Overexpression Of Superoxide Dismutase Research Articles

Role of metabolically generated reactive oxygen species for lipotoxicity in pancreatic β‐cells

Chronically elevated concentrations of non-esterified fatty acids (NEFAs) in type 2 diabetes may be involved in β-cell dysfunction and apoptosis. It has been shown that long-chain saturated NEFAs exhibit a strong cytotoxic effect upon insulin-producing cells, while short-chain as well as unsaturated NEFAs are well tolerated. Moreover, long-chain unsaturated NEFAs counteract the toxicity of palmitic acid. Reactive oxygen species (ROS) formation and gene expression analyses together with viability assays in different β-cell lines showed that the G-protein-coupled receptors 40 and 120 do not mediate lipotoxicity. This is independent from the role, which these receptors, specifically GPR40, play in the potentiation of glucose-induced insulin secretion by saturated and unsaturated long-chain NEFAs. Long-chain NEFAs are not only metabolized in the mitochondria but also in peroxisomes. In contrast to mitochondrial β-oxidation, the acyl-coenzyme A (CoA) oxidases in the peroxisomes form hydrogen peroxide and not reducing equivalents. As β-cells almost completely lack catalase, they are exceptionally vulnerable to hydrogen peroxide generated in peroxisomes. ROS generation in the respiratory chain is less important because overexpression of catalase and superoxide dismutase in the mitochondria do not provide protection. Thus, peroxisomally generated hydrogen peroxide is the likely ROS that causes pancreatic β-cell dysfunction and ultimately β-cell death.

Proteomic alterations of Escherichia coli by paraquat.

Paraquat (PQ; a widely used herbicide) exerts its harmful effect to human, mammals and microorganisms upon intracellular conversion to superoxide radical. Cellular responses against toxic paraquat remain not fully understood, especially on the adaptive metabolic changes as a consequence of oxidative burden. In this study, alterations of metabolic processes of Escherichia coli (E. coli) by paraquat were systematically investigated by two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting (PMF). In host cells, the first line mechanism was scrutinized by a remarkable induction of endogenous superoxide dismutase (E. coli SOD). The second line involved in the metabolic adaptation and compensation for energy production by up- or down-regulation of the enzymes implicated in glycolysis and tricarboxylic acid cycle. Notably, down-regulation of aconitase enzyme and changes of enzyme isoform from the acidic (pI~5.29) to the higher basidic form (pI~5.59) were detected. Meanwhile, up-regulation of fumarase approximately 4-5 folds were observed. Importantly, overexpression of human manganese superoxide dismutase (human Mn-SOD) in E. coli cells could in turn down-regulate the expression of fumarase enzyme. This observation was not found when the cells expressing human catalase were tested. Other mechanisms such as changes of purine nucleoside phosphorylase and protein transporters (D-ribose-binding protein and oligopeptide binding protein) were also accounted. However, among all the differentially expressed proteins, the fumarase enzyme is evidenced to be a major target responsible for superoxide-generating paraquat, which may further be applied as a potential biomarker for paraquat toxicity in the future.

Inhibition of central angiotensin II-induced pressor responses by hydrogen peroxide

Hydrogen peroxide (H2O2), important reactive oxygen species produced endogenously, may have different physiological actions. The superoxide anion (O2·−) is suggested to be part of the signaling mechanisms activated by angiotensin II (ANG II) and central virus-mediated overexpression of the enzyme superoxide dismutase (that dismutates O2·− to H2O2) reduces pressor and dipsogenic responses to central ANG II. Whether this result might reflect elevation of H2O2 rather than depletion of O2·− has not been addressed. Here we investigated the effects of H2O2 injected intracerebroventricularly (i.c.v.) or ATZ (3-amino-1,2,4-triazole, a catalase inhibitor) injected intravenously (i.v.) or i.c.v. on the pressor responses induced by i.c.v. injections of ANG II. Normotensive male Holtzman rats (280–320 g, n=5–13/group) with stainless steel cannulas implanted in the lateral ventricle were used. Prior injection of H2O2 (5 μmol/1 μl) or ATZ (5 nmol/1 μl) i.c.v. almost abolished the pressor responses induced by ANG II (50 ng/1 μl) also injected i.c.v. (7±3 and 5±3 mm Hg, respectively, vs. control: 19±4 mm Hg). Injection of ATZ (3.6 mmol/kg b.wt.) i.v. also reduced central ANG II-induced pressor responses. Injections of H2O2 i.c.v. and ATZ i.c.v. or i.v. alone produced no effect on baseline arterial pressure. Central ANG II, H2O2 or ATZ did not affect heart rate. The results show that central injections of H2O2 and central or peripheral injections of ATZ reduced the pressor responses induced by i.c.v. ANG II, suggesting that exogenous or endogenous H2O2 may inhibit central pressor mechanisms activated by ANG II.

