's monkey), Leontopithecus (lion tamarin), and Saguinus (tamarin) into subfamily Callitrichinae; Callitrichinae, Aotus, Cebus, and Saimiri into family Cebidae; Cacajao (uakari monkey) and Chiropotes (saki) into subtribe Chiropotina; Chiropotina and Pithecia (bearded saki) into tribe Pitheciini; Pitheciini and Callicebus into subfamily Pitheciinae; Brachyteles (woolly spider monkey), Lagothrix (woolly monkey), and Ateles into tribe Atelini; and Atelini and Alouatta into subfamily Atelinae. In addition the epsilon and IRBP results congruently grouped (but at lesser strengths) Brachyteles and Lagothrix into subtribe Brachytelina within Atelini, and also Cebus and Saimiri into subfamily Cebinae within Cebidae. Because the IRBP results weakly grouped Pitheciinae with Cebidae, whereas the epsilon results weakly grouped Pitheciinae with Atelinae, the present evidence is best represented in an interim cladistic classification of ceboids by dividing the superfamily Ceboidea into three families: Atelidae, Pitheciidae, and Cebidae.

Molecular phylogeny of the New World monkeys (Platyrrhini, primates).

Phylogenetic relationships among the 16 extant genera of Ceboidea (the New World monkeys) were examined using aligned epsilon-globin gene sequences from 19 New World monkeys (representing all 16 extant ceboid genera), and seven catarrhines (one Old World monkey and six hominoids) and tarsier as the outgroups. The consensus maximum parsimony tree found for these epsilon-globin sequences and the levels of support from parsimony and bootstrap analyses, for the clades in this tree, provided strong evidence for a cladistic classification with the following clusters. Subtribes Callitrichina (Callithrix, Cebuella), Callimiconina (Callimico), Leontopithecina (Leontopithecus), and Saguina (Saguinus) constitute subfamily Callitrichinae, and subfamilies Callitrichinae, Aotinae (Aotus), Saimiriinae (Saimiri), and Cebinae (Cebus) constitute family Cebidae. In turn, subtribes Chiropotina (Chiropotes, Cacajao) and Pitheciina (Pithecia) constitute tribe Pithecini, tribes Pitheciini and Callicebini (Callicebus) constitute subfamily Pitheciinae, tribes Atelini (Brachyteles, Lagothrix, Ateles) and Alouattini (Alouatta) constitute subfamily Atelinae, and subfamilies Pitheciinae and Atelinae constitute family Atelidae. The two families (Cebidae and Atelidae) constitute the Ceboidea, the only extant superfamily of infraorder Platyrrhini. The sister-group relationships of Brachyteles and Lagothrix, Saguinus and Leontopithecus, and Callimico with a Cebuella/Callithrix clade is not as well supported by the parsimony and bootstrap analyses. Therefore, these relationships are not incorporated in the proposed cladistic classification. On determining branch lengths for the ceboid phylogenetic tree from only the more freely evolving noncoding sequences at the epsilon-globin locus and taking the reference age of 35 million years ago (MYA) for the New World monkey-catarrhine branch point, we estimated the age of the atelid-cebid branch point as about 20 MYA, and the ages of the next branch points, those between the subfamilies in each family, as 19-16 MYA.

Genetic studies in three South American black populations.

Twenty-one genetic systems were investigated in three relatively isolated South American Black populations. Unexpected allele frequencies were found in different systems in all populations, suggesting the occurrence of genetic drift and/or founder effects. The estimates of racial admixture indicate 50% to 79% of Black ancestry, with various degrees of White (18%-28%) and Amerindian (3%-32%) ancestry.

Reconstructing history: the Amazonian Mura Indians.

We studied demographic characteristics of two Mura Indian villages. Two-hundred six inhabitants of these villages and another population were tested in relation to 30 blood and 4 saliva genetic systems. These groups exhibit high mobility and exogamy rates and high fertility but relatively low mortality and variance in number of children per woman. Hb J Oxford and albumin Maku were observed, and they show high prevalences of GPT*1 and RH*R1 but low of HP*1 compared to other South American Indians. Four electrophoretic saliva systems are reported here for the first time in a predominantly Amerindian group. The amount of polymorphism was more limited than that found in Caucasian, black, and Oriental populations. The Mura are still predominantly Indian (82%) but have African (11%) and Caucasoid (7%) admixture. Using these values, the putative ancestral Mura gene frequencies were assessed. Problems related to quantitative estimations of admixture and the factors that influence the process are discussed.