Insulin-like growth factor-1 protects preimplantation embryos from anti-developmental actions of menadione

Menadione is a naphthoquinone used as a vitamin K source in animal feed that can generate reactive oxygen species (ROS) and cause apoptosis. Here, we examined whether menadione reduces development of preimplantation bovine embryos in a ROS-dependent process and tested the hypothesis that actions of menadione would be reduced by insulin-like growth factor-1 (IGF-1). Menadione caused a concentration-dependent decrease in the proportion of embryos that became blastocysts. All concentrations tested (1, 2.5, and 5.0 mu M) inhibited development. Treatment with 100 ng/ml IGF-1 reduced the magnitude of the anti-developmental effects of the two lowest menadione concentrations. Menadione also caused a concentration-dependent increase in the percent of cells positive for the TUNEL reaction. The response was lower for IGF-1-treated embryos. The effects of menadione were mediated by ROS because (1) the anti-developmental effect of menadione was blocked by the antioxidants dithiothreitol and Trolox and (2) menadione caused an increase in ROS generation. Treatment with IGF-1 did not reduce ROS formation in menadione-treated embryos. In conclusion, concentrations of menadione as low as 1.0 mu M can compromise development of bovine preimplantation embryos to the blastocyst stage of development in a ROS-dependent mechanism. Anti-developmental actions of menadione can be blocked by IGF-1 through effects downstream of ROS generation.

Oxidative stress response to menadione and cumene hydroperoxide in the opportunistic fungal pathogen Candida glabrata

Candida glabrata is an opportunistic fungal pathogen that can cause severe invasive infections and can evade phagocytic cell clearance. We are interested in understanding the virulence of this fungal pathogen, in particular its oxidative stress response. Here we investigated C. glabrata, Saccharomyces cerevisiae and Candida albicans responses to two different oxidants: menadione and cumene hydroperoxide (CHP). In log-phase, in the presence of menadione, C. glabrata requires Cta1p (catalase), while in a stationary phase (SP), Cta1p is dispensable. In addition, C. glabrata is less resistant to menadione than C. albicans in SP. The S. cerevisiae laboratory reference strain is less resistant to menadione than C. glabrata and C. albicans; however S. cerevisiaeclinical isolates (CIs) are more resistant than the lab reference strain. Furthermore, S. cerevisiae CIs showed an increased catalase activity. Interestingly, in SP C. glabrata and S. cerevisiae are more resistant to CHP than C. albicans and Cta1p plays no apparent role in detoxifying this oxidant.

Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide

To cope with oxidative stress, Candida albicans possesses several enzymes involved in a number of biological processes, including superoxide dismutases (Sods) and glutaredoxins (Grxs). The resistance of C. albicans to reactive oxygen species is thought to act as a virulence factor. Genes such as SOD1 and GRX2, which encode for a Sod and Grx, respectively, in C. albicans are widely recognised to be important for pathogenesis. We generated a double mutant, Δgrx2/sod1, for both genes. This strain is very defective in hyphae formation and is susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, the double null mutant was susceptible to menadione and resistant to diamide. The reintegration of the SOD1 gene in the null mutant led to recovery in resistance to menadione, whereas reintegration of the GRX2 gene made the null mutant sensitive to diamide. Despite having two different roles in the responses to oxidative stress generated by chemical compounds, GRX2 and SOD1 are important for C. albicans pathogenesis because the double mutant Δgrx2/sod1 was very susceptible to neutrophil killing and was defective in hyphae formation in addition to having a lower virulence in an animal model of systemic infection.

