Recent advances in DNA vaccines for autoimmune diseases

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of ginger (Zingiber officinale Roscoe) on DNA damage and development of urothelial tumors in a mouse bladder carcinogenesis model

Extracts of the spice ginger (Zingiber officinale Roscoe) are rich in gingerols and shogaols, which exhibit antioxidant, anti-inflammatory, antifungal, anti mycobacterial, and anticarcinogenic proprieties. The present study evaluated the chemoprotective effects of a ginger extract on the DNA damage and the development of bladder cancer induced by N-butyl-N-(4-hydroxibutyl) nitrosamine (BBN)/N-methyl-N-nitrosourea (MNU) in male Swiss mice. Groups G1-G3 were given 0.05% BBN in drinking water for 18 weeks and four i.p. injections of 30 mg/kg body weight MNU at 1, 3, 10, and 18 weeks. Group G4 and G5 received only the BBN or MNU treatments, respectively, and groups G6 and G7 were not treated with BBN or MNU. Additionally, Groups G2, G3, and G6 were fed diets containing 1, 2, and 2% ginger extract, respectively, while Groups G1, G4, G5, and G7 were fed basal diet. Samples of peripheral blood were collected during the experiment for genotoxicity analysis; blood collected 4 hr after each MNU dose was used for the analysis of DNA damage with the Comet assay (assay performed on leukocytes from all groups), while reficulocytes collected 24 hr after the last MNU treatment of Groups G5-G7 were used for the micronucleus assay. At the end of the experiment, the urinary bladder was removed, fixed, and prepared for histopathological, cell proliferation, and apoptosis evaluations. Ginger by itself was not genotoxic, and it did not alter the DNA damage levels induced by the BBN/MNU treatment during the course of the exposure. The incidence and multiplicity of simple and nodular hyperplasia and transitional cell carcinoma (TCC) were increased by the BBN/MNU treatment, but dietary ginger had no significant effect on these responses. However, in Group G2 (BBN/MNU/2% ginger-treated group), there was an increased incidence of Grade 2 TCC. The results suggest that ginger extract does not inhibit the development of BBN-induced mouse bladder tumors.

Anti-clastogenic effect of beta-glucan extracted from barley towards chemically induced DNA damage in rodent cells

beta-Glucan (BG) was tested in vitro to determine its potential clastogenic and/or anti-clastogenic activity, and attempts were made to elucidate its possible mechanism of action by using combinations with an inhibitor of DNA polymerase. The study was carried out on cells deficient (CHO-k1) and cells proficient (HTC) in phases I and II enzymes, and the DNA damage was assessed by the chromosomal aberration assay. BG did not show a clastogenic effect, but was anti-clastogenic in both cell lines used, and at all concentrations tested (2.5, 5 and 10 mg/mL) in combination with damage inducing agents (methylmethane sulfonate in cell line CHO-k1, and methylmethane sulfonate or 2-aminoanthracene in cell line HTC). BG also showed a protective effect in the presence of a DNA polymerase beta inhibitor (cytosine arabinoside-3-phosphate, Ara-C), demonstrating that BG does not act through an anti-mutagenic mechanism of action involving DNA polymerase beta.

Evaluation of antimutagenic activity and mechanisms of action of beta-glucan from barley, in CHO-k1 and HTC cell lines using the micronucleus test

