OxyR acts as a repressor of catalase expression in Neisseria gonorrhoeae

It has been reported that Neisseria gonorrhoeae possesses a very high level of catalase activity, but the regulation of catalase expression has not been investigated extensively. In Escherichia coli and Salmonella enterica serovar Typhimurium, it has been demonstrated that OxyR is a positive regulator of hydrogen peroxide-inducible genes, including the gene encoding catalase. The oxyR gene from N. gonorrhoeae was cloned and used to complement an E. coli oxyR mutant, confirming its identity and function. The gene was inactivated by inserting a kanamycin resistance cassette and used to make a knockout allele on the chromosome of N. gonorrhoeae strain 1291. In contrast to E. coli, the N. gonorrhoeae oxyR::kan mutant expressed ninefold-more catalase activity and was more resistant to hydrogen peroxide killing than the wild type. These data are consistent with OxyR in N. gonorrhoeae acting as a repressor of catalase expression.

wsp Gene sequences from the Wolbachia of filarial nematodes

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences.

Inhibition of NF kappa B leads to an intracellular reducing environment in human monocyte-derived immature dendritic cells

Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.

Oral disease in animals: The Australian perspective. Isolation and characterisation of black-pigmented bacteria from the oral cavity of marsupials

A wide range of animals suffer from periodontal disease. However, there is very little reported on disease and oral micro-biota of Australian animals. Therefore, the oral cavity of 90 marsupials was examined for oral health status. Plaque samples were collected from the subgingival margins using curettes; or swabs. Plaque samples were plated onto. non-selective trypticase soy agar plates, selective trypticase soy agar, non-selective and selective Wilkens Chalgrens, Agar. Plates were incubated in an anaerobic atmosphere and examined after 7-14 days for the presence of black-brown-pigmented colonies. A combination of morphological and biochemical tests were used (colonial morphology, pigmentation, aerobic growth, Gram reaction, fluorescence under long-wave UV light (360 nm), production of catalase, enzymatic activity with fluorogenic substrates and haemagglutination of sheep red cells) to identify these organisms. Black-pigmented bacteria were cultivated from the plaque of 32 animals including six eastern grey kangaroos, a musky rat kangaroo, a whiptail and a red-necked wallaby, 18 koalas, a bandicoot and five brushtail possums. No black-pigmented colonies were cultivated from squirrel or sugar gliders or quokkas or from marsupial mice. The majority of isolates were identified as Porphyromonas gingivalis-like species with the higher prevalence of isolation from the oral cavity of macropods (the kangaroos and wallabies). Oral diseases, such as gingivitis can be found in native Australian animals with older koalas having an increase in disease indicators and black-pigmented bacteria. Non-selective Wilkens Chalgren Agar was the medium of choice for the isolation of black-pigmented bacteria. (C) 2002 Elsevier Science Ltd. All rights reserved.

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.

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.

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: Influence on protein stability and activity

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV3 5S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn(189)) associated with the point of glycan attachment (Asn(189)) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35::prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent K-m) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (E,,) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases. (c) 2005 Elsevier 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.

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.

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.

alpha-tocopherol and alpha-lipoic acid enhance the erythrocyte antioxidant defence in cyclosporine A-treated rats

