Electron Beam Infrared Nano-Ellipsometry of Individual Indium Tin Oxide Nanocrystals.

Leveraging recent advances in electron energy monochromation and aberration correction, we record the spatially resolved infrared plasmon spectrum of individual tin-doped indium oxide nanocrystals using electron energy-loss spectroscopy (EELS). Both surface and bulk plasmon responses are measured as a function of tin doping concentration from 1-10 atomic percent. These results are compared to theoretical models, which elucidate the spectral detuning of the same surface plasmon resonance feature when measured from aloof and penetrating probe geometries. We additionally demonstrate a unique approach to retrieving the fundamental dielectric parameters of individual semiconductor nanocrystals via EELS. This method, devoid from ensemble averaging, illustrates the potential for electron-beam ellipsometry measurements on materials that cannot be prepared in bulk form or as thin films.

Mitigating peroxynitrite mediated mitochondrial dysfunction in aged rat brain by mitochondria-targeted antioxidant MitoQ.

Although reactive oxygen species mediated oxidative stress is a well-documented mechanism of aging, recent evidences indicate involvement of nitrosative stress in the same. As mitochondrial dysfunction is considered as one of the primary features of aging, the present study was designed to understand the involvement of nitrosative stress by studying the impact of a mitochondria-targeted antioxidant MitoQ, a peroxynitrite (ONOO-) scavenger, on mitochondrial functions. Four groups of rats were included in this study: Group I: Young-6 months (-MitoQ), Group II: Aged-22 months (- MitoQ), Group III: Young-6 months (+ MitoQ), Group IV: Aged-22 months (+ MitoQ). The rats belonging to group III and IV were treated with oral administration of MitoQ (500 µM) daily through drinking water for 5 weeks. MitoQ efficiently suppressed synaptosomal lipid peroxidation and protein oxidation accompanied by diminution of nitrite production and protein bound 3-nitrotyrosine. MitoQ normalized enhanced caspase 3 and 9 activities in aged rat brains and efficiently reversed ONOO- mediated mitochondrial complex I and IV inhibition, restored mitochondrial ATP production and lowered mitochondrial membrane potential loss. To ascertain these findings, a mitochondrial in vitro model (iron/ascorbate) was used involving different free radical scavengers and anti-oxidants. MitoQ provided better protection compared to mercaptoethylguanidine, N-nitro-L-arginine-methyl ester and superoxide dismutase establishing the predominancy of ONOO- in the process compared to •NO and O 2•- . These results clearly highlight the involvement of nitrosative stress in aging process with MitoQ having therapeutic potential to fight against ONOO- mediated aging deficits.

10-(6-Plastoquinonyl) decyltriphenylphosphonium imparts anti-colitogenic protection through recovery of mitochondrial dysfunction in ulcerated murine colon: Implications in ulcerative colitis.

AIMS: To elucidate the impact of 10-(6-plastoquinonyl) decyltriphenylphosphonium (SkQ1) as an anti-colitogenic agent for maintenance of colon epithelial tract in ulcerated mice through recovery of mitochondrial dysfunction and mitochondrial stress by virtue of its free radical scavenging properties. MAIN METHODS: DSS induced ulcerated BALB/c mice were treated with SkQ1 for 14 days @ 30 nmol/kg/body wt./day/mice. Post-treatment, isolated colonic mitochondria were utilized for spectrophotometric and spectrofluorometric biochemical analysis of various mitochondrial functional variables including individual mitochondrial respiratory enzyme complexes. Confocal microscopy was utilized for measuring mitochondrial membrane potential in vivo. ELISA technique was adapted for measuring colonic nitrite and 3-nitrotyrosine (3-NT) content. Finally in vitro cell line study was carried out to substantiate in vivo findings and elucidate the involvement of free radicals in UC using antioxidant/free radical scavenging regimen. KEY FINDINGS: Treatment with SkQ1 in vivo reduced histopathological severity of colitis, induced recovery of mitochondrial respiratory complex activities and associated functional variables, improved oxidative stress indices and normalized mitochondrial cardiolipin content. Importantly, SkQ1 lowered nitrite concentration and 3-nitrotyrosine formation in vivo. In vitro SkQ1 restored mitochondrial functions wherein the efficacy of SkQ1 proved equal or better compared to SOD and DMSO indicating predominant involvement of O2- and OH in UC. However, NO and ONOO- also seemed to play a secondary role as MEG and L-NAME provided lesser protection as compared to SOD and DMSO. SIGNIFICANCE: SkQ1 can be considered as a potent anti-colitogenic agent by virtue of its free radical scavenging properties in treating UC.

