Differentiation of Melipona quadrifasciata L. (Hymenoptera, Apidae, Meliponini) subspecies using cytochrome b PCR-RFLP patterns

Melipona quadrifasciata quadrifasciata and M. quadrifasciata anthidioides are subspecies of M. quadrifasciata, a stingless bee species common in coastal Brazil. These subspecies are discriminated by the yellow stripe pattern of the abdominal tergites. We found Vsp I restriction patterns in the cytochrome b region closely associated to each subspecies in 155 M. quadrifasciata colonies of different geographical origin. This mitochondrial DNA molecular marker facilitates diagnosis of M. quadrifasciata subspecies matrilines and can be used to establish their natural distribution and identify hybrid colonies.

Differential diagnosis of oocysts of Hammondia-like organisms of dogs and cats by PCR-RFLP analysis of 70-kilodalton heat shock protein (HSP70) gene

Organisms of the genera Toxoplasma, Hammondia and Neospora, the Hammondia-like organisms, are closely related coccidian with similarly sized oocysts. Therefore, a diagnosis based on microscopy of oocysts in feces is not a method of choice for species identification of these important parasites. In this paper, we present a polymerase chain reaction coupled with restriction fragment length polymorphism (PCR-RFLP) method to differentially diagnose oocysts of Toxoplasma gondii from oocyst of Hammondia hammondi. Another PCR-RFLP was designed to differentiate oocysts of Hammondia heydorni from oocysts of Neospora spp. Both PCR-RFLP are based on nucleotide sequences of the Hsp70 coding gene. In conclusion, we presented two alternative molecular diagnostic assays that can be successfully applied for the differentiation of oocysts of Hammondia-like organisms shed by felids and canids.

Mitochondrial DNA Diversity of Orchid Bee Euglossa fimbriata (Hymenoptera: Apidae) Populations Assessed by PCR-RFLP

Euglossa fimbriata is a euglossine species widely distributed in Brazil and occurring primarily in Atlantic Forest remnants. In this study, the genetic mitochondrial structure of E. fimbriata from six Atlantic Forest fragments was studied by RFLP analysis of three PCR-amplified mtDNA gene segments (16S, COI-COII, and cyt b). Ten composite haplotypes were identified, six of which were exclusive and represented singleton mitotypes. Low haplotype diversity (0.085-0.289) and nucleotide diversity (0.000-0.002) were detected within samples. AMOVA partitioned 91.13% of the overall genetic variation within samples and 8.87% (I center dot(st) = 0.089; P < 0.05) among samples. Pairwise comparisons indicated high levels of differentiation among some pairs of samples (I center dot(st) = 0.161-0.218; P < 0.05). These high levels indicate that these populations of E. fimbriata, despite their highly fragmented landscape, apparently have not suffered loss of genetic variation, suggesting that this particular population is not currently endangered.

A PCR-RFLP assay for gender assignment in the three-toed sloths (Bradypus, Pilosa, Bradypodidae)

The three-toed sloths (Bradypus) are slow-moving arboreal neotropical mammals. Understanding demographic variables (such as sex ratio) of populations is a key for conservation purposes. Nevertheless, gender assignment of Bradypus is particularly challenging because of the lack of sexual dimorphism in infants and in adults, particularly B. torquatus, the most endangered of the three-toed sloths, in which sex is attributed by visual observation of the reproductively active males. Here, we standardized a method for sexing Bradypus individuals using PCR-RFLP of sex-linked genes ZFX/ZFY. This assay was validated with known-gender animals and proved accurate to assign gender on three Bradypus species.