2-Bromo-1,4-naphthoquinone: a potentially improved substitute of menadione in Apatone™ therapy

Apatone™, a combination of menadione (2-methyl-1,4-naphthoquinone, VK3) and ascorbic acid (vitamin C, VC) is a new strategy for cancer treatment. Part of its effect on tumor cells is related to the cellular pro-oxidative imbalance provoked by the generation of hydrogen peroxide (H2O2) through naphthoquinone redox cycling. In this study, we attempted to find new naphthoquinone derivatives that would increase the efficiency of H2O2 production, thereby potentially increasing its efficacy for cancer treatment. The presence of an electron-withdrawing group in the naphthoquinone moiety had a direct effect on the efficiency of H2O2 production. The compound 2-bromo-1,4-naphthoquinone (BrQ), in which the bromine atom substituted the methyl group in VK3, was approximately 10- and 19-fold more efficient than VK3 in terms of oxygen consumption and H2O2 production, respectively. The ratio [H2O2]produced / [naphthoquinone]consumed was 68 ± 11 and 5.8 ± 0.2 (µM/µM) for BrQ and VK3, respectively, indicating a higher efficacy of BrQ as a catalyst for the autoxidation of ascorbic acid. Both VK3 and BrQ reacted with glutathione (GSH), but BrQ was the more effective substrate. Part of GSH was incorporated into the naphthoquinone, producing a nucleophilic substitution product (Q-SG). The depletion of BrQ by GSH did not prevent its redox capacity since Q-SG was also able to catalyze the production of reactive oxygen species. VK3/VC has already been submitted to clinical trials for the treatment of prostate cancer and has demonstrated promising results. However, replacement of VK3 with BrQ will open new lines of investigation regarding this approach to cancer treatment.

2-Bromo-1,4-naphthoquinone: a potentially improved substitute of menadione in Apatone therapy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identification of possible targets of the Aspergillus fumigatus CRZ1 homologue, CrzA

Background: Calcineurin, a serine/threonine-specific protein phosphatase, plays an important role in the control of cell morphology and virulence in fungi. Calcineurin regulates localization and activity of a transcription factor called CRZ1. Recently, we characterize Aspergillus fumigatus CRZ1 homologue, AfCrzA. Here, we investigate which pathways are influenced by A. fumigatus AfCrzA during a short pulse of calcium by comparatively determining the transcriptional profile of A. fumigatus wild type and.AfcrzA mutant strains. Results: We were able to observe 3,622 genes modulated in at least one timepoint in the mutant when compared to the wild type strain (3,211 and 411 at 10 and 30 minutes, respectively). Decreased mRNA abundance in the Delta crzA was seen for genes encoding calcium transporters, transcription factors and genes that could be directly or indirectly involved in calcium metabolism. Increased mRNA accumulation was observed for some genes encoding proteins involved in stress response. AfCrzA overexpression in A. fumigatus increases the expression of several of these genes. The deleted strain of one of these genes, AfRcnA, belonging to a class of endogenous calcineurin regulators, calcipressins, had more calcineurin activity after exposure to calcium and was less sensitive to menadione 30 mu M, hydrogen peroxide 2.5 mM, EGTA 25 mM, and MnCl(2) 25 mM. We constructed deletion, overexpression, and GFP fusion protein for the closely related A. nidulans AnRcnA. GFP

Antimicrobial activities of Garcinia kola on oral Fusobacterium nucleatum and biofilm

The extracts from the root, bark and seed of Garcinia kola are currently used in traditional medicine in Nigeria. The aim of this study was to evaluate the inhibitory activity of crude extracts of G. kola on Fusobacterium nucleatum isolated from the oral cavity. Methanol and aqueous extracts were prepared from the seed and the minimal inhibitory concentration was evaluated by the agar dilution method, using a Wilkins-Chalgren agar supplemented with horse blood (5%), hemin (5 mu g/ml) and menadione (1 mu g/ml). Antimicrobial activity of plant extracts on microbial biofilms was determined in microtiter plates. The seed of G. kola demonstrated significant inhibitory action on F. nucleatum isolates at a concentration of 1.25 and 12.5 mg/ml for amoxicillin resistant strain. It was able to inhibit the microbial biofilm formed by the association of F. nucleatum with Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomycetemcomitans ATCC 33384 and Prevotella intermedia ATCC 2564 at a concentration of 25 mg/ml. The in-vitro inhibitory effect of G. kola on F. nucleatum population suggests a potential role for its use in oral hygiene.