Contributions and Limitations of Mitochondria-Targeted and Non-Targeted Antioxidants in the Treatment of Parkinsonism: an Updated Review.

As conventional therapeutics can only treat the symptoms of Parkinson's disease (PD), major focus of research in recent times is to slow down or prevent the progression of neuronal degeneration in PD. Non-targeted antioxidants have been an integral part of the conventional therapeutics regimen; however, their importance have lessened over time because of their controversial outcomes in clinical PD trials. Inability to permeate and localize within the mitochondria remains the main drawback on the part of non-targeted antioxidants inspite of possessing free radical scavenging properties. In contrast, mitochondrial-targeted antioxidants (MTAs), a special class of compounds have emerged having high advantages over non-targeted antioxidants by virtue of efficient pharmacokinetics and better absorption rate with capability to localize many fold inside the mitochondrial matrix. Preclinical experimentations indicate that MTAs have the potential to act as better alternatives compared to conventional non-targeted antioxidants in treating PD; however, sufficient clinical trials have not been conducted to investigate the efficacies of MTAs in treating PD. Controversial clinical outcomes on the part of non-targeted antioxidants and lack of clinical trials involving MTAs have made it difficult to go ahead with a direct comparison and in turn have slowed down the progress of development of safer and better alternate strategies in treating PD. This review provides an insight on the roles MTAs and non-targeted antioxidants have played in the treatment of PD till date in preclinical and clinical settings and discusses about the limitations of mitochondria-targeted and non-targeted antioxidants that can be resolved for developing effective strategies in treating Parkinsonism.

Melatonin ameliorates lipopolysaccharide induced brain inflammation through modulation of oxidative status and diminution of cytokine rush in Danio rerio.

Lipopolysaccharide (LPS) is known to induce inflammation and immunonomodulation in a piscine model of Danio rerio. Present study aimed to explore the ability of melatonin in attenuating LPS-induced oxidative damages using this model. In LPS-exposed fish, activation of stress marker MDA was observed in brain with corresponding augmentation of multiple pro-inflammatory cytokines (IL1ß, IL6, IL10 and TNFα). In addition, it also showed marked increase in the levels of heat shock factor (HSF) and heat shock proteins (HSPs) in association with transcription factors (NF-kB and NRF2) and mitogen-activated protein kinases (MAPKs). The changes in the levels of these mediators are highly correlated with the induction of pro-inflammatory cytokines. In melatonin-treated fishes, significant amelioration of oxidative stress was observed with reduced levels of MDA and pro-inflammatory cytokines. Melatonin also modulated expression of HSPs that facilitated the brain to overcome inflammation-induced stress by directly initiating NFkB/NRF2 translocation. In summary, melatonin effectively functions to reduce stress induced inflammatory signalling through modulation of oxidative stress and protects the brain from the neuropathological insult.

Substance P failed to reverse dextran sulfate sodium-induced murine colitis mediated by mitochondrial dysfunction: implications in ulcerative colitis.

As controversy exists about the efficacy of substance P (SP) in treating ulcerative colitis (UC) with no previous study highlighting the impact of SP on mitochondrial dysfunction in this diseased condition, it became logical to perform the present study. C57BL/6 J mice were administered with DSS @ 3.5%/gm body weight for 3 cycles of 5 days each followed by i.v. dose of SP @ 5nmole per kg for consecutive 7 days. Histopathological features were noticed in the affected colon along with colonic mitochondrial dysfunction, alterations in mitochondrial stress variables and enhanced colonic cell death. Interestingly, SP failed to reverse colitic features and proved ineffective in inhibiting mitochondrial dysfunction. Unexpectedly SP alone seemed to impart detrimental effects on some of the mitochondrial functions, enhanced lipid peroxidation and increased staining intensities for caspases 3 and 9 in the normal colon. To substantiate in vivo findings and to assess free radical scavenging property of SP, Caco-2 cells were exposed to DSS with or without SP in the presence and absence of specific free radical scavengers and antioxidants. Interestingly, in vitro treatment with SP failed to restore mitochondrial functions and its efficacy proved below par compared to SOD and DMSO indicating involvement of O2 •- and •OH in the progression of UC. Besides, catalase, L-NAME and MEG proved ineffective indicating non-involvement of H2O2, NO and ONOO- in UC. Thus, SP may not be a potent anti-colitogenic agent targeting colonic mitochondrial dysfunction for maintenance of colon epithelial tract as it lacks free radical scavenging property.

