Insights into the mechanims underlyng the antiproliferative potential of a Co(II) coordination compound bearing 1,10-Phenanthroline-5,6-Dione: DNA and protein interaction studies

The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)(2)][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 mu M) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)(2)(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 mu M, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K (b) (+/- A standard error of the mean) of (3.48 +/- A 0.03) x 10(5) M-1 and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K (b) similar to 4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.

Micronutrients intake associated with DNA damage assessed by in a human biomonitoring study

Nutrition science has evolved into a multidisciplinary field that applies molecular biology and integrates individual health with the epidemiologic investigation of population health. Nutritional genomics studies the functional interaction of food and its components, macro and micronutrients, with the genome at the molecular, cellular, and systemic level. Diet can influence cancer development in several ways, namely direct action of carcinogens in food that can damage DNA, diet components (macro or micronutrients) that can block or induce enzymes involved in activation or deactivation of carcinogenic substances. Moreover, inadequate intake of some molecules involved in DNA synthesis, repair or methylation can influence mutation rate or changes in gene expression. Several studies support the idea that diet can influence the risk of cancer; however information concerning the precise dietary factor that determines human cancer is an ongoing debate. A lot of epidemiological studies, involving food frequency questionnaires, have been developed providing important information concerning diet and cancer, however, diet is a complex composite of various nutrients (macro and micronutrients) and non-nutritive food constituents that makes the search for specific factors almost limitless.

Cobalt complexes bearing scorpionate ligands: Synthesis, characterization, cytotoxicity and DNA cleavage

A number of novel, water-stable redox-active cobalt complexes of the C-functionalized tripodal ligands tris(pyrazolyl)methane XC(pz)(3) (X = HOCH2, CH2OCH2Py or CH2OSO2Me) are reported along with their effects on DNA. The compounds were isolated as air-stable solids and fully characterized by IR and FIR spectroscopies, ESI-MS(+/-), cyclic voltammetry, controlled potential electrolysis, elemental analysis and, in a number of cases, also by single-crystal X-ray diffraction. They showed moderate cytotoxicity in vitro towards HCT116 colorectal carcinoma and HepG2 hepatocellular carcinoma human cancer cell lines. This viability loss is correlated with an increase of tumour cell lines apoptosis. Reactivity studies with biomolecules, such as reducing agents, H2O2, plasmid DNA and UV-visible titrations were also performed to provide tentative insights into the mode of action of the complexes. Incubation of Co(II) complexes with pDNA induced double strand breaks, without requiring the presence of any activator. This pDNA cleavage appears to be mediated by O-centred radical species.

Potential infectivity of blood from HBsAG asymptomatic carriers due to the presence of HBV-DNA and comparison with other markers of hbv infection

Serum samples from 356 HBsAg positive asymptomatic carriers, which were titrated by reverse passive hemagglutination, were analysed for the presence of HBV-DNA, HBsAg and IgM anti-HBc. The samples were divided in three classes, according to the titers of HBsAg and IgM anti-HBc and the distribution of HBV-DNA and HBsAg among these classes was studied. In the high titer class of HBsAg, 65% of samples have one or both markers against only 19% in the low titer class. From the total of 356 samples, 121 gave positive results for IgM anti-HBc (33.9%). From these, 38.9% of HBV-DNA and 47.9% of HBeAg were observed, whereas in samples with absence of IgM anti-HBc, 18.3% and 16.6% were respectively found. A higher frequency of agreement between all these markers was found in the class of high titers of HBsAg; however, HBV-DNA was detected in the low titer class of HBsAg and little or no IgM anti-HBc, showing potential blood infectivity even in HBsAg positive borderline samples.

Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon.

Nucleic Acid Research (2007) Vol.37 N. 14 4755-4766

Immunization aganist hepatitis B in children from endemic zone: evaluation of the antibody response against the DNA recombinant vaccine (Engerix B-20 mcg)

A previous seroepidemiological study in the rural zone of Vargem Alta (ES) SouthEast of Brazil, showed a prevalence of up to 9% of hepatitis B surface antigen (HBsAg) in some areas. One hundred susceptible children aging 1 to 5 years old were selected and immunized with a recombinant DNA hepatitis B vaccine (Smith-Kline 20 mcg) using the 0-1-6 months vaccination schedule. Blood samples were collected at the time of the first vaccine dose (month 0) in order to confirm susceptible individuals and 1,3,6 and 8 months after the first dose , to evaluate the antibody response. Our results showed that two and five months after the second dose, 79% and 88% of children seroconverted respectively, reaching 97% after the third dose. The levels of anti-HBs were calculated in milli International Units/ml (mIU/ml) and demonstrated the markedly increase of protective levels of antibodies after the third dose. These data showed a good immunogenicity of the DNA recombinant hepatitis B vaccine when administered in children of endemic areas.

DNA nanoprobes for molecular detection

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Biológica – especialidade Engenharia Genética, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Random amplified polymorphic DNA (RAPD) analysis of Lutzomyia longipalpis laboratory populations

The phlebotomine sand fly Lutzomyia longipalpis has been incriminated as a vector of American visceral leishmaniasis, caused by Leishmania chagasi. However, some evidence has been accumulated suggesting that it may exist in nature not as a single but as a species complex. Our goal was to compare four laboratory reference populations of L. longipalpis from distinct geographic regions at the molecular level by RAPD-PCR. We screened genomic DNA for polymorphic sites by PCR amplification with decamer single primers of arbitrary nucleotide sequences. One primer distinguished one population (Marajó Island, Pará State, Brazil) from the other three (Lapinha Cave, Minas Gerais State, Brazil; Melgar, Tolima Department, Colombia and Liberia, Guanacaste Province, Costa Rica). The population-specific and the conserved RAPD-PCR amplified fragments were cloned and shown to differ only in number of internal repeats.

