Targeting the Transposase Domain of the DNA Repair Component Metnase to Enhance Chemotherapy

Previous studies have shown that the DNA repair component Metnase (SETMAR) mediates resistance to DNA damaging cancer chemotherapy. Metnase has a nuclease domain that shares homology with the Transposase family. We therefore virtually screened the tertiary Metnase structure against the 550,000 compound ChemDiv library to identify small molecules that might dock in the active site of the transposase nuclease domain of Metnase. We identified eight compounds as possible Metnase inhibitors. Interestingly, among these candidate inhibitors were quinolone antibiotics and HIV integrase inhibitors, which share common structural features. Previous reports have described possible activity of quinolones as antineoplastic agents. Therefore, we chose the quinolone ciprofloxacin for further study, based on its wide clinical availability and low toxicity. We found that ciprofloxacin inhibits the ability of Metnase to cleave DNA and inhibits Metnase-dependent DNA repair. Ciprofloxacin on its own did not induce DNA damage, but it did reduce repair of chemotherapy-induced DNA damage. Ciprofloxacin increased the sensitivity of cancer cell lines and a xenograft tumor model to clinically relevant chemotherapy. These studies provide a mechanism for the previously postulated antineoplastic activity of quinolones, and suggest that ciprofloxacin might be a simple yet effective adjunct to cancer chemotherapy. Cancer Res; 72(23); 6200-8. (C) 2012 AACR.

Active site mutants of human secreted Group IIA Phospholipase A(2) lacking hydrolytic activity retain their bactericidal effect

The Human Secreted Group IIA Phospholipase A(2) (hsPIA2GIIA) presents potent bactericidal activity, and is considered to contribute to the acute-phase immune response. Hydrolysis of inner membrane phospholipids is suggested to underlie the bactericidal activity, and we have evaluated this proposal by comparing catalytic activity with bactericidal and liposome membrane damaging effects of the G30S, H48Q and D49K h5PLA2GIIA mutants. All mutants showed severely impaired hydrolytic activities against mixed DOPC:DOPG liposome membranes, however the bactericidal effect against Micrococcus luteus was less affected, with 50% killing at concentrations of 1, 3, 7 and 9 mu g/mL for the wild-type, D49K, H48Q and G30S mutants respectively. Furthermore, all proteins showed Ca2+-independent damaging activity against Liposome membranes demonstrating that in addition to the hydrolysis-dependent membrane damage, the hsPLA2GIIA presents a mechanism for permeabilization of phospholipid bilayers that is independent of catalytic activity, which may play a role in the bactericidal function of the protein (C) 2011 Elsevier Masson SAS. All rights reserved.

Characterization of anti-silencing factor 1 in Leishmania major

Anti-silencing factor 1 (ASF1) is a histone chaperone that contributes to the histone deposition during nucleosome assembly in newly replicated DNA. It is involved in chromatin disassembly, transcription activation and in the cellular response to DNA damage. In Leishmania major the ASF1 gene (LmASF1) is located in chromosome 20 and codes for a protein showing 67% of identity with the Trypanosoma brucei TbASF1a. Compared to orthologous proteins, LmASF1 conserves the main residues relevant for its various biological functions. To study ASF1 in Leishmania we generated a mutant overexpressing LmASF1 in L. major. We observed that the excess of LmASF1 impaired promastigotes growth rates and had no impact on cell cycle progress. Differently from yeast, ASF1 overproduction in Leishmania did not affect expression levels of genes located on telomeres, but led to an upregulation of proteins involved in chromatin remodelling and physiological stress, such as heat shock proteins, oxidoreductase activity and proteolysis. In addition, we observed that LmASF1 mutant is more susceptible to the DNA damaging agent, methyl methane sulphonate, than the control line. Therefore, our study suggests that ASF1 from Leishmania pertains to the chromatin remodelling machinery of the parasite and acts on its response to DNA damage.

