Defenses against oxidative stress in Neisseria gonorrhoeae and Neisseria meningitidis: Distinctive systems for different lifestyles

Defenses against oxidative stress are crucial for the survival of the pathogens Neisseria meningitidis and Neisseria gonorrhoeae. An Mn(II) uptake system is involved in manganese (Mn)-dependent resistance to superoxide radicals in N. gonorrhoeae. Here, we show that accumulation of Mn also confers resistance to hydrogen peroxide killing via a catalase-independent mechanism. An mntC mutant of N. meningitidis is susceptible to oxidative killing, but supplementation of growth media with Mn does not enhance the organism's resistance to oxidative killing. N. meningitidis is able to grow in the presence of millimolar levels of Mn ion, in contrast to N. gonorrhoeae, whose growth is retarded at Mn concentrations >100 mumol/L, indicating that Mn homeostasis in the 2 species is probably quite different. N. meningitidis superoxide dismutase B plays a role in protection against oxidative killing. However, a sodC mutant of N. meningitidis is no more sensitive to oxidative killing than is the wild type. A cytochrome c peroxidase (Ccp) is present in N. gonorrhoeae but not in N. meningitidis. Investigations of a ccp mutant revealed a role for Ccp in protection against hydrogen peroxide killing. These differences in oxidative defenses in the pathogenic Neisseria are most likely a result of their localization in different ecological niches.

AP-2 transcription factor family member expression, activity, and regulation in human epidermal keratinocytes in vitro

The AP-2 transcription factor family is presumed to play an important role in the regulation of the keratinocyte squamous differentiation program; however, limited functional data are available to support this. In the present study, the activity and regulation of AP-2 were examined in differentiating human epidermal keratinocytes. We report that (1) AP-2 transcriptional activity decreases in differentiated keratinocytes but remains unchanged in differentiation-insensitive squamous cell carcinoma cell lines, (2) diminished AP-2 transcriptional activity is associated with a loss of specific DNA-bound AP-2 complexes, and (3) there is an increase in the ability of cytoplasmic extracts, derived from differentiated keratinocytes, to phosphorylate AP-2alpha and AP-2beta when cells differentiate. In contrast, extracts from differentiation-insensitive squamous cell carcinoma cells are unable to phosphorylate AP-2 proteins. Finally, the phosphorylation of recombinant AP-2alpha by cytosolic extracts from differentiated keratinocytes is associated with decreased AP-2 DNA-binding activity. Combined, these data indicate that AP-2 trans-activation and DNA-binding activity decrease as keratinocytes differentiate, and that this decreased activity is associated with an enhanced ability to phosphorylate AP-2alpha and beta.

Mutation in mitochondrial DNA as a cause of presbyacusis

Much of the hearing loss that occurs in old age is likely to be due to the long-term deterioration of the mitochondria in the different structures of the cochlea. The current review surveys some of the basic information on mitochondria and mitochondrial DNA, as a background to their possible involvement in presbyacusis. It is likely that oxygen radicals damage mitochondrial DNA and other components of the mitochondria, such as their proteins and lipids. This further compromises both oxidative phosphorylation and the repair processes in mitochondria, setting up a vicious cycle of degradation. Evidence is presented from inherited point mutations on the possibly most critical sites for mutations in mitochondrial DNA associated with hearing loss. It is suggested that random sorting and clonal expansion of mutations both maintain the integrity of the pool of mitochondrial DNA molecules and give rise to the apoptosis that leads to loss of vulnerable cells, and hence to deafness. It is moreover suggested that apoptosis of the vulnerable cells of the inner ear may to some extent be preventable, or at least delayed. Copyright (C) 2004 S. Karger AG, Basel.

Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display

Objective. To identify differentially expressed genes in synovial fibroblasts and examine the effect on gene expression of exposure to TNF-alpha and IL-1beta. Methods. Restriction fragment differential display was used to isolate genes using degenerate primers complementary to the lysophosphatidic acid acyl transferase gene family. Differential gene expression was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry using a variety of synovial fibroblasts, including cells from patients with osteoarthritis and self-limiting parvovirus arthritis. Results. Irrespective of disease process, synovial fibroblasts constitutively produced higher levels of IL-6 and monocyte chemoattractant protein 1 (MCP-1) (CCL2) than skin fibroblasts. Seven genes were differentially expressed in synovial fibroblasts compared with skin fibroblasts. Of these genes, four [tissue factor pathway inhibitor 2 (TFPI2), growth regulatory oncogene beta (GRObeta), manganese superoxide dismutase (MnSOD) and granulocyte chemotactic protein 2 (GCP-2)] were all found to be constitutively overexpressed in synoviocytes derived from patients with osteoarthritis. These four genes were only weakly expressed in other synovial fibroblasts (rheumatoid and self-limiting parvovirus infection). However, expression in all types of fibroblasts was increased after stimulation with TNF-alpha and IL-1beta. Three other genes (aggrecan, biglycan and caldesmon) were expressed at higher levels in all types of synovial fibroblasts compared with skin fibroblasts even after stimulation with TNF-alpha and IL-1. Conclusions. Seven genes have been identified with differential expression patterns in terms of disease process (osteoarthritis vs rheumatoid arthritis), state of activation (resting vs cytokine activation) and anatomical location (synovium vs skin). Four of these genes, TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6), were selectively overexpressed in osteoarthritis fibroblasts rather than rheumatoid fibroblasts. While these differences may represent differential behaviour of synovial fibroblasts in in vitro culture, these observations suggest that TFPI2, GRObeta (CXCL2), MnSOD and GCP-2 (CXCL6) may represent new targets for treatments specifically tailored to osteoarthritis.

Global changes in gene expression observed at the transition from growth to stationary phase in Listeria monocytogenes ScottA batch culture

Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually. The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L. monocytogenes adapts to environmental change are yet to be fully elucidated. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. We have utilized a proteomic approach to investigate the response of L. monocytogenes batch cultures to the transition from exponential to stationary growth phase. Proteomic analysis showed that batch cultures of L. monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone. Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase. We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase. The significance of these findings in terms of functionality and the mechanistic picture are discussed.

Nitric oxide donors inhibit 5-hydroxytryptamine (5-HT) uptake by the human 5-HT transporter (SERT)

1 The aim was to test the hypothesis that nitric oxide ( NO) donor drugs can inhibit the 5-hydroxytryptamine (5-HT) transporter, SERT. 2 The NO donors, MAHMA/NO ( a NONOate; (Z)-1-[N-methyl-N-[6-(N-methylammoniohexyl)amino]]diazen- 1-ium-1,2-diolate), SIN-1 ( a sydnonimine; 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride), FK409 ( an oxime; (+/-)-(4-ethyl-2E-(hydroxyimino)-5-nitro-3E-hexenamide)) and peroxynitrite, but not Angeli's salt ( source of nitroxyl anion) or sodium nitrite, caused concentration-dependent inhibition of the specific uptake of [H-3]- 5-HT in COS-7 cells expressing human SERT. 3 Superoxide dismutase (150 U ml(-1)) plus catalase ( 1200 U ml(-1)), used to remove superoxide and hence prevent peroxynitrite formation, prevented the inhibitory effect of SIN-1 ( which generates superoxide) but not of MAHMA/NO or FK409. 4 The inhibitory effects of the NO donors were not affected by the free radical scavenger, hydroxocobalamin (1 mM) or the guanylate cyclase inhibitor, ODQ (1H-[ 1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one; 3 muM). 5 L-Cysteine ( 1 mM; source of excess thiol residues) abolished or markedly reduced the inhibitory effects of MAHMA/NO, SIN-1, FK409 and peroxynitrite. 6 It is concluded that inhibition of SERT by the NO donors cannot be attributed exclusively to NO free radical nor to nitroxyl anion. It does not involve guanosine-3',5'-cyclic monophosphate, but may involve nitrosation of cysteine residues on the SERT protein. Peroxynitrite mediates the effect of SIN-1, but not the other drugs. 7 Data in mice with hypoxic pulmonary hypertension suggest that SERT inhibitors may attenuate pulmonary vascular remodelling. Thus, NO donors may be useful in pulmonary hypertension, not only as vasodilators, but also because they inhibit SERT, provided they display this effect in vivo at appropriate doses.

The effects of pre-weaning undernutrition on the expression levels of free radical deactivating enzymes in the mouse brain

A mild degree of undernutrition brought about by restricting the amount of food in the diet is known to alter the life span of an animal. It has been hypothesised that this may be related to the effects of undernutrition on an animals anti-oxidant defense system. We have therefore, used real-time PCR (rt-PCR) techniques to determine the levels of mRNA expression for manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase (Cu/ZnSOD), glutathione peroxidase 1 (GPx 1) and catalase in the brains of Quackenbush mice undernourished from conception until 21-post-natal days of age. It was found that 21- and 61-day-old undernourished mice had a deficit in the expression of Cu/ZnSOD in both the cerebellum and forebrain regions compared to age-matched controls. The expression of MnSOD was found to be greater in the cerebellum, but not the forebrain region, of 21-day-old undernourished mice. There were no significant differences in the expression of GPx 1 and catalase between control and undernourished or previously undernourished mice. Our results confirm that undernutrition during the early life of a mouse may disrupt some of the enzymes involved in the anti-oxidant defense systems.