Toxoplasma gondii isolates: Multi locus RFLP-PCR genotyping from human patients in Sao Paulo State, Brazil identified distinct genotypes

This study investigated the genetic characteristics of Toxoplasma gondii samples collected from 62 patients with toxoplasmosis in Sao Paulo State, Brazil. DNA samples were isolated from blood, cerebrospinal fluid and amniotic fluids of 25 patients with cerebral toxoplasmosis and AIDS, two patients with acute toxoplasmosis, 12 patients with ocular toxoplasmosis, six newborns with congenital toxoplasmosis and 17 pregnant women with acute infection. Diagnosis of toxoplasmosis was based in clinical, radiological and laboratory features. Genotyping was performed using multilocus PCR-RFLP genetic markers including SAG1, SAG2, 5`- and 3`-SAG2, alt.SAG2, SAG3, BTUB, GRA6, C22-8, c29-2, L358, PK1 and Apico. Among the 62 clinical samples, 20 (32%) were successfully genotyped at eight or more genetic loci and were grouped to three distinct genotypes. Eighteen samples belonged to ToxoDB Genotype #65 and the other two samples were identified as ToxoDB Genotypes #6 and #71, respectively (http://toxodb.org/toxo/). Patients presenting Genotypes #6 and #71 had severe and atypical cerebral toxoplasmosis, characterized by diffuse encephalitis without extensive brain lesions. These results indicate that T. gondii Genotype #65 may have a high frequency in causing human toxoplasmosis in Sao Paulo State, Brazil. This unusual finding highlights the need to investigate the possible association of parasite genotypes with human toxoplasmosis. (C) 2011 Elsevier Inc. All rights reserved.

Methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and high plasma homocysteine in chronic hepatitis C (CHC) infected patients from the Northeast of Brazil

Background/Aim: Hyperhomocysteinemia due to Methylenetetrahydrofolate Reductase (MTHFR) gene, in particular the C677T (Ala222Val) polymorphism were recently associated to steatosis and fibrosis. We analyzed the frequency of MTHFR gene in a cross-sectional study of patients affected by Chronic Hepatitis C (CHC) from Northeast of Brazil. Method: One hundred seven-four untreated patients with CHC were genotyped for the C677T MTHFR. Genomic DNA was extracted from peripheral blood cells and the C677T MTHFR polymorphism was identified by PCR-RFLP. The homocysteine (Hcy) levels were determined by chemiluminescence method. All patients were negative for markers of Wilson's disease, hemochromatosis and autoimmune diseases and have current and past daily alcohol intake less than 100 g/week. Results: Among subjects infected with CHC genotype non-1 the frequency of MTHFR genotypes TT was 9.8% versus 4.4% genotype 1 (p = 0.01). Nevertheless, association was found between the MTHFR genotype TT x CT/CC polymorphism and the degree of steatosis and fibrosis in both hepatitis C genotype (p < 0.05). A significant difference was found on plasma Hcy levels in patients with steatosis regardless of HCV genotype (p = 0.03). Conclusion: Our results indicate that plasma Hcy levels is highly prevalent in subjects with chronic hepatits C with steatosis regardless of HCV genotype and vitamin deficiency. The presence of genotype TT of MTHFR C677T polymorphism was more common in CHC genotype non-1 infected patient regardless of histopathological classification and genotype TT+CT frequencies were significant in the presence of fibrosis grade 1+2 and of steatosis in CHC infected patients from the northeast of Brazil regardless of HCV genotype. The genetic susceptibility of MTHFR C677T polymorphism should be confirmed in a large population.

GSTP1 Ile105Val polymorphism in astrocytomas and glioblastomas

Glutathione S-transferases (GSTs) constitute a superfamily of ubiquitous multifunctional enzymes that are involved in the cellular detoxification of a large number of endogenous and exogenous chemical agents that have electrophilic functional groups. People who have deficiencies in this family of genes are at increased risk of developing some types of tumors. We examined GSTP1 Ile105Val polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. Patients who had the Val allele of the GSTP1 Ile105Val polymorphism had an increased risk of tumor development (odds ratio = 8.60; 95% confidence interval = 4.74-17.87; P < 0.001). Overall survival of patients did not differ significantly. We suggest that GSTP1 Ile105Val polymorphisms are involved in susceptibility to developing astrocytomas and glioblastomas.