Virulence attributes and genetic variability of oral Candida albicans and Candida tropicalis isolates

The wide spectrum of candidiasis and its clinical importance encourage the research with the purpose of clarifying the mechanisms of pathogenicity and identification of virulence factors of Candida sp. Therefore, the aim of this study was to verify the adhesion capacity, protease activity and genotypic diversity of oral C. albicans and C. tropicalis isolates. The adhesion ability to the extracellular matrix glycoproteins laminin and fibronectin was evaluated using the ELISA technique. The research of proteases was carried out in agar plate containing bovine albumin and through a quantitative method in buffer solution containing haemoglobin. Intra and interspecies polymorphisms was verified through random amplified polymorphic DNA (RAPD) technique. All C. albicans and C. tropicalis isolates binded to immobilised laminin and fibronectin. Ca33 and Ct13 isolates had relative adhesion index significantly higher than the other isolates for both glycoproteins (P < 0.001). Protease activity was observed in all isolates of C. albicans using either the semi-quantitative or quantitative assay. The protease activity of C. tropicalis was better detected through the quantitative assay. The genotypic diversity by RAPD revealed a heterogeneous population in both species. Nevertheless, C. tropicalis presented higher genetic variability than C. albicans strains.

NF-κB Drives the Synthesis of Melatonin in RAW 264.7 Macrophages by Inducing the Transcription of the Arylalkylamine-N-Acetyltransferase (AA-NAT) Gene

We demonstrate that during inflammatory responses the nuclear factor kappa B (NF-kappa B) induces the synthesis of melatonin by macrophages and that macrophage-synthesized melatonin modulates the function of these professional phagocytes in an autocrine manner. Expression of a DsRed2 fluorescent reporter driven by regions of the aa-nat promoter, that encodes the key enzyme involved in melatonin synthesis (arylalkylamine-N-acetyltransferase), containing one or two upstream kappa B binding sites in RAW 264.7 macrophage cell lines was repressed when NF-kappa B activity was inhibited by blocking its nuclear translocation or its DNA binding activity or by silencing the transcription of the RelA or c-Rel NF-kappa B subunits. Therefore, transcription of aa-nat driven by NF-kappa B dimers containing RelA or c-Rel subunits mediates pathogen-associated molecular patterns (PAMPs) or pro-inflammatory cytokine-induced melatonin synthesis in macrophages. Furthermore, melatonin acts in an autocrine manner to potentiate macrophage phagocytic activity, whereas luzindole, a competitive antagonist of melatonin receptors, decreases macrophage phagocytic activity. The opposing functions of NF-kappa B in the modulation of AA-NAT expression in pinealocytes and macrophages may represent the key mechanism for the switch in the source of melatonin from the pineal gland to immune-competent cells during the development of an inflammatory response.

Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-kappa B pathway in 3T3-L1 adipocytes

Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by differentiated adipocytes and are involved in energy homeostasis, insulin sensitivity, and the anti-inflammatory response. These two adipokines are reduced in obese subjects, which favors increased activation of nuclear factor kappa B (NF-kappa B) and leads to elevation of pro-inflammatory adipokines. However, the effects of adiponectin and IL-10 on NF-kappa B DNA binding activity (NF-kappa Bp50 and NF-kappa Bp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4) pathway, such as MYD88 and TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of lipopolysaccharide-treated 3T3-L1 adipocytes for 24 h elevated IL-6 levels; activated the NF-kappa B pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased the nuclear activity of NF-kappa B (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited the elevation of IL-6 levels and activated NF-kappa B (p50 and p65) DNA binding. Taken together, the present results provide evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response in adipocytes. In addition, inhibition of NF-kappa B signaling pathways may be an excellent strategy for the treatment of inflammation in obese individuals. (C) 2011 Elsevier Ltd. All rights reserved.

Characterization of suramin binding sites on the human group IIA secreted phospholipase A(2) by site-directed mutagenesis and molecular dynamics simulation

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the h5PLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

The bactericidal effect of human secreted group IID phospholipase A(2) results from both hydrolytic and non-hydrolytic activities

The Human Secreted Group IID Phospholipase A(2) (hsPLA2GIID) may be involved in the human acute immune response. Here we have demonstrated that the hsPLA2GIID presents bactericidal and Ca2+-independent liposome membrane-damaging activities and we have compared these effects with the catalytic activity of active-site mutants of the protein. All mutants showed reduced hydrolytic activity against DOPC:DOPG liposome membranes, however bactericidal effects against Escherichia coli and Micrococcus luteus were less affected, with the D49K mutant retaining 30% killing of the Gram-negative bacteria at a concentration of 10 mu g/mL despite the absence of catalytic activity. The H48Q mutant maintained Ca2+-independent membrane-damaging activity whereas the G30S and D49K mutants were approximately 50% of the wild-type protein, demonstrating that phospholipid bilayer permeabilization by the hsPLA2GIID is independent of catalytic activity. We suggest that this Ca2+-independent damaging activity may play a role in the bactericidal function of the protein. (C) 2012 Elsevier Masson SAS. All rights reserved.