Effect of lead on oxidative stress, Na+K+ATPase activity and mitochondrial electron transport chain activity of the brain of Clarias batrachus L.

The present invivo study was designed to elucidate the toxic effect of lead on oxidative stress, Na(+)K(+)ATPase and mitochondrial electron transport chain activity of the brain of Clarias batrachus. The fish were exposed to 10 and 20% of the derived 96 h LC(50) value, 37.8 and 75.6 mg L(-1), respectively, and sampled on 20, 40 and 60 days. Exposure of fish brain to lead demonstrated an increased production of reactive oxygen species, increased lipid peroxidation, loss of protein thiol groups in synaptosomal fraction with the decreased activity of Na(+)K(+)ATPase, partial inactivation of mitochondrial electron transport chain activity and energy depletion. However, no change in protein carbonyl content in synaptosomal fraction was observed due to lead exposure. Concluding the results of our investigation we suggest that lead exposure induces oxidative stress in the brain of Clarias batrachus and the decline in Na(+)K(+)ATPase activity was presumeably mediated by the combined action of lipid peroxidation and deficient mitochondrial electron transport chain activity.

Role of free radical scavenging activity of vasoactive intestinal peptide in the attenuation of mitochondrial dysfunction to ameliorate dextran sulphate sodium-induced colitis in mice: Implications in ulcerative colitis.

OBJECTIVE: To evaluate the efficacy of vasoactive intestinal peptide (VIP) in treating ulcerative colitis (UC), targeting colonic mitochondrial dysfunction by virtue of its free radical scavenging properties for maintenance of colon mucosal integrity. METHODS: A murine model was administered with dextran sodium sulfate (DSS) to induce colitis in C57BL/6J mice at 3.5%/g bodyweight for 3 cycles of 5 days each, followed by an intraperitoneal dose of VIP at 0.5 nmol/L per mouse per day for 10 days. The post-treatment mice were sacrificed and their colon samples were utilized for further analysis. To substantiate the in vivo findings and identify the reactive species involved in progression of UC, Caco-2 cells were subjected to DSS (5%) for 24 hours at 37 °C with or without VIP (10 nmol/L) in the presence or absence of specific free radical scavengers and antioxidants. RESULTS: Treatment with VIP reduced histopathological severity of colitis and cell death markers in murine model, leading to partial recovery of inhibited mitochondrial respiratory complexes, altered mitochondrial membrane potential and lowered adenosine triphosphate generation. Interestingly, in vitro treatment with VIP restored mitochondrial functions and its efficacy was equal to super oxide dismutase and dimethyl sulfoxide, indicating involvement of superoxide free radical (O2 •-) and hydroxyl radical (•OH) in progression of UC. However, catalase, Nω-nitro-l-arginine methyl ester and mercaptoethylguanidine were ineffective, indicating non-involvement of hydrogen peroxide, nitric oxide and ONOO- in UC. CONCLUSION: By virtue of its free radical scavenging properties VIP can act as a potent anti-colitogenic agent, reversing colonic mitochondrial dysfunction for treating UC.

Ultraviolet and Infrared luminescent Au-rich nanostructure growth in SiO2 by burrowing and inverse Oswald ripening process.

We study the evolution of nanoparticle morphology through successive stages when Au-Si bilayer on SiO2 is irradiated with 500 keV Xe-ions and resulting luminescence in the UV, Visible and infrared range. An array of nanoscale island morphology is developed on the silica surface in the initial stage of evolution which undergoes gradual burrowing in the silica matrix accompanied by elongation of large ones in the direction of incident ions under cumulative ion irradiation. Burrowing is found to occur in order to minimize the surface free energy of the nanoparticles. Numerical simulation based on the unified thermal spike model shows formation of molten latent tracks due to ions energy release which drive the dewetting of the metal layer and further give mobility to nanoparticle atoms leading to burrowing in the later stage of evolution and elongation of large nanoparticles. Satellite nanoparticles are observed to grow around Au nanoparticles embedded in silica through nucleation of Au atoms dissolved in the matrix by incident ions. The varying diameters of the Au satellite nanoparticles seem to result in luminescence in the UV and infrared range. The observed structure may find application in surface enhanced Raman scattering, catalysis, and LEDs.