SOD1 and MitoTEMPO partially prevent mitochondrial permeability transition pore opening, necrosis, and mitochondrial apoptosis after ATP depletion recovery

Generation of excessive reactive oxygen species (ROS) leads to mitochondrial dysfunction, apoptosis, and necrosis in renal ischemia–reperfusion (IR) injury. Previously we showed that lentiviral vector-mediated overexpression of superoxide dismutase-1 (SOD1) in proximal tubular epithelial cells (LLC-PK 1) reduced cytotoxicity in an in vitro model of IR injury. Here, we examined the effects of SOD1 overexpression on mitochondrial signaling after ATP depletion–recovery (ATP-DR). To examine the role of mitochondrial ROS, a subset of cells was treated with the mitochondrial antioxidant MitoTEMPO. ATP-DR-mediated increase in mitochondrial calcium, loss of mitochondrial membrane potential, and increase in mitochondrial permeability transition pore (MPTP) were attenuated by SOD1 and MitoTEMPO ( P < 0.01). SOD1 prevented ATP-DR-induced mitochondrial Bax translocation, although the release of proapoptotic proteins from mitochondria was not prevented by SOD1 alone and required the presence of both SOD1 and MitoTEMPO. SOD1 suppressed the increase in c-jun phosphorylation, suggesting that JNK signaling regulates Bax translocation to mitochondria via ROS. ATP-DR-mediated changes in MPTP and mitochondrial signaling increased necrosis and apoptosis, both of which were partially attenuated by SOD1 and MitoTEMPO. These studies show that SOD1 and MitoTEMPO preserve mitochondrial integrity and attenuate ATP-DR-mediated necrosis and apoptosis.

MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor β1: a novel role of miR-382

We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor β1 (TGFβ1), a model of epithelial–mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFβ1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 miRNAs and 16 proteins. Of the miRNAs and proteins that exhibited reciprocal changes in expression, 77 pairs met the sequence criteria for miRNA–target interactions. Knockdown of miR-382, which was up-regulated by TGFβ1, attenuated TGFβ1-induced loss of the epithelial marker E-cadherin. miR-382 was confirmed by 3′-untranslated region reporter assay to target five genes that were downregulated at the protein level by TGFβ1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFβ1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFβ1-induced loss of the epithelial marker. The study provided experimental evidence in the form of reciprocal expression at the protein level for a large number of predicted miRNA-target pairs and discovered a novel role of miR-382 and SOD2 in the loss of epithelial characteristics induced by TGFβ1.

Induction of Cell Death through Alteration of Oxidants and Antioxidants in Lung Epithelial Cells Exposed to High Energy Protons

Radiation affects several cellular and molecular processes, including double strand breakage and modifications of sugar moieties and bases. In outer space, protons are the primary radiation source that poses a range of potential health risks to astronauts. On the other hand, the use of proton irradiation for tumor radiation therapy is increasing, as it largely spares healthy tissues while killing tumor tissues. Although radiation-related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton irradiation remain poorly understood. Therefore, in this study, we irradiated rat lung epithelial cells with different doses of protons and investigated their effects on cell proliferation and death. Our data show an inhibition of cell proliferation in proton-irradiated cells with a significant dose-dependent activation and repression of reactive oxygen species and antioxidants glutathione and superoxide dismutase, respectively, compared with control cells. In addition, the activities of apoptosis-related genes such as caspase-3 and -8 were induced in a dose-dependent manner with corresponding increased levels of DNA fragmentation in proton-irradiated cells compared with control cells. Together, our results show that proton irradiation alters oxidant and antioxidant levels in cells to activate the apoptotic pathway for cell death.