Silencing of mitochondrial alternative oxidase gene of Aspergillus fumigatus enhances reactive oxygen species production and killing of the fungus by macrophages

We previously demonstrated that conidia from Aspergillus fumigatus incubated with menadione and paraquat increases activity and expression of cyanide-insensitive alternative oxidase (AOX). Here, we employed the RNA silencing technique in A. fumigatus using the vector pALB1/aoxAf in order to down-regulate the aox gene. Positive transformants for aox gene silencing of A. fumigatus were more susceptible both to an imposed in vitro oxidative stress condition and to macrophages killing, suggesting that AOX is required for the A. fumigatus pathogenicity, mainly for the survival of the fungus conidia during host infection and resistance to reactive oxygen species generated by macrophages.

Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase

Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress. Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione. GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively). A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value. However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes. Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage. Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride. These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione. Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels. Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.

Grx5 glutaredoxin plays a central role in protection against protein oxidative damage in Saccharomyces cerevisiae

Glutaredoxins are members of a superfamily of thiol disulfide oxidoreductases involved in maintaining the redox state of target proteins. In Saccharomyces cerevisiae, two glutaredoxins (Grx1 and Grx2) containing a cysteine pair at the active site had been characterized as protecting yeast cells against oxidative damage. In this work, another subfamily of yeast glutaredoxins (Grx3, Grx4, and Grx5) that differs from the first in containing a single cysteine residue at the putative active site is described. This trait is also characteristic for a number of glutaredoxins from bacteria to humans, with which the Grx3/4/5 group has extensive homology over two regions. Mutants lacking Grx5 are partially deficient in growth in rich and minimal media and also highly sensitive to oxidative damage caused by menadione and hydrogen peroxide. A significant increase in total protein carbonyl content is constitutively observed in grx5cells, and a number of specific proteins, including transketolase, appear to be highly oxidized in this mutant. The synthetic lethality of the grx5 and grx2 mutations on one hand and ofgrx5 with the grx3 grx4 combination on the other points to a complex functional relationship among yeast glutaredoxins, with Grx5 playing a specially important role in protection against oxidative stress both during ordinary growth conditions and after externally induced damage. Grx5-deficient mutants are also sensitive to osmotic stress, which indicates a relationship between the two types of stress in yeast cells.

Cyanocobalamin is a Superoxide Scavenger and Neuroprotectant in Neuronal Cells

Les dommages au nerf optique (neuropathie optique) peuvent entraîner la perte permanente de la vision ou la cécité causée par la mort des cellules ganglionnaires de la rétine (CGR). Nous avons identifié qu’une surproduction de l'anion superoxyde constitue un événement moléculaire critique précédant la mort cellulaire induite par des lésions. Récemment, Suarez-Moreira et al (JACS 131:15078, 2009) ont démontré que la vitamine B12 peut capter l’anion superoxyde aussi efficacement que l’enzyme superoxyde dismutase. La carence en vitamine B12 peut conduire à une neuropathie optique causée par des mécanismes inconnus. Nous avons étudié la relation entre la captation de superoxyde par la cyanocobalamine (forme de vitamine B12 la plus abondante) et ses propriétés neuroprotectrices dans les cellules neuronales. La cyanocobalamine aux concentrations de 10 μM et 100 μM a réduit le taux de production de superoxyde respectivement par 34% et 79% dans les essais sans-cellule. Dans les cellules RGC-5 traités avec la ménadione, les concentrations de cyanocobalamine supérieures à 10 nM ont diminué l’anion superoxyde à des valeurs similaires à celles traitées par PEG-SOD. La cyanocobalamine aux concentrations de 100 μM et 1 μM a réduit la mort des cellules RGC-5 exposées à la ménadione par 20% et 32%, respectivement. Chez les rats avec section du nerf optique unilatérale, une dose intravitréenne de 667 μM de cyanocobalamine a réduit le nombre de CGRs exposées au superoxyde. Cette dose a également augmenté le taux de survie des CGRs comparativement aux rats injectés avec la solution témoin. Ces données suggèrent que la vitamine B12 peut être un neuroprotecteur important, et sa carence nutritionnelle pourrait causer la mort de CGRs. La vitamine B12 pourrait aussi potentiellement être utilisée comme une thérapie pour ralentir la progression de la mort CGR chez les patients avec les neuropathies optiques caractérisés par une surproduction de superoxyde.

