Functional characterization and localization of AQP3 in the human colon

Water channels or aquaporins (AQPs) have been identified in a large variety of tissues. Nevertheless, their role in the human gastrointestinal tract, where their action is essential for the reabsorption and secretion of water and electrolytes, is still unclear. The purpose of the present study was to investigate the structure and function of water channels expressed in the human colon. A cDNA fragment of about 420 bp with a 98% identity to human AQP3 was amplified from human stomach, small intestine and colon by reverse transcription polymerase chain reaction (RT-PCR) and a transcript of 2.2 kb was expressed more abundantly in colon than in jejunum, ileum and stomach as indicated by Northern blots. Expression of mRNA from the colon of adults and children but not from other gastrointestinal regions in Xenopus oocytes enhanced the osmotic water permeability, and the urea and glycerol transport in a manner sensitive to an antisense AQP3 oligonucleotide, indicating the presence of functional AQP3. Immunocytochemistry and immunofluorescence studies in human colon revealed that the AQP3 protein is restricted to the villus epithelial cells. The immunostaining within these cells was more intense in the apical than in the basolateral membranes. The presence of AQP3 in villus epithelial cells suggests that AQP3 is implicated in water absorption across human colonic surface cells.

Genotyping of group A rotavirus samples from Brazilian children by probe hybridization

The G genotyping of 74 group A rotavirus samples was done by RNA-DNA hybridization (dot-blot) using oligonucleotide probes for the VP7 gene region of the human rotavirus serotypes/genotypes 1, 2, 3 and 4. Thirty-one samples could be genotyped by dot-blot showing the following results: G1 = 16, G4 = 6, G3 = 5, and G2 = 4. The data show circulation of genotypes G1-G4 and the predominance of G1. The knowledge of genotypes provides important information concerning rotavirus circulation in Central Brazil.

Design and applications of modified oligonucleotides

Oligonucleotides have a wide range of applications in fields such as biotechnology, molecular biology, diagnosis and therapy. However, the spectrum of uses can be broadened by introducing chemical modifications into their structures. The most prolific field in the search for new oligonucleotide analogs is the antisense strategy, where chemical modifications confer appropriate characteristics such as hybridization, resistance to nucleases, cellular uptake, selectivity and, basically, good pharmacokinetic and pharmacodynamic properties. Combinatorial technology is another research area where oligonucleotides and their analogs are extensively employed. Aptamers, new catalytic ribozymes and deoxyribozymes are RNA or DNA molecules individualized from a randomly synthesized library on the basis of a particular property. They are identified by repeated cycles of selection and amplification, using PCR technologies. Modified nucleotides can be introduced either during the amplification procedure or after selection.

Rapid, reliable and inexpensive quality assessment of biotinylated cRNA

The interpretation of oligonucleotide array experiments depends on the quality of the target cRNA used. cRNA target quality is assessed by quantitative analysis of the representation of 5' and 3' sequences of control genes using commercially available Test arrays. The Test array provides an economically priced means of determining the quality of labeled target prior to analysis on whole genome expression arrays. This manuscript validates the use of a duplex RT-PCR assay as a faster (6 h) and less expensive (6 were chosen and classified as degraded cRNAs, and 31 samples with a ß-actin 3'/5' ratio <6 were selected as good quality cRNAs. Blinded samples were then used for the RT-PCR assay. After gel electrophoresis, optical densities of the amplified 3' and 5' fragments of ß-actin were measured and the 3'/5' ratio was calculated. There was a strong correlation (r² = 0.6802) between the array and the RT-PCR ß-actin 3'/5' ratios. Moreover, the RT-PCR 3'/5' ratio was significantly different (P < 0.0001) between undegraded (mean ± SD, 0.34 ± 0.09) and degraded (1.71 ± 0.83) samples. None of the other parameters analyzed, such as i) the starting amount of RNA, ii) RNA quality assessed using the Bioanalyzer Chip technology, or iii) the concentration and OD260/OD280 ratio of the purified biotinylated cRNA, correlated with cRNA quality.