Resurrecting Hope for Antioxidant Treatment of Cardiovascular Disease

Oxidative stress has been associated with cardiovascular disease and the development of hypertension in both humans and in animal models. Antioxidant therapy has been successful in ameliorating disease in animals, but the results from human studies have been largely disappointing. Despite these negative results, a wealth of evidence suggests that oxidative stress is involved in the development of cardiovascular disease both in humans and animal models. Because global attenuation of cellular reactive oxygen species (ROS) may not be advantageous, more recent therapeutic strategies target specific subcellular sources of oxidative stress. Most studies implicate plasmalemmal membrane-bound NADPH oxidase as a key mediator of ROS generation in cardiovascular disease. However, in the current issue of Circulation Research , Dikalova et al1 demonstrate that specific attenuation of mitochondrial derived oxidants prevents the development of angiotensin II–induced hypertension in the mouse. Perhaps more interesting is the mechanism: inhibition of a mitochondrial ROS-triggered activation of NADPH oxidase, interrupting a vicious cycle of ROS production. The novel approach of targeting the inciting source of ROS (mitochondria) to prevent accelerated oxidant production may hold more promise for humans by eliminating pathological but not homeostatic sources of ROS. Seminal observations by Harrison et al2 showed evidence of increased oxidative stress within the systemic vasculature in an Angiotensin II–induced rat model of hypertension, piquing interest in a possible etiologic role for ROS in this condition. In animal models, stimulating ROS production can induce hypertension,2,3 whereas treatment with a variety of antioxidants including vitamin C and E, or targeted inhibition of enzymatic oxidant production, can reduce blood pressure.3–6 Hypertensive patients demonstrate increased NAD(P)H oxidase activity7 with elevated levels of hydrogen peroxide in plasma.8 Antioxidant treatment leads to enhanced vasodilation in both forearm and coronary arteries of human hypertensive subjects,9,10 indicating a functional vasomotor …

Lung extracellular superoxide dismutase overexpression lessens bleomycin-induced pulmonary hypertension and vascular remodeling.

Interstitial lung disease is a devastating disease in humans that can be further complicated by the development of secondary pulmonary hypertension. Accumulating evidence indicates that the oxidant superoxide can contribute to the pathogenesis of both interstitial lung disease and pulmonary hypertension. We used a model of pulmonary hypertension secondary to bleomycin-induced pulmonary fibrosis to test the hypothesis that an imbalance in extracellular superoxide and its antioxidant defense, extracellular superoxide dismutase, will promote pulmonary vascular remodeling and pulmonary hypertension. We exposed transgenic mice overexpressing lung extracellular superoxide dismutase and wild-type littermates to a single dose of intratracheal bleomycin, and evaluated the mice weekly for up to 35 days. We assessed pulmonary vascular remodeling and the expression of several genes critical to lung fibrosis, as well as pulmonary hypertension and mortality. The overexpression of extracellular superoxide dismutase protected against late remodeling within the medial, adventitial, and intimal layers of the vessel wall after the administration of bleomycin, and attenuated pulmonary hypertension at the same late time point. The overexpression of extracellular superoxide dismutase also blocked the early up-regulation of two key genes in the lung known to be critical in pulmonary fibrosis and vascular remodeling, the transcription factor early growth response-1 and transforming growth factor-β. The overexpression of extracellular superoxide dismutase attenuated late pulmonary hypertension and significantly improved survival after exposure to bleomycin. These data indicate an important role for an extracellular oxidant/antioxidant imbalance in the pathogenesis of pulmonary vascular remodeling associated with secondary pulmonary hypertension attributable to bleomycin-induced lung fibrosis.