Changes in CREB activation in the prefrontal cortex and hippocampus blunt ethanol-induced behavioral sensitization in adolescent mice

Drug dependence is a major health problem in adults and has been recognized as a significant problem in adolescents. We previously demonstrated that repeated treatment with a behaviorally sensitizing dose of ethanol in adult mice induced tolerance or no sensitization in adolescents and that repeated ethanol-treated adolescents expressed lower Fos and Egr-1 expression than adult mice in the prefrontal cortex (PFC). In the present work, we investigated the effects of acute and repeated ethanol administration on cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) DNA-binding activity using the electrophoretic mobility shift assay (EMSA) and the phosphorylated CREB (pCREB)/CREB ratio using immunoblotting in both the PFC and hippocampus in adolescent and adult mice. Adult mice exhibited typical locomotor sensitization after 15 days of daily treatment with 2.0 g/kg ethanol, whereas adolescent mice did not exhibit sensitization. Overall, adolescent mice displayed lower CREB binding activity in the PFC compared with adult mice, whereas opposite effects were observed in the hippocampus. The present results indicate that ethanol exposure induces significant and differential neuroadaptive changes in CREB DNA-binding activity in the PFC and hippocampus in adolescent mice compared with adult mice. These differential molecular changes may contribute to the blunted ethanol-induced behavioral sensitization observed in adolescent mice.

The Gracilariaceae Germplasm Bank of the University of So Paulo, Brazil-a DNA barcoding approach

The University of So Paulo Gracilariaceae Germplasm Bank has 50 strains collected mostly in Brazil, but also elsewhere in the world. This bank has been used as a source of material for research developed locally and abroad. With over 200 species, some of which have high economic value, the family Gracilariaceae has been extensively studied. Nonetheless, taxonomic problems still persist by the existence of cryptic species, phenotypic plasticity, and broad geographic distribution. In the case of algae kept in culture for long periods of time, the identification is even more problematic as a consequence of considerable morphological modification. Thus, the use of molecular markers has been shown to be an efficient tool to elucidate taxonomic issues in the group. In this work, we sequenced the 5'-end of the cox1 gene for 41 strains and the universal plastid amplicon (UPA) plastid region for 45 strains, covering all 50 strains in the bank. In addition, the rbcL for representatives of the cox1/UPA clusters was sequenced for 14 strains. The original species identification based on morphology was compared with the molecular data obtained in this work, resulting in the identification of 13 different species. Our analyses indicate that cox1 and UPA are suitable markers for the delineation of species of Gracilariales in the germplasm bank. The addition of DNA barcode tags to the samples in the Gracilariaceae germplasm bank and the molecular identification of the species will make this bank even more useful for future research as the species can be easily traced and confirmed.

INVESTIGAÇÃO ELETROQUÍMICA E CALORIMÉTRICA DA INTERAÇÃO DE NOVOS AGENTES ANTITUMORAIS BISCATIÔNICOS COM DNA

ELECTROCHEMICAL AND CALORIMETRIC INVESTIGATION OF INTERACTION OF NOVEL BISCATIONIC ANTICANCER AGENTS WITH DNA. Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-alpha,omega-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Glutathione Peroxidase 1 Pro198Leu Polymorphism in Brazilian Alzheimer's Disease Patients: Relations to the Enzyme Activity and to Selenium Status

Background/Aims: Oxidative stress plays a central role in Alzheimer's disease (AD). Pro198Leu cytosolic glutathione peroxidase (GPx1) polymorphism seems to be associated with a lower activity of this enzyme, but there are no studies with AD patients. Thus, the aim was to determine the frequency of the GPx1 Pro198Leu polymorphism in AD patients and to verify its relation to glutathione peroxidase (GPx) activity and selenium (Se) status. Methods:The study was carried out in a group of AD elderly (n = 28) compared to a control group (n = 29). Blood Se concentrations were measured through hydride generation atomic absorption spectroscopy. GPx activity was determined using a commercial kit, and the polymorphism using amplified DNA sequencing. Results:The distribution of genotypes was not different between groups. The variant allele frequency was 0.179 (AD group) and 0.207 (control group). Although no differences regarding GPx activity were found between individuals with different genotypes, lower blood Se levels were found in Pro/Pro AD patients compared to Pro/Pro control subjects, which was not found in the Pro/Leu groups. Moreover, the association between the erythrocyte Se concentration and GPx activity was affected by the Pro198Leu genotype. Conclusions: Results indicate that this polymorphism had apparently affected Se status in AD patients and that more studies in this field are necessary. Copyright (c) 2012 S. Karger AG, Basel