Polymorphisms and DNA methylation of gene TP53 associated with extra-axial brain tumors

The p53 tumor suppressor gene is the most frequently mutated gene in human cancer; this gene is mutated in up to 50% of human tumors. It has a critical role in the cell cycle, apoptosis and cell senescence, and it participates in many crucial physiological and pathological processes. Polymorphisms of p53 have been suggested to be associated with genetically determined susceptibility in various types of cancer. Another process involved with the development and progression of tumors is DNA hypermethylation. Aberrant methylation of the promoter is an alternative epigenetic change in genetic mechanisms, leading to tumor suppressor gene inactivation. In the present study, we examined the TP53 Arg72Pro and Pro47Ser polymorphisms using PCR-RFLP and the pattern of methylation of the p53 gene by methylation-specific PCR in 90 extra-axial brain tumor samples. Patients who had the allele Pro of the TP53 Arg72Pro polymorphism had an increased risk of tumor development ( odds ratio, OR = 3.23; confidence interval at 95%, 95% CI = 1.71-6.08; P = 0.003), as did the allele Ser of TP53 Pro47Ser polymorphism (OR = 1.28; 95% CI = 0.03-2.10; P = 0.01). Comparison of overall survival of patients did not show significant differences. In the analysis of DNA methylation, we observed that 37.5% of meningiomas, 30% of schwannomas and 52.6% of metastases were hypermethylated, suggesting that methylation is important for tumor progression. We suggest that TP53 Pro47Ser and Arg72Pro polymorphisms and DNA hypermethylation are involved in susceptibility for developing extra-axial brain tumors.

VIABILITY OF SPORULATED OOCYSTS OF NEOSPORA CANINUM AFTER EXPOSURE TO DIFFERENT PHYSICAL AND CHEMICAL TREATMENTS

The aim of the present study was to evaluate the viability of Neospora caninum sporulated oocysts after various chemical and physical treatments. Bioassays in gerbils and molecular techniques (PCR-RFLP) were used for identification of the oocysts shed by experimentally infected dogs. Sporulated oocysts were purified and divided into 11 treatment groups as follows: absolute ethanol for 1 hr; 20 C for 6 hr; 4 C for 6 hr; 60 C for 1 min; 100 C for 1 min; 10% formaldehyde for 1 hr; 10% ammonia for 1 hr; 2% iodine for 1 hr; 10% sodium hypochlorite for I hr; 70% ethanol for I hr; and one group was left untreated and kept as a positive control. All chemical treatments were performed at room temperature (37 C). A total of 33 gerbils, or 3 gerbils per treatment, were used for bioassays. After treatment, the oocysts were divided into aliquots of 1,000 oocysts and orally administered to gerbils. After 63 days, the gerbils were anesthetized and killed with 0.2 ml of T61; blood and tissue samples were collected for serological (IFAT and western blotting), molecular (real-time PCR), histopathology, and immunohistochemical tests. Treatments were considered effective only if all 5 detection techniques tested negative. High temperatures at 100 C for 1 min and 10% sodium hypochlorite for 1 hr were the only treatments that met this condition, effectively inactivating all oocysts.

Allele-Specific Expression of the MAOA Gene and X Chromosome Inactivation in In Vitro Produced Bovine Embryos

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8-to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. Mol. Reprod. Dev. MoL Reprod. Dev. 77: 615-621, 2010. (C) 2010 Wiley-Liss, Inc.

Evaluation of nutritional and genetic determinants of total homocysteine, methylmalonic acid and S-adenosylmethionine/S-adenosylhomocysteine values in Brazilian childbearing-age women