Epigenetic Attenuation of Mitochondrial Superoxide Dismutase 2 in Pulmonary Arterial Hypertension

Excessive proliferation and impaired apoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs) contribute to vascular obstruction in patients and fawn-hooded rats (FHRs) with PA hypertension (PAH). Expression and activity of mitochondrial superoxide dismutase-2 (SOD2), the major generator of H(2)O(2), is known to be reduced in PAH; however, the mechanism and therapeutic relevance of this are unknown. SOD2 expression in PASMCs is decreased in PAH patients and FHRs with PAH. FHR PASMCs have higher proliferation and lower apoptosis rates than Sprague-Dawley rat PASMCs. Moreover, FHR PASMCs have hyperpolarized mitochondria, low H(2)O(2) production, and reduced cytoplasmic and mitochondrial redox state. Administration of SOD2 small interfering RNA to normal PASMCs recapitulates the FHR PAH phenotype, hyperpolarizing mitochondria, decreasing H(2)O(2), and inhibiting caspase activity. Conversely, SOD2 overexpression in FHR PASMCs or therapy with the SOD-mimetic metalloporphyrin Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) reverses the hyperproliferative PAH phenotype. Importantly, SOD-mimetic therapy regresses PAH in vivo. Investigation of the SOD2 gene revealed no mutation, suggesting a possible epigenetic dysregulation. Genomic bisulfite sequencing demonstrates selective hypermethylation of a CpG island in an enhancer region of intron 2 and another in the promoter. Differential methylation occurs selectively in PAs versus aortic SMCs and is reversed by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, restoring both SOD2 expression and the ratio of proliferation to apoptosis. Expression of the enzymes that mediate gene methylation, DNA methyltransferases 1 and 3B, is upregulated in FHR lungs. Tissue-specific, epigenetic SOD2 deficiency initiates and sustains a heritable form of PAH by impairing redox signaling and creating a proliferative, apoptosis-resistant PASMC. SOD augmentation regresses experimental PAH. The discovery of an epigenetic component to PAH may offer new therapeutic targets.

403 Mitochondrial superoxide dismutase overexpression changes neuroendocrine, proliferation and apoptosis resistance features in prostate cancer cells

403 Mitochondrial superoxide dismutase overexpression changes neuroendocrine, proliferation and apoptosis resistance features in prostate cancer cells

Pluronic-modified superoxide dismutase 1 attenuates angiotensin II-induced increase in intracellular superoxide in neurons

Overexpressing superoxide dismutase 1 (SOD1; also called Cu/ZnSOD), an intracellular superoxide (O 2 •−)-scavenging enzyme, in central neurons inhibits angiotensin II (AngII) intraneuronal signaling and normalizes cardiovascular dysfunction in diseases associated with enhanced AngII signaling in the brain, including hypertension and heart failure. However, the blood–brain barrier and neuronal cell membranes impose a tremendous impediment for the delivery of SOD1 to central neurons, which hinders the potential therapeutic impact of SOD1 treatment on these diseases. To address this, we developed conjugates of SOD1 with poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) block copolymer (Pluronic) (SOD1–P85 and SOD1–L81), which retained significant SOD1 enzymatic activity. The modified SOD1 effectively scavenged xanthine oxidase/hypoxanthine-derived O 2 •−, as determined by HPLC and the measurement of 2-hydroxyethidium. Using catecholaminergic neurons, we observed an increase in neuronal uptake of SOD1–Pluronic after 1, 6, or 24 h, compared to neurons treated with pure SOD1 or PEG–SOD1. Importantly, without inducing neuronal toxicity, SOD1–Pluronic conjugates significantly inhibited AngII-induced increases in intraneuronal O 2 •− levels. These data indicate that SOD1–Pluronic conjugates penetrate neuronal cell membranes, which results in elevated intracellular levels of functional SOD1. Pluronic conjugation may be a new delivery system for SOD1 into central neurons and therapeutically beneficial for AngII-related cardiovascular diseases.