Exercise and the endothelial cell

Regular exercise is known to be effective in the prevention and treatment of cardiovascular disease. Among the cardioprotectant mechanisms influenced by exercise, the endothelium is becoming recognised as a major target. Preservation of endothelial cell structure is vital for frictionless blood flow, prevention of macrophage and lipid infiltration and, ultimately, optimal vascular function. Exercise causes various kinds of mechanical, chemical and thermal stresses, and repeated exposure to these stresses may precondition the endothelial cell to future stresses through a number of different mechanisms. This review discusses stress-induced changes in endothelial cell morphology, biochemistry and components of platelet activation and cell adhesion that impact on endothelial cell structure. An enhanced understanding of the effects of exercise on the endothelial cell will assist in directing future research into the prevention of cardiovascular disease. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Cyclosporine A-induced changes to erythrocyte redox balance is time course-dependent

Cyclosporine A-treated transplant recipients develop pronounced cardiovascular disease and have increased oxidative stress and altered antioxidant capacity in erythrocytes and plasma. These experiments investigated the time-course of cyclosporine A-induced changes to redox balance in plasma and erythrocytes. Rats were randomly assigned to either a control or cyclosporine A-treated group. Treatment animals received 25 mg/kg of cyclosporine A via intraperitoneal injection for either 7 days or a single dose. Control rats were injected with the same volume of the vehicle. Three hours after the final injections, plasma was analysed for total antioxidant status, a-tocopherol, malondialdehyde, and creatinine. Erythrocytes were analysed for reduced glutathione (GSH), alpha-tocopherol, methaemoglobin, malondialdehyde, and the activities of superoxide dismutase, catalase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G6PD). Cyclosporine A administration for 7 days resulted in a significant increase (P < 0.05) in plasma malondialdehyde, methaemoglobin, and superoxide dismutase and catalase activities. There was a significant decrease (P < 0.05) in erythrocyte GSH concentration and G6PD activity in cyclosporine A animals. There were no significant differences (P > 0.05) between groups following a single dose of cyclosporine A in any of the measures. In summary, cyclosporine A alters erythrocyte redox balance after 7 days administration, but not after a single dose.

Investigation of oxidative stress defenses of Neisseria gonorrhoeae by using a human polymorphonuclear leukocyte survival assay

Neisseria gonorrhoeae has well-characterized oxidative stress defense systems that protect against oxidative killing in in vitro assays. In contrast, mutant strains of N. gonorrhoeae lacking oxidative stress defenses are identical to the wild type when tested in an ex vivo survival assay using human polymorphonuclear leukocytes.

Cyclosporine A induced changes to plasma and erythrocyte antioxidant defences

Organ transplant recipients develop pronounced cardiovascular disease, and decreased antioxidant capacity in plasma and erythrocytes is associated with the pathogenesis of this disease. These experiments tested the hypothesis that the immunosuppressant cyclosporine A (CsA) alters erythrocyte redox balance and reduces plasma antioxidant capacity. Female Sprague-Dawley rats were randomly assigned to a control or CsA treated group. Treatment animals received 25 mg/kg/day of CsA via intraperitoneal injection for 18 days. Control rats were injected with the same volume of the vehicle. Three hours after the final CsA injection, rats were exsanguinated and plasma analysed for total antioxidant status (TAS), alpha-tocopherol, malondialdehyde (MDA), and creatinine. Erythrocytes were analysed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glucose-6-phosphate dehydrogenase (G6PD) activities, alpha-tocopherol, and MDA. CsA administration resulted in a significant (P < 0.05) decrease in plasma TAS and significant increases (P < 0.05) in plasma creatinine and MDA. Erythrocyte CAT was significantly (P < 0.05) increased in CsA treated rats compared to controls. There were no significant differences (P > 0.05) in erythrocyte SOD, GPX, G6PD, alpha-tocopherol or MDA between groups. In summary, CsA alters erythrocyte antioxidant defence and decreases plasma total antioxidant capacity.