Background: Cobalamin (Cbl) and folate deficiencies and gene polymorphism of key enzymes or carriers can impair homocysteine metabolism and may change the serum values of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). We investigated the nutritional and genetic determinants for total homocysteine (tHcy), methylmalonic acid (MMA) and SAM/SAH in healthy Brazilian childbearing-age women. Methods: Serum concentrations of Cbl, folate, red blood cell folate, ferritin, tHcy, MMA, SAM, SAH and other metabolites were measured in 102 healthy unrelated women. The genotypes for MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G, TC2 C776G, TC2 A67G and RFCI A80G gene polymorphisms were identified by PCR-RFLP. Results: Serum folate and Cbl were inversely correlated with tHcy and serum MMA, respectively. Cbl deficiency was associated with increased MMA and reduced alpha-aminobutyrate, serine and N-methylglycine concentrations. No variable was associated with SAM/SAH ratio. In addition, gene polymorphisms were not selected as determinants for tHcy, MMA and SAM/SAH ratio. Iron, Cbl and folate deficiencies were found respectively in 30.4%, 22.5% and 2.0% of individuals studied. Conclusions: There was a high frequency of Cbl and iron deficiency in this group of childbearing-age women. Serum folate and Cbl were the determinants of serum tHcy and MMA concentration, respectively. (c) 2007 Elsevier B.V. All rights reserved.

Increased MMA concentration and body mass index are associated with spontaneous abortion in Brazilian women A pilot study

Background: The pathophysiology of spontaneous abortion is complex and may involve the interaction of genetic and environmental factors. We evaluated the predictors of spontaneous abortion in Brazilian pregnant women. The effects of age, gestational age. body mass index (BMI), cigarette smoking, alcohol ingestion, use of multivitamins and concentrations of vitamins (folate, cobalamin and vitamin 136) and vitamin-dependent metabolites were analyzed. Methods: Study population included 100 healthy women that attended pre-natal care in 2 health centers of Sao Paulo, Brazil, and in whom pregnancy outcome was known. Folate and cobalamin status was measured in blood specimens collected between 4 and 16 weeks. The genotypes for 8 gene polymorphisms were evaluated by PCR-RFLP. Results: Eighty-eight women had normal pregnancy outcome (Group 1), while 12 experienced a miscarriage after blood collection (Group 2). Increased methylmalonic acid (MMA) concentrations were found in Group 2 (median [25th-75th percentile]=274 [149-425] nmol/l) relative to Group 1 (138 [98-185]) (P<0.01). No differences between the groups were observed for serum cobalamin, serum or red cell folate, and serum total homocysteine or allele frequencies for 8 polymorphisms. In a conditional logistic regression analysis including age, gestational age, serum creatinine, MMA, cystathionine, body mass index (BMI), cigarette smoking, alcohol ingestion and use of multivitamins the risk of abortion was significantly associated with MMA (OR [95% CI] = 3.80 [1.36, 10.62] per quartile increase in MMA), BMI (OR [95% CI] = 5.49 [1.29,23.39] per quartile) and gestational age (OR [95% CI] = 0.10 [0.01, 0.77] per increase of interval in gestational age). Conclusions: Increased serum MMA and BMI concentrations are associated with spontaneous abortion in Brazilian women. (C) 2009 Elsevier B.V. All rights reserved.

Relationships between gene polymorphisms of folate-related proteins and vitamins and metabolites in pregnant women and neonates

Background: The methylenetetrahydrofolate reductase (MTHFR), glutamate carboxypeptidase II (GCPII) and reduced folate carrier (RFC1) gene polymorphisms were associated with folate status. We investigated the effects of these polymorphisms on serum folate (SF) and folate-related metabolites in mothers and their neonates. Methods: Cobalamin (Cbl), SF, total homocysteine (tHcy), methylmalonic acid (MMA), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in 275 healthy women and their neonates. MTHFR C677T, GCPII C1561T and RFC1 A80G polymorphisms were determined by PCR-RFLP. Results: Maternal tHcy was affected individually by MTHFR C677T and GCPII C1561T polymorphisms and by combined genotypes MTHFR 677TT/GCPII 1561CC and MTHFR 677TT/RFC1 80AG. The MTHFR and RFC1 polymorphisms were not associated with variations in vitamins or SAM, SAH and MMA in neonates. Neonatal tHcy was predicted directly by maternal tHcy and inversely by maternal SF, neonatal Cbl and neonatal RFC1 80G allele (AG+GG genotypes). Maternal MMA and SAM/SAH were predicted by creatinine and Cbl, respectively. Neonatal MMA was predicted by maternal MMA and GCPII 1561T allele (CT+TT genotypes) and by neonatal Cbl. Conclusions: Maternal tHcy was affected by MTHFR C677T, RFC1 A80G and GCPII C1561T polymorphisms. Maternal GCPII C1561T variant was associated with neonatal MMA. Neonatal RFC1 A80G polymorphism influenced tHcy in neonates. (C) 2008 Elsevier B.V. All rights reserved.