Extended lifespan of Drosophila parkin mutants through sequestration of redox-active metals and enhancement of anti-oxidative pathways

The mechanisms underlying neuron death in Parkinson's disease are unknown, but both genetic defects and environmental factors are implicated in its pathogenesis. Mutations in the parkin gene lead to autosomal recessive juvenile Parkinsonism (AR-JP). Here we report that compared to control flies, Drosophila lacking parkin show significantly reduced lifespan but no difference in dopamine neuron numbers when raised on food supplemented with environmental pesticides or mitochondrial toxins. Moreover, chelation of redox-active metals, anti-oxidants and overexpression of superoxide dismutase 1 all significantly reversed the reduced longevity of parkin-deficient flies. Finally, parkin deficiency exacerbated the rough eye phenotype of Drosophila caused by overexpression of the copper importer B (Ctr1B). Taken together, our results demonstrate an important function of parkin in the protection against redox-active metals and pesticides implicated in the etiology of Parkinson's disease. They also corroborate that oxidative stress, perhaps as a consequence of mitochondrial dysfunction, is a major determinant of morbidity in parkin mutant flies.

Abstract 4008: p75NTR is overexpressed in vestibular schwannomas and protects cells from apoptosis due to suppressed c-Jun N-terminal kinase (JNK) activity

Abstract Vestibular schwannomas (VSs) represent benign Schwann cell (SC) neoplasms arising from the eighth cranial nerve and account for 8-10% of all intracranial neoplasms. Denervation by axotomy leads to increased p75NTR expression, JNK activation, and eventual death of SCs. In contrast to SCs, VS cells survive in the absence of axons and here we investigated the interactions of p75NTR and JNK signaling on the survival response of cultured primary human VS cells. Using qRT-PCR and western blotting, we find that p75NTR is over-expressed in VS cells compared with normal nerve. However, in contrast to normal SCs, treatment of cultured VS cells with pro-NGF, a high affinity ligand for p75NTR, failed to induce apoptosis. We confirmed that VS cells express sortilin, a co-receptor with p75NTR to bind pro-NGF and induce apoptosis. We further show that VS cells display persistent JNK phosphorylation and activity compared with normal SCs. Treatment of VS cultures with the JNK inhibitors, I-JIP or SP600125, results in decreased proliferation, increased apoptosis, and increased accumulation of mitochondrial superoxides. Overexpression of mitochondrial superoxide dismutase prevents apoptosis due to suppressed JNK. These results suggest that persistent JNK contributes to the survival of VS cells, at least in part, by suppressing accumulation of mitochondrial superoxides. Treatment of VS cultures with proNGF protected the cells from apoptosis due to JNK inhibition. This protective effect is specific to proNGF and not seen with other neural derived growth factors such as neuregulin-1 (NRG-1). Indeed treatment with NRG-1 increased apoptosis in cultures with suppressed JNK activity. Further, VS cells lack Trk expression supporting the notion that the protective effect of proNGF is mediated by p75NTR. These results suggest that, in contrast to normal SCs which undergo apoptosis in response to p75NTR and JNK signaling, these signals promote VS cell survival. Such differences may account for the ability of VS cells to proliferate and survive in the absence of axons. Support: DOD NF050193 and NIDCD R01DC009801 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4008.

Absence of Mitochondrial Toxicity in Hearts of Transgenic Mice Treated with Abacavir