Equol: a comparison of the effects of the racemic compound with that of the purified S-enantiomer on the growth, invasion, and DNA integrity of breast and prostate cells in vitro

It has been postulated that the R- and S-equol enantiomers have different biological properties given their different binding affinities for the estrogen receptor. S-(-)equol is produced via the bacterial conversion of the soy isoflavone daidzein in the gut. We have compared the biological effects of purified S-equol to that of racemic (R and S) equol on breast and prostate cancer cells of varying receptor status in vitro. Both racemic and S-equol inhibited the growth of the breast cancer cell line MDA-MB-231 (> or = 10 microM) and the prostate cancer cell lines LNCaP (> or = 5 microM) and LAPC-4 (> or = 2.5 microM). The compounds also showed equipotent effects in inhibiting the invasion of MDA-MB-231 and PC-3 cancer cells through matrigel. S-equol (1, 10, 30 microM) was unable to prevent DNA damage in MCF-7 or MCF-10A breast cells following exposure to 2-hydroxy-4-nonenal, menadione, or benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide. In contrast, racemic equol (10, 30 microM) prevented DNA damage in MCF-10A cells following exposure to 2-hydroxy-4-nonenal or menadione. These findings suggest that racemic equol has strong antigenotoxic activity in contrast to the purified S-equol enantiomer implicating the R-, rather than the S-enantiomer as being responsible for the antioxidant effects of equol, a finding that may have implications for the in vivo chemoprotective properties of equol.

The mitochondrial transcription factor A functions in mitochondrial base excision repair

Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids TFAM plays an important role in mitochondrial transcription and replication TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected in mitochondria, whereas base excision repair (BER) has been comprehensively characterized in these organelles The BER proteins are associated with the inner membrane in mitochondria and thus with the mitochondrial nucleoid, where TFAM is also situated However, a function for TFAM in BER has not yet been investigated This study examines the role of TFAM in BER In vitro studies with purified recombinant TFAM indicate that it preferentially binds to DNA containing 8-oxoguanines, but not to abasic sites, uracils, or a gap in the sequence TFAM inhibited the in vitro incision activity of 8-oxoguanine DNA glycosylase (OGG1), uracil-DNA glycosylase (UDG), apurinic endonuclease 1 (APE1), and nucleotide incorporation by DNA polymerase gamma (pol gamma) On the other hand, a DNA binding-defective TFAM mutant, L58A, showed less inhibition of BER in vitro Characterization of TFAM knockdown (KD) cells revealed that these lysates had higher 8oxoG incision activity without changes in alpha OGG1 protein levels TFAM KD cells had mild resistance to menadione and increased damage accumulation in the mtDNA when compared to the control cells In addition, we found that the tumor suppressor p53, which has been shown to interact with and alter the DNA binding activity of TFAM, alleviates TFAM-Induced inhibition of BER proteins Together, the results suggest that TFAM modulates BER in mitochondria by virtue of its DNA binding activity and protein interactions Published by Elsevier B V

Microbiota associated with chronic osteomyelitis of the jaws

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

ZmPUMP encodes a maize mitochondrial uncoupling protein that is induced by oxidative stress

A cDNA clone (designated ZmPUMP) encoding an uncoupling protein (UCP) from maize (Zea mays) was identified by searching for homologous sequences among the expressed sequence tags of the GenBank database. The ZmPUMP cDNA contains a single open reading frame of 933 nucleotides encoding 310 amino acids. Several features identified the predicted ZmPUMP protein as a member of the mitochondrial UCP subfamily of mitochondrial carriers. Expression studies demonstrated that ZmPUMP is ubiquitously expressed in maize tissues and its transcript level is not altered in early stages of embryo germination. In contrast to known UCP genes, ZmPUMP is not responsive to cold stress. Instead its expression is increased in response to H 2O 2- or menadione-induced oxidative stress. © 2003 Elsevier Science Ireland Ltd. All rights reserved.