Due to the need to identify new antimutagenic agents and to determine their mechanism of action, the present study examined the mechanism of action of the P-glucan with regard to antimutagenicity using the micronucleus assay in CHO-kl and HTC cell lines. The mutagenicity experiments were performed with three different concentrations of P-glucan (5, 10, and 20 mu g/mL), in wich only the highest dose showed mutagenic activity. In the antimutagenicity experiments, the same concentrations of P-glucan were combined with a mutagenic agent, methylmethane sulfonate, or 2-aminoanthracene, using four different treatment protocols: pre-treatment, simultaneous treatment (simple and with pre-incubation), and post-treatment. The results indicate that the CHO-kl cell line treated with MMS presented a chemopreventive activity for all the doses of P-glucan in the different treatment protocols, except for the lowest dose in post-treatment. When HTC cell line treated with MMS is analysed, a chemopreventive activity can be verified for the highest dose in both pre- and post-treatment. For the simple simultaneous treatment, the three doses demonstrated efficacy, while for the simultaneous treatment with pre-incubation only the intermediate concentration was effective. In HTC treated with 2AA both the lowest dose in the pre-treatment protocol and the post-treatment protocol did not show efficacy in preventing DNA damage. The evaluation of the different protocols and the damage decrease percentages observed suggest that P-glucan has both desmutagenic and bioantimutagenic activity. It is necessary, however, to note that efficacy and mechanism of action are subject to variation when compared the two cell lines, since in HTC, representing a drug-metabolizing system, this substance can show a diminished chemopreventive capacity. (c) 2006 Elsevier Ltd. All rights reserved.

Protective effects of beta-glucan extracted from Agaricus brasiliensis against chemically induced DNA damage in human lymphocytes

beta-Glucans (BGs) are polysaccharides that are found in the cell walls of organisms such as bacteria, fungi, and some cereals. The objective of the present study was to investigate the genotoxic and antigenotoxic effects of BG extracted from the mushroom Agaricus brasiliensis (=Agaricus blazei Murrill ss. Heinemann). The mutagenic activity of BG was tested in single-cell gel electrophoresis assays with human peripheral lymphocytes. In addition, the protective effects against the cooked food mutagen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) and (+/-)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), which is the main metabolite of B[a]P, and against ROS (H2O2)-induced DNA damage, were studied. The results showed that the compound itself was devoid of mutagenic activity, and that a significant dose-dependent protective effect against damage induced by hydrogen peroxide and Trp-P-2 occurred in the dose range 20-80 mu g/ml. To investigate the prevention of Trp-P-2-induced DNA damage, a binding assay was carried out to determine whether BG inactivates the amine via direct binding. Since no such interactions were observed, it is likely that BG interacts with enzymes involved in the metabolism of the amine.

Protective effect of beta-glucan extracted from Saccharomyces cerevisiae, against DNA damage and cytotoxicity in wild-type (k1) and repair-deficient (xrs5) CHO cells

A large number of functional foods, including those that contain P-glucan, have been shown to prevent the development of cancer and other chronic diseases. The aim of the present study was to elucidate its mechanism of action, as well as to understand its effects as an antigenotoxic, anticlastogenic agent, and to determine its capacity to preserve cell viability. The investigation was carried out in the CHO-k1 and CHO-xrs5 cell lines. The cytokinesis-blocked micronucleus assay indicated that the different doses of beta-glucan examined (5, 10, 20 and 40 mu g/ml) did not show clastogenic effects. In the CHO-k1 cell line, a chemopreventive effect could be observed in all the protocols tested: pre-treatment (% reduction of 35.0-57.3), simultaneous treatment (simple - 5 reduction of 19.7-55.6 and with pre-incubation - of 42.7-56.4) and post-treatment (% reduction of 17.9-37.6). This finding indicates mechanisms of action involving desmutagenesis and bio-antimutagenesis, albeit the latter having a lesser role. However, in the repair-deficient CHO-xrs5 cells, beta-glucan did not show a protective effect with post-treatment (% reduction of 2.96), thus supporting the involvement of bioantimutagenesis. The comet assay in CHO-k1 cells demonstrated that beta-glucan has neither a genotoxic nor an antigenotoxic effect. Cell viability tests indicated that beta-glucan preserves cell viability in both cell lines, preventing apoptotic events. These findings suggest that beta-glucan, when present in foods, could provide them with nutraceutical characteristics and act as a dietary supplement, or that P-glucan could be used in new drug development. (c) 2006 Elsevier Ltd. All rights reserved.