Epidermal growth factor receptor and proliferating cell nuclear antigen in astrocytomas.

AIMS: The involvement of various growth factors, growth factor receptors and proliferative markers in the molecular pathogenesis of astrocytic neoplasms are being studied extensively. Epidermal Growth Factor Receptor (EGFR) gene overexpression occurs in nearly 50% of cases of glioblastoma. Since EGFR and proliferating cell nuclear antigen (PCNA) are involved in mitogenic signal transduction and cellular proliferation pathway, we have studied the correlation between the expression of EGFR and PCNA labeling index in astrocytic tumors. MATERIALS AND METHODS: We investigated the immunohistochemical expression of EGFR and PCNA using the appropriate monoclonal antibodies in 40 cases of astrocytic tumors of which 21 cases were glioblastoma, eight cases were Grade III or anaplastic astrocytomas and six cases were Grade II or diffuse astrocytomas and five cases were Grade I or pilocytic astrocytomas. RESULTS: Both the EGFR expression and PCNA labeling index increase with increasing grades of astrocytomas with a significantly high percentage of cells showing positive staining for both EGFR and PCNA in GBM and Grade III astrocytomas compared to Grade II astrocytomas. The expression levels of both EGFR and PCNA were low in Grade I or pilocytic astrocytomas. CONCLUSIONS: A significant correlation was found between EGFR overexpression and PCNA labeling index in Grade III and Grade II astrocytomas and glioblastoma. These suggest that the tumor proliferation, at least in higher grades of astrocytomas is dependent in some measure on EGF and EGFR-related signaling pathways.

Colonic levels of vasoactive intestinal peptide decrease during infection and exogenous VIP protects epithelial mitochondria against the negative effects of IFNγ and TNFα induced during Citrobacter rodentium infection.

Citrobacter rodentium infection is a model for infection with attaching and effacing pathogens, such as enteropathogenic Escherichia coli. The vasoactive intestinal peptide (VIP) has emerged as an anti-inflammatory agent, documented to inhibit Th1 immune responses and successfully treat animal models of inflammation. VIP is also a mucus secretagogue. Here, we found that colonic levels of VIP decrease during murine C. rodentium infection with a similar time dependency as measurements reflecting mitochondrial function and epithelial integrity. The decrease in VIP appears mainly driven by changes in the cytokine environment, as no changes in VIP levels were detected in infected mice lacking interferon gamma (IFNγ). VIP supplementation alleviated the reduction of activity and levels of mitochondrial respiratory complexes I and IV, mitochondrial phosphorylation capacity, transmembrane potential and ATP generation caused by IFNγ, TNFα and C. rodentium infection, in an in vitro mucosal surface. Similarly, VIP treatment regimens that included the day 5-10 post infection period alleviated decreases in enzyme complexes I and IV, phosphorylation capacity, mitochondrial transmembrane potential and ATP generation as well as increased apoptosis levels during murine infection with C. rodentium. However, VIP treatment failed to alleviate colitis, although there was a tendency to decreased pathogen density in contact with the epithelium and in the spleen. Both in vivo and in vitro, NO generation increased during C. rodentium infection, which was alleviated by VIP. Thus, therapeutic VIP administration to restore the decreased levels during infection had beneficial effects on epithelial cells and their mitochondria, but not on the overall infection outcome.

Mitochondrial respiratory chain inhibition and Na+K+ATPase dysfunction are determinant factors modulating the toxicity of nickel in the brain of indian catfish Clarias batrachus L.