Polymorphism of the promoter region and exon 1 of the CTLA4 gene in endemic pemphigus foliaceus (fogo selvagem)

Endemic pemphigus foliaceus (EPF) is an autoimmune bullous skin disease characterized by acantholysis and antibodies against a desmosomal protein, desmoglein 1. Genetic and environmental factors contribute to development of this multifactorial disease. HLA class II and some cytokine gene polymorphisms are the only genetic markers thus far known to be associated with susceptibility to or protection from EPF. The cytotoxic T-lymphocyte antigen-4 gene (CTLA4) encodes a key immunoreceptor molecule that regulates and inhibits T-cell proliferation. It participates in the regulatory process controlling autoreactivity and therefore has been considered a strong candidate gene in autoimmune diseases. In the search for genes that might influence EPF pathogenesis, we analyzed variants of the CTLA4 gene in a sample of 118 patients and 291 controls from a Brazilian population. This is the first study investigating the possible role of polymorphisms of the 2q33 chromosomal region in differential susceptibility to pemphigus foliaceus. Promoter region and exon 1 single nucleotide polymorphisms -318 (C,T) and 49 (A,G) were genotyped using sequence-specific oligonucleotide probes after amplification by the polymerase chain reaction. The allelic and genotypic frequencies did not differ significantly between the patient and the control groups (-318T: 9.8 and 10.9%, 49G: 33.0 and 35.2% were the allelic frequencies in patients and controls, respectively). In addition, no significant difference was found when the patient and control population samples were stratified by the presence of HLA-DRB1 alleles. We conclude that the CTLA4 -318 (C,T) and 49 (A,G) polymorphisms do not play a major role in EPF development.

Apolipoprotein CIII polymorphism and triglyceride levels of a Japanese population living in Southern Brazil

Apolipoprotein CIII (apo-CIII) participates in the regulation of triglyceride-rich lipoprotein metabolism. Several polymorphic sites have been detected within and around the apo-CIII gene. Here, we examined the relationship between apo-CIII SstI polymorphism (CC, CG, GG genotypes) and plasma triglyceride (TG) levels in a group of 159 Japanese individuals living in Southern Brazil. The sample was divided into a group of Japanese descendants (N = 51) with high TG (HTG; >200 mg/dL) and a group of Japanese descendants (N = 108) with normal TG (NTG; <200 mg/dL). TG and total cholesterol levels were analyzed by an enzymatic method using the Labtest-Diagnostic kit and high- and low-density lipoproteins by a direct method using the Labtest-Diagnostic kit and DiaSys Diagnostic System International kit, respectively. A 428-bp sequence of apo-CIII gene was amplified using oligonucleotide primers 5' GGT GAC CGA TGG CTT CAG TTC CCT GA 3' and 5' CAG AAG GTG GAT AGA GCG CTG GCC T 3'. The PCR products were digested with a restriction endonuclease SstI. Rare G allele was highly prevalent in our study population (0.416) compared to Caucasians (0.00-0.11). G allele was almost two times more prevalent in the HTG group compared to the NTG group (P < 0.001). The genotype distribution was consistent with the Hardy-Weinberg equilibrium. There was a significant association between rare G allele and HTG in Japanese individuals living in Southern Brazil as indicated by one-way ANOVA, P < 0.05.

A majority of Brazilian patients with rheumatoid arthritis HLA-DRB1 alleles carry both the HLA-DRB1 shared epitope and anti-citrunillated peptide antibodies

