Possible implication of Mdm2 as a prognostic marker in invasive laryngeal carcinoma

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Galectin-3 Overexpression in Invasive Laryngeal Carcinoma, Assessed by Computer-assisted Analysis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Serum ferritin and transferrin saturation levels in β0 and β + thalassemia patients

There have been few studies on the mutations that cause heterozygous beta-thalassemia and how they affect the iron profile. One hundred and thirty-eight individuals were analyzed, 90 thalasemic β0 and 48 thalasemic β+, identified by classical and molecular methods. Mutations in the hemochromatosis (HFE) gene, detected using PCR-RFLP, were found in 30.4% of these beta-thalassemic patients; heterozygosity for H63D (20.3%) was the most frequent. Ferritin levels and transferrin saturation were similar in beta-thalassemics with and without mutations in the HFE gene. Ferritin concentrations were significantly higher in men and in individuals over 40 years of age. Transferrin saturation also was significantly higher in men, but only in those without HFE gene mutations. There was no significant difference in the iron profile among the β0 and β+ thalassemics, with and without HFE gene mutations. The frequency of ferritin values above 200 ng/mL in women and 300 ng/mL in men was also similar in β0 and β+ thalassemics (P > 0.72). Our conclusion is that ferritin levels are variable in the beta-thalassemia, trait regardless of the type of beta-globin mutation. Furthermore, HFE gene polymorphisms do not change the iron profile in these individuals. ©FUNPEC-RP www.funpecrp.com.br.

Influence of δβ-thalassemia or regulatory elements in individuals with increased fetal Hb levels in the São Paulo northwest population

Fetal hemoglobin (Hb F), formed by two alpha globin chains (α) and two gamma chains (γ) (α2 γ2), has reduced expression in adults, ranging from 0 to 1% of total hemoglobin. Increased levels of Hb F are due to mutations in the β-globin family, which cause hereditary persistence of fetal hemoglobin (HPFH) and delta-beta thalassemia (δβ-thalassemia).The control of the production takes place by the regulatory region and regions outside the β-globin family, among them 2q16, 6q23, 8q, and Xp22.2.The aims of this study were to determine the presence and frequency of two mutations for δβ-thalassemia, the XmnI polymorphism and β-globin haplotypes in healthy individuals with increased Hb F in the State of São Paulo. We analyzed 60 samples of peripheral blood of healthy adults, without complaints of anemia. The samples were separated into two groups according to Hb F level: group I - 34 samples with Hb F ranging from 2 to 15% and group II - 26 samples with Hb F over 15%. In relation to the polymorphisms examined, we found three heterozygous individuals (5%) for Spanish δβ-thalassemia, belonging to group I, whose Hb F levels were within the normal range.The Sicilian δβ-thalassemia mutation was not found, indicating the need to study other polymorphisms related to the increase of Hb F in adult life.The frequency of XmnI polymorphism was 33.3% and the mean Hb F levels were 15.48 ± 11.69%.The frequency observed in our study for this polymorphic site is higher than that found in the literature for healthy subjects.This polymorphism was more prevalent in individuals with Hb F levels below 15%. For four samples positive for this polymorphism, the Hb F levels were explained by the presence of HPFH and Spanish δβ-thalassemia mutations, so that the presence of the XmnI polymorphic site was not a determinant in the overexpression of γ-globin genes. Regarding β-globin haplotypes, 18 alleles and 27 distinct genotypic patterns were found.The pattern Atp1/Atp2 was the mostfrequent genotype (13.72%).Of the 18 alleles, 13 showed atypical patterns.The results show that the haplotype V was the most frequent (27.45%), followed by atypical Atp2 (13.72%) and Atp1 (11.76%), and that there was a higher correlation with the presence of HPFH and XmnI polymorphism.The high frequency of haplotype V in our samples and high frequency of atypical haplotypes may reflect a high rate of miscegenation in this population, suggesting an ethnic characteristic for the Brazilian population, requiring the evaluation of population genetic markers to corroborate this hypothesis. © FUNPEC-RP.