Nickel is a potential neurotoxic pollutant inflicting damage in living organisms, including fish, mainly through oxidative stress. Previous studies have demonstrated the impact of nickel toxicity on mitochondrial function, but there remain lacunae on the damage inflicted at mitochondrial respiratory level. Deficient mitochondrial function usually affects the activities of important adenosinetriphosphatases responsible for the maintenance of normal neuronal function, namely Na+K+ATPase, as explored in our study. Previous reports demonstrated the dysfunction of this enzyme upon nickel exposure but the contributing factors for the inhibition of this enzyme remained unexplored. The main purpose of this study was to elucidate the impact of nickel neurotoxicity on mitochondrial respiratory complexes and Na+K+ATPase in the piscine brain and to determine the contributing factors that had an impact on the same. Adult Clarias batrachus were exposed to nickel treated water at 10% and 20% of the 96 h LC50 value (41 mg.l-1) respectively and sampled on 20, 40 and 60 days. Exposure of fish brain to nickel led to partial inhibition of complex IV of mitochondrial respiratory chain, however, the activities of complex I, II and III remained unaltered. This partial inhibition of mitochondrial respiratory chain might have been sufficient to lower mitochondrial energy production in mitochondria that contributed to the partial dysfunction of Na+K+ATPase. Besides energy depletion other contributing factors were involved in the dysfunction of this enzyme, like loss of thiol groups for enzyme activity and lipid peroxidation-derived end products that might have induced conformational and functional changes. However, providing direct evidence for such conformational and functional changes of Na+K+ATPase was beyond the scope of the present study. In addition, immunoblotting results also showed a decrease in Na+K+ATPase protein expression highlighting the impact of nickel neurotoxicity on the expression of the enzyme itself. The implication of the inhibition of mitochondrial respiration and Na+K+ATPase dysfunction was the neuronal death as evidenced by enhanced caspase-3 and caspase-9 activities. Thus, this study established the deleterious impact of nickel neurotoxicity on mitochondrial functions in the piscine brain and identified probable contributing factors that can act concurrently in the inhibition of Na+K+ATPase. This study also provided a vital clue about the specific areas that the therapeutic agents should target to counter nickel neurotoxicity.

Dopamine but not 3,4-dihydroxy phenylacetic acid (DOPAC) inhibits brain respiratory chain activity by autoxidation and mitochondria catalyzed oxidation to quinone products: implications in Parkinson's disease.

This study reveals that, in contrast to dopamine (DA), 3,4 dihydroxyphenylacetic acid (DOPAC) during in vitro incubation up to 2 h causes only marginal inhibition of rat brain mitochondrial respiratory chain activity, a minimal loss of protein free thiols and very little quinoprotein adduct formation. The damaging effects of DA on brain mitochondria are, however, conspicuous and apparently mediated by quinone oxidation products generated by autoxidation of DA as well as catalyzed by a mitochondrial activity, inhibitable by clorgyline (2.5-10 microM) and cyanide (1 mM).

Annealing Induced Morphology of Silver Nanoparticles on Pyramidal Silicon Surface and Their Application to Surface-Enhanced Raman Scattering.

This paper reports on a simple and cost-effective process of developing a stable surface-enhanced Raman scattering (SERS) substrate based on silver (Ag) nanoparticles deposited on silicon (Si) surface. Durability is an important issue for preparing SERS active substrate as silver nanostructures are prone to rapid surface oxidation when exposed to ambient conditions, which may result in the loss of the enhancement capabilities in a short period of time. Here, we employ the galvanic displacement method to produce Ag nanoparticles on Si(100) substrate prepatterned with arrays of micropyramids by chemical etching, and subsequently, separate pieces of such substrates were annealed in oxygen and nitrogen environments at 550 °C. Interestingly, while nitrogen-annealed Si substrates were featured by spherical-shaped Ag particles, the oxygen annealed Si substrates were dominated by the formation of triangular shape particles attached with the spherical one. Remarkably, the oxygen-annealed substrate thus produced shows very high SERS enhancement compared to the either unannealed or nitrogen annealed substrate. The hitherto unobserved coexistence of triangular morphology with the spherical one and the gap between the two (source of efficient hot-spots) are the origin of enhanced SERS activity for the oxygen-annealed Ag particle-covered Si substrate as probed by the combined finite-difference time domain (FDTD) simulation and cathodoluminesensce (CL) experiment. As the substrate has already been annealed in an oxygen environment, further probability of oxidation is reduced in the present synthesis protocol that paves the way for making a novel long-lived thermally stable SERS substrate.

Studies on acute and chronic toxicity of cadmium to freshwater snail Lymnaea acuminata (Lamarck) with special reference to behavioral and hematological changes.