The aim of this study was to determine the effects of dietary antioxidant supplementation with alpha-tocopherol and alpha-lipoic acid on cyclosporine A (cyclosporine)-induced alterations to erythrocyte and plasma redox balance. Rats were randomly assigned to either control, antioxidant (alpha-tocopherol 1000 IU/kg diet and alpha-lipoic acid 1.6 g/kg diet), cyclosporine (25 mg/kg/day), or cyclosporine + antioxidant treatments. Cyclosporine was administered for 7 days after an 8 week feeding period. Plasma was analysed for alpha-tocopherol, total antioxidant capacity, malondialdehyde, and creatinine. Erythrocytes were analysed for glutathione, methaemoglobin, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, alpha-tocopherol and malondialdehye. Cyclosporine administration caused a significant decrease in superoxide dismutase activity (P < 0.05 control versus cyclosporine) and this was improved by antioxidant supplementation (P < 0.05 cyclosporine versus cyclosporine + antioxidant; P < 0.05 control versus cyclosporine + antioxidant). Animals receiving cyclosporine and antioxidants showed significantly increased (P < 0.05) catalase activity compared to both groups not receiving cyclosporine. Cyclosporine administration induced significant increases in plasma malondialdehyde and creatinine concentration (P < 0.05 control versus cyclosporine). Antioxidant supplementation prevented the cyclosporine induced increase in plasma creatinine (P < 0.05 cyclosporine versus cyclosporine + antioxidant; P > 0.05 control versus cyclosporine + antioxidant), however, supplementation did not alter the cyclosporine induced increase in plasma malondialdehyde concentration (P > 0.05 cyclosporine versus cyclosporine + antioxidant). Antioxidant supplementation resulted in significant increases (P < 0.05) in plasma and erythrocyte alpha-tocopherol in both of the supplemented groups compared to non-supplemented groups. In conclusion, dietary supplementation with alpha-tocopherol and alpha-lipoic acid enhanced the erythrocyte antioxidant defence and reduced nephrotoxicity in cyclosporine treated animals.

Antioxidant supplementation enhances erythrocyte antioxidant status and attenuates cyclosporine-induced vascular dysfunction

The aim of this study was to determine the effects of dietary antioxidant supplementation with a-tocopherol and a-lipoic acid on cyclosporine-induced alterations to erythrocyte and plasma redox balance, and cyclosporine-induced endothelial and smooth muscle dysfunction. Rats were randomly assigned to either control, antioxidant, cyclosporine or cyclosporine + antioxidant treatments. Cyclosporine A was administered for 10 days after an 8-week feeding period. Plasma was analyzed for alpha-tocopherol, total antioxidant capacity, malondialdehyde and creatinine. Erythrocytes were analyzed for glutathione, methemoglobin, superoxide dismutase, catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase, alpha-tocopherol and malondialdehye. Vascular endothelial and smooth muscle function was determined in vitro. Antioxidant supplementation resulted in significant increases in erythrocyte a-tocopherol concentration and glutathione peroxidase activity in both of the antioxidant-supplemented groups. Cyclosporine administration caused significant decreases in glutathione concentration, methemoglobin concentration and superoxide dismutase activity. Antioxidant supplementation attenuated the cyclosporine-induced decrease in superoxide dismutase activity. Cyclosporine therapy impaired both endothelium-independent and -dependent relaxation of the thoracic aorta, and this was attenuated by antioxidant supplementation. In summary, dietary supplementation with alpha-tocopherol and alpha-lipoic acid attenuated the cyclosporine-induced decrease in erythrocyte superoxide dismutase activity and attenuated cyclosporine-induced vascular dysfunction.

Diel 'tuning' of coral metabolism: physiological responses to light cues

Hermatypic-zooxanthellate corals track the diel patterns of the main environmental parameters temperature, UV and visible light - by acclimation processes that include biochemical responses. The diel course of solar radiation is followed by photosynthesis rates and thereby elicits simultaneous changes in tissue oxygen tension due to the shift in photosynthesis/respiration balance. The recurrent patterns of sunlight are reflected in fluorescence yields, photosynthetic pigment content and activity of the two protective enzymes superoxide dismutase (SOD) and catalase (CAT), enzymes that are among the universal defenses against free radical damage in living tissue. All of these were investigated in three scleractinian corals: Favia favus, Plerogyra sinuosa and Goniopora lobata. The activity of SOD and CAT in the animal host followed the course of solar radiation, increased with the rates of photosynthetic oxygen production and was correlated with a decrease in the maximum quantum yield of photochemistry in Photosystem H (PSII) (Delta F'/F-m'). SOD and CAT activity in the symbiotic algae also exhibited a light intensity correlated pattern, albeit a less pronounced one. The observed rise of the free-radical-scavenger enzymes, with a time scale of minutes to several hours, is an important protective mechanism for the existence and remarkable success of the unique cnidarian-dinoflagellate associations, in which photosynthetic oxygen production takes place within animal cells. This represents a facet of the precarious act of balancing the photosynthetic production of oxygen by the algal symbionts with their destructive action on all living cells, especially those of the animal host.