Atorvastatin effects on SREBF1a and SCAP gene expression in mononuclear cells and its relation with lowering-lipids response

Background: The transcription factors SREBP1 and SCAP are involved in intracellular cholesterol homeostasis. Polymorphisms of these genes have been associated with variations on serum lipid levels and response to statins that are potent cholesterol-lowering drugs. We evaluated the effects of atorvastatin on SREBF1a and SCAP mRNA expression in peripheral blood mononuclear cells (PBMC) and a possible association with gene polymorphisms and lowering-cholesterol response. Methods: Fifty-nine hypercholesterolemic patients were treated with atorvastatin (10 mg/day for 4 weeks). Serum lipid profile and mRNA expression in PBMC were assessed before and after the treatment. Gene expression was quantified by real-time PCR using GAPD as endogenous reference and mRNA expression in HepG2 cells as calibrator. SREBF1 -36delG and SCAP A2386G polymorphisms were detected by PCR-RFLP. Results: Our results showed that transcription of SREBF1a and SCAP was coordinately regulated by atorvastatin (r=0.595, p<0.001), and that reduction in SCAP transcription was associated with the 2386AA genotype (p=0.019). Individuals who responded to atorvastatin with a downregulation of SCAP had also a lower triglyceride compared to those who responded to atorvastatin with an upregulation of SCAP. Conclusion: Atorvastatin has differential effects on SREBF1a and SCAP mRNA expression in PBMC that are associated with baseline transcription levels, triglycerides response to atorvastatin and SCAP A2386G polymorphism. (c) 2008 Elsevier B.V. All rights reserved.

ABCB1 and ABCC1 expression in peripheral mononuclear cells is influenced by gene polymorphisms and atorvastatin treatment

This study investigated the effects of atorvastatin on ABCB1 and ABCC1 mRNA expression on peripheral blood mononuclear cells (PBMC) and their relationship with gene polymorphisms and lowering-cholesterol response. one hundred and thirty-six individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). Blood samples were collected for serum lipids and apolipoproteins measurements and DNA and RNA extraction. ABCB1 (C3435T and G2677T/A) and ABCC1 (G2012T) gene polymorphisms were identified by polymerase chain reaction-restriction (PCR)-RFLP and mRNA expression was measured in peripheral blood mononuclear cells by singleplex real-time PCR. ABCB1 polymorphisms were associated with risk for coronary artery disease (CAD) (p < 0.05). After atorvastatin treatment, both ABCB1 and ABCC1 genes showed 50% reduction of the mRNA expression (p < 0.05). Reduction of ABCB1 expression was associated with ABCB1 G2677T/A polymorphism (p = 0.039). Basal ABCB1 mRNA in the lower quartile (<0.024) was associated with lower reduction rate of serum low-density lipoprotein (LDL) cholesterol (33.4 +/- 12.4%) and apolipoprotein B (apoB) (17.0 +/- 31.3%) when compared with the higher quartile (>0.085: LDL-c = 40.3 +/- 14.3%; apoB = 32.5 +/- 10.7%; p < 0.05). ABCB1 substrates or inhibitors did not affect the baseline expression, while ABCB1 inhibitors reversed the effects of atorvastatin on both ABCB1 and ABCC1 transporters. In conclusion, ABCB1 and ABCC1 mRNA levels in PBMC are modulated by atorvastatin and ABCB1 G2677T/A polymorphism. and ABCB1 baseline expression is related to differences in serum LDL cholesterol and apoB in response to atorvastatin. (C) 2008 Elsevier Inc. All rights reserved.