Stable assemblies of cationic bilayer fragments and CpG oligonucleotide with enhanced immunoadjuvant activity in vivo

The cationic lipid dioctadecyldimethylammonium bromide (DODAB) and the CpG oligonucleotide (CpG) have been separately used as potent immunoadjuvants driving Th1 responses. Here DODAB bilayer fragments (BF) and CpG (5 -TTGACGTTCG-3) assemblies have their physical properties and immunoadjuvant activity determined using ovalbumin (OVA) as a model antigen. At 0.1 mg/mL OVA, the dependence of DODAB BF/OVA size and zeta-potential on time and [DODAB] establishes 0.1 mMDODAB as suitable for obtaining stable and cationic DODAB BF/OVA assemblies. At 0.1 mMDODAB, 0.1 mg/mL OVA and 0.006 mMCpG, the zeta-potential is zero. At [CpG]>0.006 mM, good colloidal stability for the anionic assemblies is due to charge overcompensation. At 0.020 mM CpG, these DODAB BF/OVA/CpG assemblies are highly effective in vivo generating responses similar to those elicited by the stable and cationic DODAB BF/OVA. The anti-OVA DTH reaction and the secretion of IFN-gamma and IL-12 are 6, 42 and 9 times larger for the DODAB BF/OVA/CpG-immunized mice than the same responses by OVA-immunized mice, respectively. This work shows for the first time that charge of small assemblies is not important to determine the immune response. (C) 2011 Elsevier B. V. All rights reserved.

EROD activity and genotoxicity in the seabob shrimp Xiphopenaeus kroyeri exposed to benzo[a]pyrene (BaP) concentrations

Seabob shrimp Xiphopenaeus kroyeri is a marine species that lives in shallow waters of coastal environments, often impacted by polycyclic aromatic hydrocarbons (PAH) pollution. In the present study, seabob shrimp were exposed for 96 h to benzo[a]pyrene (BaP) at the nominal concentrations of 100, 200, 400 and 800 microg.L-1. Animals of the control groups were exposed either to clean water or to the BaP-carrier (DMSO). At the end of the exposures, muscle tissues were sampled for BaP uptake assessment and hepatopancreas and hemolymph for EROD enzyme activity and hemocytes DNA damage, respectively. EROD activity and DNA damage increased significantly as a function of BaP exposure concentrations. Significant correlations between BaP uptake and both EROD activity and DNA damage suggest that they can be used as suitable tools for integrated levels of study on the biomarkers of PAH exposure.

Evaluation of the mutagenic activity of chrysin, a flavonoid inhibitor of the aromatization process

Chrysin is one of the natural flavonoids present in plants, and large amounts are present in honey and propolis. In addition to anticancer, antioxidation, and anti-inflammatory activities, chrysin has also been reported to be an inhibitor of aromatase, an enzyme converting testosterone into estrogen. The present study evaluated the mutagenicity of this flavonoid using micronucleus (MN) with HepG2 cells and Salmonella. Cell survival after exposure to different concentrations of chrysin was also determined using sulforhodamine B (SRB) colorimetric assay in HepG2 cells and the influence of this flavonoid on growth of cells in relation to the cell cycle and apoptosis. TheMN test showed that from 1 to 15 mu M of this flavonoid mutagenic activity was noted in HepG2 cells. The Salmonella assay demonstrated a positive response to the TA100 Salmonella strain in the presence or absence of S9, suggesting that this compound acted on DNA, inducing base pair substitution before or after metabolism via cytochrome P-450. The SRB assay illustrated that chrysin promoted growth inhibition of HepG2 cells in both periods studied (24 and 48 h). After 24 h of exposure it was noted that the most significant results were obtained with a concentration of 50 mu M, resulting in 83% inhibition and SubG0 percentage of 12%. After 48 h of incubation cell proliferation inhibition rates (97% at 50 mu M) were significantly higher. Our results showed that chrysin is a mutagenic and cytotoxic compound in cultured human HepG2 cells and Salmonella typhimurium. Although it is widely accepted that flavonoids are substances beneficial to health, one must evaluate the risk versus benefit relationship and concentrations of these substances to which an individual may be exposed.