DETECTION OF HTLV-I ANTIBODIES AND DNA IN BLOOD SAMPLE OF A PATIENT WITH MYELOPATHY IN NIGERIA

We describe a case of human T-lymphotropic virus type I associated myelopathy in a 50-year old woman in Nigeria. The patient presented with progressive loss of tone to the two lower limbs and later inability to walk. The HTLV-I antibody presence in the plasma collected from the patient was repeatedly detected by enzyme immunoassays (Abbott HTLV-I EIA and Coulter SELECT-HTLV I/II) and confirmed by Western blot technique. In addition, HTLV-I DNA was amplified from the genomic DNA isolated from the peripheral blood mononuclear cells of the patient by the polymerase chain reaction technique. This finding is significant being the first report of association of HTLV-I with myelopathy in Nigeria.

FETO-PLACENTARY PATHOLOGY IN HUMAN PARVOVIRUS B19 INFECTION

In view of the scarce references concerning the histological data in congenital parvovirus human B19 infection, we intend to provide a description of the pathological features observed in six autopsies.The virus was detected by DNA hybridization (ISH-DBH),PCR and electronmicroscopy (EM) in paraffin-embedded feto-placentary tissues.These cases constitute a subset from 86 Non Immunologic Hydrops Fetalis (NIHF) cases, in which a systemic complex of inflammatory/degenerative lesions of unknown etiology was visualized by optical microscopy. In one case a syphilitic process was detected, typefying a double infection. All fetuses showed a similar pathology - hydrops, hepato-splenomegaly, lung hypoplasia and erythroblastemia, the specific histological feature being the presence of intranuclear inclusions in the erythroid progenitors, in the erythropoietic visceral tissue and in blood marrow. Complex cardiopathy allied to abnormal lung lobulation and polisplenia were observed once; in 2 cases endocardial fibroelastosis was diagnosed. The pulmonary lesions were represented by dysmaturity allied to interstitial mononuclear infiltration. The hepatic consisted of cholestasis, portal fibrosis, canalicular proliferation, hemossiderosis, focal necroses and giant cell transformation. The central nervous system lesions were predominantly anoxic although the autolysis impaired a correct diagnosis.

HEPATITIS B VIRUS INFECTION PROFILE IN CENTRAL BRAZILIAN HEMODIALYSIS POPULATION

Hepatitis B has proved to be a major health hazard in hemodialysis patients. In order to investigate the hepatitis B virus (HBV) infection profile in the hemodialysis population of Goiânia city - Central Brazil, all dialysis patients (N=282) were studied. The prevalence of any HBV marker (HBsAg, anti-HBs, and anti-HBc) was 56.7% (95% CI: 51.1-62.7), ranging from 33.3% to 77.7% depending on dialysis unit. HBV-DNA was detected in 67.6% and 88.2% of the HBsAg-positive serum samples, in 91.3% and 100% of the HBsAg/HBeAg-positive samples, and in 18.2% and 63.6% of the HBsAg/anti-HBe-reactive sera by hybridization and PCR, respectively. The length of time on hemodialysis was significantly associated with HBV seropositivity. Only 10% of the patients reported received hepatitis B vaccination. The findings of a high HBV infection prevalence in this population and the increased risk for HBV infection on long-term hemodialysis suggest the environmental transmission, emphasizing the urgent need to evaluate strategies of control and prevention followed in these units.

Genetic variation between susceptible and non-susceptible snails to Schistosoma infection using random amplified polymorphic DNA analysis (RAPDs)

Susceptibility of snails to infection by certain trematodes and their suitability as hosts for continued development has been a bewildering problem in host-parasite relationships. The present work emphasizes our interest in snail genetics to determine what genes or gene products are specifically responsible for susceptibility of snails to infection. High molecular weight DNA was extracted from both susceptible and non-susceptible snails within the same species Biomphalaria tenagophila. RAPD was undertaken to distinguish between the two types of snails. Random primers (10 mers) were used to amplify the extracted DNA by the polymerase chain reaction (PCR) followed by polyacrylamide gel electrophoresis (PAGE) and silver staining. The results suggest that RAPD represents an efficient means of genome comparison, since many molecular markers were detected as genetic variations between susceptible and non-susceptible snails.

Arbitrarily primed PCR fingerprinting of RNA and DNA in Entamoeba histolytica

Differences were detected in the gene expression of strains of E. histolytica using RNA (RAP-PCR) and DNA fingerprinting (RAPD). Analysis of the electrophoretic profiles of the gels revealed some polymorphic markers that could be used in the individual characterization of the strains. The 260 bands generated by using five different primers for RAP-PCR, as well as RAPD, were employed in the construction of dendograms. The dendogram obtained based on the RAPD products permitted the distinction of symptomatic and asymptomatic isolates, as well the correlation between the polymorphism exhibited and the virulence of the strains. The dendogram obtained for the RAP-PCR products did not show a correlation with the virulence of the strains but revealed a high degree of intraspecific transcriptional variability that could be related to other biological features, whether or not these are involved in the pathogenesis of amebiasis.

Gold nanoparticle-DNA conjugates for oligonucleotide vectorization towards gene silencing

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Estudo do efeito de radiação ionizante em metaloproteínas que ligam DNA

Dissertação para obtenção do Grau de Mestre em Biotecnologia