Molluscs have long been regarded as promising bioindicator and biomonitoring subjects for heavy metals as molluscs are highly tolerant to heavy metals and exhibit high accumulation in their body. In spite of several previous studies about the impact of cadmium on molluscs, little information exists in literatures concerning the toxic effects of cadmium on Lymnaea acuminata, especially pertaining to behavioral and hematological changes as these are considered effective bioindicators and biomonitoring variables for detecting heavy metals in polluted water bodies. In the present study, the median lethal concentrations of cadmium chloride to snail, Lymnaea acuminata, were estimated to be 9.66, 7.69, 6.26, and 5.54 mg/L at 24, 48, 72, and 96 h, respectively. For behavioral studies, variable test concentrations of cadmium from 0.00 to 10 mg/L were used. The clumping tendency, crawling activity, and touch reflex in the exposed snails were gradually decreased with higher concentrations at 72 and 96 h. For measuring the hemocyte numbers in the circulating hemolymph of snail during chronic cadmium exposure, two sublethal doses of cadmium (10 and 20% 96-h LC50-0.55 and 1.11 mg/L, respectively) were used. A significant variation (p < 0.05) from the control at all exposure times (7, 14, 21, and 28 days) was recorded at 1.11 mg/L concentration. The total count of circulating hemocytes was significantly reduced (p < 0.05) compared to the controls at both concentrations of cadmium exposure at all time periods except 14 and 21 days exposure at 0.55 mg/L where values were non-significantly increased. In comparison between two sublethal doses, blood cells were significantly (p < 0.05) lowered at 1.11 mg/L cadmium treatment. Considering the behavioral and hematological data, it seems possible to forecast the physiological state of snails in cadmium-contaminated water bodies and these findings can be used in determining the safe disposal level of cadmium in aquatic ecosystem.

Neuroprotective Efficacy of Mitochondrial Antioxidant MitoQ in Suppressing Peroxynitrite-Mediated Mitochondrial Dysfunction Inflicted by Lead Toxicity in the Rat Brain.

Lead (Pb) is one of the most pollutant metals that accumulate in the brain mitochondria disrupting mitochondrial structure and function. Though oxidative stress mediated by reactive oxygen species remains the most accepted mechanism of Pb neurotoxicity, some reports suggest the involvement of nitric oxide (•NO) and reactive nitrogen species in Pb-induced neurotoxicity. But the impact of Pb neurotoxicity on mitochondrial respiratory enzyme complexes remains unknown with no relevant report highlighting the involvement of peroxynitrite (ONOO-) in it. Herein, we investigated these effects in in vivo rat model by oral application of MitoQ, a known mitochondria-specific antioxidant with ONOO- scavenging activity. Interestingly, MitoQ efficiently alleviated ONOO--mediated mitochondrial complexes II, III and IV inhibition, increased mitochondrial ATP production and restored mitochondrial membrane potential. MitoQ lowered enhanced caspases 3 and 9 activities upon Pb exposure and also suppressed synaptosomal lipid peroxidation and protein oxidation accompanied by diminution of nitrite production and protein-bound 3-nitrotyrosine. To ascertain our in vivo findings on mitochondrial dysfunction, we carried out similar experiments in the presence of different antioxidants and free radical scavengers in the in vitro SHSY5Y cell line model. MitoQ provided better protection compared to mercaptoethylguanidine, N-nitro-L-arginine methyl ester and superoxide dismutase suggesting the predominant involvement of ONOO- compared to •NO and O2•-. However, dimethylsulphoxide and catalase failed to provide protection signifying the noninvolvement of •OH and H2O2 in the process. The better protection provided by MitoQ in SHSY5Y cells can be attributed to the fact that MitoQ targets mitochondria whereas mercaptoethylguanidine, N-nitro-L-arginine methyl ester and superoxide dismutase are known to target mainly cytoplasm and not mitochondria. Taken together the results from the present study clearly brings out the potential of MitoQ against ONOO--induced toxicity upon Pb exposure indicating its therapeutic potential in metal toxicity.

Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implications for Parkinson's disease.

Several studies on mitochondrial functions following brief exposure (5-15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1-0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of *OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).

Enhancement in cellular Na+K+ATPase activity by low doses of peroxynitrite in mouse renal tissue and in cultured HK2 cells.