Detecção de papilomavírus humano (HPV) em material biológico de pacientes com carcinoma epidermóide de orofaringe

Pós-graduação em Odontologia - FOA

Hemoglobin D-Punjab: origin, distribution and laboratory diagnosis

This review discusses hemoglobin D-Punjab, also known as hemoglobin D-Los Angeles, one of the most common hemoglobin variants worldwide. It is derived from a point mutation in the beta-globin gene (HBB: c.364G>C; rs33946267) prevalent in the Punjab region, Northwestern Indian. Hemoglobin D-Punjab can be inherited in heterozygosis with hemoglobin A causing no clinical or hematological alterations, or in homozygosis, the rarest form of inheritance, a condition that is commonly not related to clinical symptomatology. Moreover, this variant can exist in association with other hemoglobinopathies, such as thalassemias; the most noticeable clinical alterations occur when hemoglobin D-Punjab is associated to hemoglobin S. The clinical manifestations of this association can be similar to homozygosis for hemoglobin S. Although hemoglobin D-Punjab is a common variant globally with clinical importance especially in cases of double heterozygosis, hemoglobin S/D-Punjab is still understudied. In Brazil, for example, hemoglobin D-Punjab is the third most common hemoglobin variant. Thus, this paper summarizes information about the origin, geographic distribution, characterization and occurrence of hemoglobin D-Punjab haplotypes to try to improve our knowledge of this variant. Moreover, a list of the main techniques used in its identification is provided emphasizing the importance of complementary molecular analysis for accurate diagnosis.

O sofrimento gera luta: o impacto da anemia falciforme e da vivência do adoecimento no desenvolvimento psíquico de portadores da doença

Pós-graduação em Saúde Coletiva - FMB

O papel da angiogênese e da hemólise na úlcera de perna de pessoas com anemia falciforme

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Hb S-Sao Paulo: A new sickling hemoglobin with stable polymers and decreased oxygen affinity

Hb S-Sao Paulo (SP) [HBB:c.20A > T p.Glu6Val: c.196A > G p.Lys65Glu] is a new double-mutant hemoglobin that was found in heterozygosis in an 18-month-old Brazilian male with moderate anemia. It behaves like Hb S in acid electrophoresis, isoelectric focusing and solubility testing but shows different behavior in alkaline electrophoresis, cation-exchange HPLC and RP-HPLC. The variant is slightly unstable, showed reduced oxygen affinity and also appeared to form polymers more stable than the Hb S. Molecular dynamics simulation suggests that the polymerization is favored by interfacial electrostatic interactions. This provides a plausible explanation for some of the reported experimental observations. (C) 2012 Elsevier Inc. All rights reserved.

Epidemiological study of HPV in oral mucosa through PCR

The Human Papillomavirus (HPV) belongs to the Papillomaviridae family and has a capsid and a single DNA strand. Its infection occurs mainly through sexual intercourse, having an important tropism for skin and mucosal cells. Aim: To evaluate the HPV presence in normal oral mucosa of asymptomatic subjects and; in parallel, to correlate social behavioral habits with the virus. Materials and Methods: Contemporary cohort cross-sectional study. The HPV was found by PCR, using general primers MY09/11 in 125 oral mucosa samples submitted to DNA extraction and PCR to search for the beta-globin gene in order to assess the quality of the extracted DNA. In parallel, we carried out a study of behavioral issues associated with the patients. Results: All the samples had a positive diagnosis of the beta-hemoglobin gene. HPV was diagnosed in 23.2% of the samples analyzed. Conclusion: The virus was present in 29 of the 125 patients, without them having any clinical-pathological manifestation associated with the HPV. As to the social behavior of the patients, we concluded that oral sex is statistically correlated to the virus, and besides the HPV has been statistically more present in female patients.

Possible implication of Mdm2 as a prognostic marker in invasive laryngeal carcinoma

Laryngeal squamous cell carcinoma is one of the most common malignant neoplasms of the head and neck. In Brazil, laryngeal tumors represent 2% of all cancers and are associated with approximately 3,000 deaths annually. Human papillomavirus (HPV) has been reported to play an important role in the etiology of laryngeal cancer. The aim of the present study was to evaluate the expression of p53, p27, and Mdm2 in laryngeal carcinomas. Sixty-three larynx biopsies were selected for the study, including 9 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastasis and 27 laryngeal carcinomas with metastasis. Twenty-seven cervical lymph nodes from patients with metastatic lesions were also evaluated. The expression levels of p53, p27, and Mdm2 were assessed by immunohistochemistry using a computer-assisted system. HPV detection and typing were performed using PCR, and the HPV types that were evaluated included HPV 6, 11, 16, 18, 31 and 33. Out of 63 patients, 53 (84.1%) were positive for beta-globin (internal control), and 10 (15.9%) were beta-globin negative and therefore excluded from the evaluation. Thus, 7 (13.2%) out of 53 patients were HPV positive, and 46 (86.8%) out of 53 patients were HPV negative. Statistically significant differences (p < 0.05) in Mdm2 expression levels were observed in the in situ laryngeal carcinoma samples compared with the laryngeal carcinoma samples with metastasis. No statistically significant differences (p > 0.05) in either p53 or p27 expression levels were detected. These findings suggest that Mdm2 may be associated with the invasiveness and aggressiveness of laryngeal carcinomas.

Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes

Many diseases affect pre-mRNA splicing, and alternative splicing is a major source of proteome diversity and an important mechanism for modulating gene expression. The ability to regulate a specific splicing event is therefore desirable; for example, to understand splicing-associated pathologies. We have developed methods based on modified U7 snRNAs, which allow us to induce efficient skipping or inclusion of selected exons. Here, we have adapted these U7 tools to a regulatable system that relies on a doxycycline-sensitive version of the Kruppel-associated box (KRAB)/KAP1 transcriptional silencing. Co-transduction of target cells with two lentiviral vectors, one carrying the KRAB protein and the other the regulatable U7 cassette, allows a tight regulation of the modified U7 snRNA. No leakage is observed in the repressed state, whereas full induction can be obtained with doxycycline in ng ml(-1) concentrations. Only a few days are necessary for a full switch, and the induction/repression can be repeated over several cycles without noticeable loss of control. Importantly, the U7 expression correlates with splicing correction, as shown for the skipping of an aberrant beta-globin exon created by a thalassaemic mutation and the promotion of exon 7 inclusion in the human SMN2 gene, an important therapeutic target for spinal muscular atrophy.

Single-stranded DNA-binding proteins regulate the abundance of LIM domain and LIM domain-binding proteins.

The LIM domain-binding protein Ldb1 is an essential cofactor of LIM-homeodomain (LIM-HD) and LIM-only (LMO) proteins in development. The stoichiometry of Ldb1, LIM-HD, and LMO proteins is tightly controlled in the cell and is likely a critical determinant of their biological actions. Single-stranded DNA-binding proteins (SSBPs) were recently shown to interact with Ldb1 and are also important in developmental programs. We establish here that two mammalian SSBPs, SSBP2 and SSBP3, contribute to an erythroid DNA-binding complex that contains the transcription factors Tal1 and GATA-1, the LIM domain protein Lmo2, and Ldb1 and binds a bipartite E-box-GATA DNA sequence motif. In addition, SSBP2 was found to augment transcription of the Protein 4.2 (P4.2) gene, a direct target of the E-box-GATA-binding complex, in an Ldb1-dependent manner and to increase endogenous Ldb1 and Lmo2 protein levels, E-box-GATA DNA-binding activity, and P4.2 and beta-globin expression in erythroid progenitors. Finally, SSBP2 was demonstrated to inhibit Ldb1 and Lmo2 interaction with the E3 ubiquitin ligase RLIM, prevent RLIM-mediated Ldb1 ubiquitination, and protect Ldb1 and Lmo2 from proteasomal degradation. These results define a novel biochemical function for SSBPs in regulating the abundance of LIM domain and LIM domain-binding proteins.

Molecular mechanisms of chondrocyte-specific expression on the mouse pro$\alpha$1(II) collagen gene

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro$\alpha1$(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a $\beta$-galactosidase reporter gene. A construction containing a 3000-bp promoter and a 3020-bp intron 1 fragment directed high levels of $\beta$-galactosidase expression specifically to chondrocytes. Successive deletions of intron 1 delineated a 48-bp fragment which targeted $\beta$-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. When the Col2a1 promoter was replaced with a minimal $\beta$-globin promoter, the 48-bp intron 1 sequence was still able to target expression of the transgene to chondrocytes, specifically. Therefore a 48-bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression to a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression. Nuclear proteins present selectively in mouse primary chondrocytes and rat chondrosarcoma cells bind to the three putative HMG (High-Mobility-Group) domain protein binding sites in this 48-bp sequence and the chondrocyte-specific proteins likely bind the DNA through minor groove. Together, my results indicate that a 48-bp sequence in Col2a1 intron 1 controls chondrocyte-specific expression in vivo and suggest that chondrocytes contain specific nuclear proteins involved in enhancer activity. ^

Antisense properties of tricyclo-DNA.

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.