The objective of the present study was to evaluate the contribution of the shared epitope (SE), the rheumatoid arthritis (RA) protection model, and the occurrence of anti-cyclic citrullinated peptide (anti-CCP) antibodies in RA patients from a genetically diverse population. One hundred and forty Brazilian RA patients and 161 matched controls were typed for HLA-DRB1 alleles using amplified DNA hybridized with sequence-specific oligonucleotide probes or primers. Patients were stratified according to the presence or absence of SE (DRB1*0401, *0404, *0405, *0101, *1001, and *1402), of the DERAA alleles (DRB1*0103, *0402, *1102, *1103, *1301, *1302, and *1304), and X (all other alleles). Anti-CCP antibodies were measured by ELISA. The combined frequency of SE-positive alleles was significantly greater (76.4 vs 23.6%, P < 0.0001) than the controls. The SE/SE and SE/X genotypes were over-represented (P < 0.0001, OR = 6.02) and DERAA/X was under-represented in RA patients (P < 0.001, OR = 0.49), whereas the frequencies of the SE/DERAA, X/X and X/DERAA genotypes were not significantly different from controls. The frequency of anti-CCP antibodies was higher in SE-positive patients than in SE-negative patients (64.6 vs 44.7%, P = 0.03; OR = 2.25). Although the Brazilian population is highly miscegenated, the results of this study support the findings observed in most genetically homogeneous populations with RA; however, they are not mutually exclusive but rather complementary. The participation of DRB1-DERAA alleles in protection against RA was also observed (OR = 0.4; 95%CI = 0.23-0.68).

Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening

The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.

Clinicopathological significance of PTPN12 expression in human breast cancer

Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a recently identified tumor suppressor gene (TSG) that is frequently compromised in human triple-negative breast cancer. In the present study, we investigated the expression of PTPN12 protein by patients with breast cancer in a Chinese population and the relationship between PTPN12 expression levels and patient clinicopathological features and prognosis. Additionally, we explored the underlying down-regulation mechanism from the perspective of an epigenetic alteration. We examined PTPN12 mRNA expression in five breast cancer cell lines using semi-quantitative reverse-transcription PCR, and detected PTPN12 protein expression using immunohistochemistry in 150 primary invasive breast cancer cases and paired adjacent non-tumor tissues. Methylation-specific PCR was performed to analyze the promoter CpG island methylation status of PTPN12. PTPN12 was significantly down-regulated in breast cancer cases (48/150) compared to adjacent noncancerous tissues (17/150; P < 0.05). Furthermore, low expression of PTPN12 showed a significant positive correlation with tumor size (P = 0.047), lymph node metastasis (P = 0.001), distant metastasis (P = 0.009), histological grade (P = 0.012), and survival time (P = 0.019). Additionally, promoter CpG island hypermethylation occurs more frequently in breast cancer cases and breast cancer cell lines with low PTPN12 expression. Our findings suggest that PTPN12 is potentially a methylation-silenced TSG for breast cancer that may play an important role in breast carcinogenesis and could potentially serve as an independent prognostic factor for invasive breast cancer patients.

Placental hydroxymethylation vsmethylation at the imprinting control region 2 on chromosome 11p15.5

In addition to methylated cytosines (5-mCs), hydroxymethylcytosines (5-hmCs) are present in CpG dinucleotide-enriched regions and some transcription regulator binding sites. Unlike methylation, hydroxymethylation does not result in silencing of gene expression, and the most commonly used methods to study methylation, such as techniques based on restriction enzymatic digestion and/or bisulfite modification, are unable to distinguish between them. Genomic imprinting is a process of gene regulation where only one member of an allelic pair is expressed depending on the parental origin. Chromosome 11p15.5 has an imprinting control region (ICR2) that includes a differentially methylated region (KvDMR1) that guarantees parent-specific gene expression. The objective of the present study was to determine the presence of 5-hmC at the KvDMR1 in human placentas. We analyzed 16 third-trimester normal human placentas (chorionic villi). We compared two different methods based on real-time PCR after enzymatic digestion. The first method distinguished methylation from hydroxymethylation, while the other method did not. Unlike other methylation studies, subtle variations of methylation in ICRs could represent a drastic deregulation of the expression of imprinted genes, leading to important phenotypic consequences, and the presence of hydroxymethylation could interfere with the results of many studies. We observed agreement between the results of both methods, indicating the absence of hydroxymethylation at the KvDMR1 in third-trimester placentas. To the best of our knowledge, this is the first study describing the investigation of hydroxymethylation in human placenta using a genomic imprinting model.