Azurin of pathogenic Neisseria spp. is involved in defense against hydrogen peroxide and survival within cervical epithelial cells

Laz, a lipid-modified azurin of the human pathogens Neisseria gonorrhoeae and Neisseria meningitidis, is involved in defense against oxidative stress and copper toxicity; laz mutant strains are hypersensitive to hydrogen peroxide and copper. The N. gonorrhoeae laz mutant also has decreased survival in an ex vivo primary human ectocervical epithelial assay.

Cytochrome P450-mediated metabolism of haloperidol and reduced haloperidol to pyridinium metabolites

Haloperidol ( HP) has been reported to undergo cytochrome P450 (P450)-mediated metabolism to potentially neurotoxic pyridinium metabolites; however, the chemical pathways and specific enzymes involved in these reactions remain to be identified. The aims of the current study were to (i) fully identify the cytochrome P450 enzymes capable of metabolizing HP to the pyridinium metabolite, 4-(4-chlorophenyl)- 1-(4-fluorophenyl)-4-oxobutylpyridinium (HPP+), and reduced HP (RHP) to 4-(4-chlorophenyl)- 1-(4-fluorophenyl)-4-hydroxybutylpyridinium (RHPP+); and (ii) determine whether 4-(4-chlorophenyl)- 1-(4-fluorophenyl)-4-oxobutyl-1,2,3,6-tetrahydropyridine (HPTP) and 4-(4-chlorophenyl)1-( 4-fluorophenyl)-4-hydroxybutyl-1,2,3,6-tetrahydropyridine (RHPTP) were metabolic intermediates in these pathways. In vitro studies were conducted using human liver microsomal preparations and recombinant human cytochrome P450 enzymes (P450s 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19 2D6, 2E1, 3A4, 3A5, and 3A7) expressed in bicistronic format with human NADPH cytochrome P450 reductase in Escherichia coli membranes. Pyridinium formation from HP and RHP was highly correlated across liver preparations, suggesting the same enzyme or enzymes were responsible for both reactions. Cytochrome P450s 3A4, 3A5, and 3A7 were the only recombinant enzymes which demonstrated significant catalytic activity under optimized conditions, although trace levels of activity could be catalyzed by NADPHP450 reductase alone. NADPH-P450 reductase-mediated activity was inhibited by reduced glutathione but not catalase or superoxide dismutase, suggesting O-2-dependent oxidation. No evidence was obtained to support the contention that HPTP and RHPTP are intermediates in these pathways. K-m values for HPP+ (34 +/- 5 mu M) and RHPP+ (64 +/- 4 mu M) formation by recombinant P450 3A4 agreed well with those obtained with human liver microsomes, consistent with P450 3A4 being the major catalyst of pyridinium metabolite formation in human liver.

An evaluation of potential mechanism-based inactivation of human drug metabolizing cytochromes P450 by monoamine oxidase inhibitors, including isoniazid

To characterize potential mechanism-based inactivation (MBI) of major human drug-metabolizing cytochromes P450 (CYP) by monoamine oxidase (MAO) inhibitors, including the antitubercular drug isoniazid. Human liver microsomal CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A activities were investigated following co- and preincubation with MAO inhibitors. Inactivation kinetic constants (K-I and k(inact)) were determined where a significant preincubation effect was observed. Spectral studies were conducted to elucidate the mechanisms of inactivation. Hydrazine MAO inhibitors generally exhibited greater inhibition of CYP following preincubation, whereas this was less frequent for the propargylamines, and tranylcypromine and moclobemide. Phenelzine and isoniazid inactivated all CYP but were most potent toward CYP3A and CYP2C19. Respective inactivation kinetic constants (K-I and k(inact)) for isoniazid were 48.6 mu M and 0.042 min(-1) and 79.3 mu M and 0.039 min(-1). Clorgyline was a selective inactivator of CYP1A2 (6.8 mu M and 0.15 min(-1)). Inactivation of CYP was irreversible, consistent with metabolite-intermediate complexation for isoniazid and clorgyline, and haeme destruction for phenelzine. With the exception of phenelzine-mediated CYP3A inactivation, glutathione and superoxide dismutase failed to protect CYP from inactivation by isoniazid and phenelzine. Glutathione partially slowed (17%) the inactivation of CYP1A2 by clorgyline. Alternate substrates or inhibitors generally protected against CYP inactivation. These data are consistent with mechanism-based inactivation of human drug-metabolizing CYP enzymes and suggest that impaired metabolic clearance may contribute to clinical drug-drug interactions with some MAO inhibitors.