In the normal condition, endogenous formation of peroxynitrite (ONOO-) from the interaction of nitric oxide and superoxide has been suggested to play a renoprotective role. However, the exact mechanism associated with renoprotection by this radical compound is not yet clearly defined. AlthoughONOO- usually inhibits renal tubular Na(+)K(+)ATPase (NKA) activity at high concentrations (micromolar to millimolar range [µM-mM], achieved in pathophysiological conditions), the effects at lower concentrations (nanomolar range [nM], relevant in normal condition) remain unknown. To examine the direct effect ofONOO- onNKAactivity, preparations of cellular membrane fraction from mouse renal tissue and from culturedHK2 cells (human proximal tubular epithelial cell lines) were incubated for 10 and 30 min each with different concentrations ofONOO- (10 nmol/L-200 µmol/L).NKAactivity in these samples (n = 5 in each case) was measured via a colorimetric assay capable of detecting inorganic phosphate. At high concentrations (1-200 µmol/L),ONOO- caused dose-dependent inhibition ofNKAactivity (-3.0 ± 0.6% and -36.4 ± 1.4%). However,NKAactivity remained unchanged at 100 and 500 nmol/LONOO- concentration, but interestingly, at lower concentrations (10 and 50 nmol/L),ONOO- caused small but significant increases in theNKAactivity (3.3 ± 1.1% and 3.1 ± 0.6%). Pretreatment with aONOO- scavenger, mercaptoethylguanidine (MEG; 200 µmol/L), prevented these biphasic responses toONOO-. This dose-dependent biphasic action ofONOO(-)onNKAactivity may implicate that this radical compound helps to maintain sodium homeostasis either by enhancing tubular sodium reabsorption under normal conditions or by inhibiting it during oxidative stress conditions.

IL-4 Protects the Mitochondria Against TNFα and IFNγ Induced Insult During Clearance of Infection with Citrobacter rodentium and Escherichia coli.

Citrobacter rodentium is a murine pathogen that serves as a model for enteropathogenic Escherichia coli. C. rodentium infection reduced the quantity and activity of mitochondrial respiratory complexes I and IV, as well as phosphorylation capacity, mitochondrial transmembrane potential and ATP generation at day 10, 14 and 19 post infection. Cytokine mRNA quantification showed increased levels of IFNγ, TNFα, IL-4, IL-6, and IL-12 during infection. The effects of adding these cytokines, C. rodentium and E. coli were hence elucidated using an in vitro colonic mucosa. Both infection and TNFα, individually and combined with IFNγ, decreased complex I and IV enzyme levels and mitochondrial function. However, IL-4 reversed these effects, and IL-6 protected against loss of complex IV. Both in vivo and in vitro, the dysfunction appeared caused by nitric oxide-generation, and was alleviated by an antioxidant targeting mitochondria. IFNγ -/- mice, containing a similar pathogen burden but higher IL-4 and IL-6, displayed no loss of any of the four complexes. Thus, the cytokine environment appears to be a more important determinant of mitochondrial function than direct actions of the pathogen. As IFNγ and TNFα levels increase during clearance of infection, the concomitant increase in IL-4 and IL-6 protects mitochondrial function.

Dietary supplementation with N-acetylcysteine, alpha-tocopherol and alpha-lipoic acid prevents age related decline in Na(+),K (+)-ATPase activity and associated peroxidative damage in rat brain synaptosomes.

This study has shown that in aged rat brain (22-24 months) crude synaptosomes in comparison to that in young animals (4-6 months), a striking decrease in the activity of Na(+),K(+)-ATPase occurs along with decreased K (m) and V (max) but without any change in enzyme content as seen by immunoblotting. This is associated with an accumulation of peroxidative damage products in aged brain. When rats are given antioxidant supplementation in the diet with a combination of N-acetylcysteine, alpha-tocopherol and alpha-lipoic acid daily from 18 months onwards and sacrificed at 22-24 months for experimentation, the age associated decrease of Na(+),K(+)-ATPase activity, alterations of its kinetic parameters and accumulation of peroxidative damage products in brain synaptosomes are prevented nearly completely. Because of the critical importance of Na(+),K(+)-ATPase in neuronal functions, the results of this study may be of potential implications in controlling age-related functional deficits of the brain.