Cytogenetic analysis was performed in peripheral blood lymphocytes from hospital workers chronically exposed to ionizing radiation in comparison to matched non-exposed individuals. The accumulated absorbed doses calculated for the radiation workers ranged from 9.5 to 209.4 mSv. The endpoints used were chromosomal aberrations (CA), micronuclei (MN), and sister chromatid exchanges (SCE). The frequencies of CA/100 cells observed for the exposed group were significantly (P=0.018) higher than in the control group: 3.2 and 2.6, respectively. Similarly, the mean numbers of SCE per cell were statistically higher (P=0.025) in the exposed group (6.2) in comparison with the control group (5.8). In the case of micronuclei analysis, no significant (P=0,06) difference between both groups was found, but these data should be cautiously interpreted since an increase in the frequencies of MN was found for radiation workers (3.0 MN/100 cells), compared to the control group (2.6 MN/100 cells) and this increase occur in parallel to CA and SCE frequencies. The difference between the results could be explained by the nature of CA and MN generation. The increased frequencies of CA and SCE in radiation workers indicate the cumulative effect of low-level chronic exposure to ionizing radiation, and the relevance of conducting cytogenetic analysis in parallel to physical dosimetry in the working place.

BackgroundThe development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.MethodsThe study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.ResultsWe observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (ARID4A, CALR, GNB2L1, RNF10, SQSTM1, USP9X) were validated by real time PCR.ConclusionsA significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.

Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.

The mode of action of annexin A1 (ANXA1) is poorly understood. By using rapid subtraction hybridization we studied the effects of human recombinant ANXA1 and the N-terminal ANXA1 peptide on gene expression in a human larynx cell line. Three genes showed strong downregulation after treatment with ANXA1. In contrast, expression of CCR10, a seven transmembrane G-protein coupled receptor for chemokine CCL27 involved in mucosal immunity, was increased. Moreover the reduction in CCR10 expression induced by ANXA1 gene deletion was rescued by intravenous treatment with low doses of ANXA1. These findings provide new evidence that ANXA1 modulates gene expression.

ABSTRACT.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 β 0 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 β 0 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 β 0 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.

Chromosomal instability involving telomeric DNA sequences was studied in mouse Balb/3T3 fibroblasts transfected with a mutated human c-Ha-ras-1 gene (B61 cells) and spontaneously immortalized normal parental cells (A31 cells), using fluorescence in situ hybridization (FISH). FISH analysis with a telomeric probe revealed high frequencies of chromosome alterations involving telomeric regions, mainly stable and unstable Robertsonian fusion-like configurations (RLC) (0.25 and 1.95/cell in A31 and B61 cells, respectively) and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS") (5.9 and 17.5/cell for A31 and B61 cells, respectively). Interstitial telomeric sequences (ITS) were also detected at both non-telomeric sites and in the centromeres of RLC. The frequencies of RLCs with ITS located in the centromeres were 3-fold higher in B61 compared with A31 cells. We demonstrated a high level of chromosome instability involving telomeric DNA sequences in ras-transfected cells overexpressing ras mRNA, which could be a consequence of rapid cell cycle progression associated with a deficient telomere capping mechanism.

The organophosphorus insecticide Nuvacron (Monocrotophos) is a very toxic agent widely utilized in Brazilian agriculture. To evaluate the clastogenic potential of this insecticide, in vivo and in vitro micronucleus (MN) assay experiments were carried out on Swiss mice and on Chinese hamster ovary (CHO) cells, respectively. Nuvacron administered at doses of 2.5 and 5.0 mg/kg induced a statistically significant increase in the frequencies of MN detected in polychromatic bone marrow erythrocytes from animals (six/group) treated ip 24 h before. Exponentially growing CHAO cells were treated continuously (16h) with Nuvacron diluted in water to final concentrations of 1, 10, 100, 200, and 400 mug/ml. Three experiments were carried out using the cytokinesis-block method and a total of 6000 binucleated cells were scored to determine MN frequencies. A statistically significant increase in the frequencies of MN was observed for the cells treated with 1 and 10 mug/ ml Nuvacron. A marked decrease in cell proliferation rates was observed for CHO cultures treated with higher concentrations. These data demonstrate that Nuvacron has a genotoxic effect on both in vivo and in vitro mammalian test systems

In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.Keywords: Cryptosporidium, glycoprotein GP60, polyclonal antibody, diagnosis. ResumoNeste trabalho, foi desenvolvido um método de expressão da glicoproteína GP60 de Cryptosporidium hominis em Escherichia coli visando produzir anticorpos IgY anti-GP60 em galinhas para utilização em estudos futuros com os objetivos de diagnóstico, prevenção e tratamento da criptosporidiose. A sequência completa de nucleotídeos do gene gp60 de C. hominis foi códon-otimizada para expressão em E. coli e sintetizada no vetor pET28-a. A subclonagem foi realizada em várias estirpes diferentes de E. coli BL21. Os ensaios de concentração do indutor IPTG, temperatura e tempo foram realizados e analisados por SDS-PAGE. As condições ótimas de expressão foram observadas em temperatura de 37 °C, incubação durante a noite e 1 mM de IPTG. A purificação da proteína foi realizada por cromatografia de afinidade utilizando o sistema de cromatografia AKTA Pure e a coluna Hi-Trap™ HP (GE Healthcare). A proteína recombinante GP60 (rGP60) foi utilizada para imunizar galinhas poedeiras para produzir IgY policlonal anti-rGP60. Verificou-se por Western blot e por imunofluorescência indireta que o anticorpo policlonal apresentou reatividade com a rGP60 e com esporozoítos de Cryptosporidium parvum, respectivamente. A rGP60 e a IgY anti-rGP60 geradas neste estudo podem ser utilizadas como modelos para o desenvolvimento de ensaios para pesquisa e diagnóstico da criptosporidiose.Palavras-chave: Cryptosporidium, glicoproteína GP60, anticorpo policlonal, diagnóstico.