Nuclear alterations associated to programmed cell death in larval salivary glands of Apis mellifera (Hymenoptera : Apidae)

The silk glands of bees are a good model for the study of cell death in insects. With the objective to detect the nuclear features during glandular regression stage, larvae at the last instar and pre-pupae were collected and their silk glands were dissected and processed for ultrastructural analysis and histologically for cytochemical and imunocytochemical analysis. The results showed that the cellular nuclei exhibited characteristics of death by atypical apoptosis as well as autophagic cell death. Among the apoptosis characteristic were: nuclear strangulation with bleb formation in some nuclei, DNA fragmentation in most of the nuclei and nucleolar fragmentation. Centripetal chromatin compaction was observed in many nuclei, forming a perichromatin halo differing from typical apoptotic nuclei. With regards to the characteristics of autophagic-programmed cell death, most relevant was the delay in the collapse of many nuclei. (C) 2006 Elsevier Ltd. All rights reserved.

Evaluation of the antigenotoxicity of polysaccharides and beta-glucans from Agaricus blazei, a model study with the single cell gel electrophoresis/Hep G2 assay

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1-16% decrease in Alu element and 18-60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.

Gene expression profiling reveals molecular marker candidates of laryngeal squamous cell carcinoma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of the Bilayer Composition on the Binding and Membrane Disrupting Effect of Polybia-MP1, an Antimicrobial Mastoparan Peptide with Leukemic T-Lymphocyte Cell Selectivity

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação da perda da tolerância à dessecação e da quantidade de dna nuclear em sementes de Peltophorum dubium (spreng.) taubert durante e após a germinação

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

COMPENSATORY LUNG GROWTH: LUNG PROTEIN,DNA and RNA CONTENTS IN TRILOBECTOMIZED RATS

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Compensatory lung growth: protein, DNA and RNA lung contents in undernourished trilobectomized rats

OBJETIVO: demonstrar se ocorre crescimento pulmonar compensatório (CPC) representado pelos conteúdos de proteínas, DNA e RNA no rato adulto jovem, subnutrido, submetido à trilobectomia pulmonar. MÉTODOS: Utilizamos 137 ratos Wistar, machos, distribuídos por sorteio, em 9 grupos, submetidos a três tratamentos (controle, toracotomia, trilobectomia), sacrificados em três momentos (7, 30 e 90 dias). Na trilobectomia foram extirpados os lobos médio, acessório e caudal direitos. Variáveis estudadas: conteúdos pulmonares de proteínas, DNA e RNA. RESULTADOS: No lobo cranial e pulmão esquerdo o conteúdo protéico foi maior nos trilobectomizados. Ocorreu CPC insuficiente para suprir a perda desta variável, sendo menor nos pulmões dos trilobectomizados. O incremento nos conteúdos de DNA do lobo cranial e pulmão esquerdo dos trilobectomizados foram suficientes para compensar a perda desta variável, resultando num conteúdo de DNA dos pulmões semelhante aos controle. O conteúdo de RNA, nos trilobectomizados, foi maior no lobo cranial e pulmão esquerdo, com maior eficiência no primeiro, insuficiente para que se aproximassem aos obtidos nos demais grupos, ficando menores. CONCLUSÃO: Nos trilobectomizados ocorreu CPC, provavelmente com hiperplasia celular e pouca hipertrofia, devido a grande compensação do DNA e pequena do RNA. Esta foi a grande diferença quando comparamos este resultado ao obtido com animais nutridos, que apresentavam hipertrofia pronunciada.

CYTOPHOTOMETRIC EVALUATION OF CORPORA ALLATA DNA DURING THE DEVELOPMENT OF LARVAE OF WORKERS AND QUEENS (APIS-MELLIFERA L, HYMENOPTERA, APIDAE)

Cytophotometric observation of corpora allata from workers and queens of A. mellifera showed significant variations in DNA content from the 2nd to the 4th instar of both castes, but the differences between castes are present only in the 2nd instar.The corpora allata are characterized by the presence of diploid, as well as polyploid cells, distributed in classes that are well established since the earliest developmental stages. However the frequency of cell nuclei in the higher classes of ploidy increases with development. This occurrence is responsible for the increase of the average DNA content in older larvae.