Abacavir (ABC) is a guanosine nucleoside reverse transcriptase inhibitor (NRTI) with potent antiretroviral activity. Since NRTIs exhibit tissue-specific inhibition of mitochondrial DNA (mtDNA) synthesis, the ability of ABC to inhibit mtDNA synthesis in vivo was evaluated. Inbred wild-type (WT) and transgenic mice (TG) treated with ABC (3.125 mg/d p. o., 35 days) were used to define mitochondrial oxidative stress and cardiac function. Chosen TGs exhibited overexpression of HIV-1 viral proteins (NL4-3Deltagag/pol, non-replication competent), hemizygous depletion or overexpression of mitochondrial superoxide dismutase (SOD2(+/-) knock-out (KO) or MnSOD OX, respectively), overexpression of mitochondrially targeted catalase (MCAT), or double "knockout" deletion of aldehyde dehydrogenase activity (ALDH2 KO). Impact on mtDNA synthesis was assessed by comparing changes in mtDNA abundance between ABC-treated and vehicle-treated WTs and TGs. No changes in mtDNA abundance occurred from ABC treatment in any mice, suggesting no inhibition of mtDNA synthesis. Left ventricle (LV) mass and LV end-diastolic dimension (LVEDD) were defined echocardiographically and remained unchanged as well. These results indicate that treatment with ABC has no visible cardiotoxicity in these adult mice exposed for 5 weeks compared to findings with other antiretroviral NRTI studies and support some claims for its relative safety.

Abacavir (ABC) Has No Cardiac Mitochondrial Toxicity in Multiple Trangenic Murine Models Compared to Zidovudine (AZT)

ABC and AZT are both nucleoside reverse transcriptase inhibitors (NRTIs) central to effective antiretroviral therapy for HIV/AIDS. Despite therapeutic success, toxicities associated with NRTI‐induced mitochondrial dysfunction have been reported. Genetically engineered mice with knock‐out (KO) or overexpression (OX) of mitochondrial superoxide dismutase (SOD2+/− KO and SOD2‐OX, respectively), mitochondrially targeted catalase (mCAT‐TG), aldehyde dehydrogenase 2 (ALDH2 KO), alcohol dehydrogenase (ADH‐TG), or wild‐type littermates (C57/BL6, WT) were treated with AZT (0.22 mg/d) or ABC (3.125 mg/d) for 35 days. Mitochondrial DNA (mtDNA) abundance and left‐ventricle (LV) mass and LV end‐diastolic dimension were determined. AZT induced cardiac oxidative stress and LV dysfunction in WTs. Effects worsened in KOs while OX and TGs prevented or attenuated AZT‐induced cardiac oxidative stress. Conversely, ABC had no effect on any cohorts, including WTs, suggesting no associated cardiac oxidative stress with ABC. These data support the clinical use of ABC as replacement for AZT in preventing cardiac oxidative stress.

Protein Aggregation in a Mutant Deficient in YajL, the Bacterial Homolog of the Parkinsonism-associated Protein DJ-1

YajL is the closest prokaryotic homolog of the parkinsonism-associated protein DJ-1 (40% sequence identity and similar three-dimensional structure), a protein of unknown function involved in the cellular response to oxidative stress. We report here that a yajL mutant of Escherichia coli displays an increased sensitivity to oxidative stress. It also exhibits a protein aggregation phenotype in aerobiosis, but not in anaerobiosis or in aerobic cells overexpressing superoxide dismutase, suggesting that protein aggregation depends on the presence of reactive oxygen species produced by respiratory chains. The protein aggregation phenotype of the yajL mutant, which can be rescued by the wild-type yajL gene, but not by the corresponding cysteine 106 mutant allele, is similar to that of multiple mutants deficient in superoxide dismutases and catalases, although intracellular hydrogen peroxide levels were not increased in the yajL mutant, suggesting that protein aggregation in this strain does not result from a hydrogen peroxide detoxification defect. Aggregation-prone proteins included 17 ribosomal proteins, the ATP synthase beta subunit, flagellin, and the outer membrane proteins OmpA and PAL; all of them are part of multiprotein complexes, suggesting that YajL might be involved in optimal expression of these complexes, especially during oxidative stress. YajL stimulated the renaturation of urea-unfolded citrate synthase and the solubilization of the urea-unfolded ribosomal proteins S1 and L3 and was more efficient as a chaperone in its oxidized form than in its reduced form. The mRNA levels of several aggregated proteins of the yajL mutant were severely affected, suggesting that YajL also acts at the level of gene expression. These two functions of YajL might explain the protein aggregation phenotype of the yajL mutant.

Astrocyte targeted overexpression of Hsp72 or SOD2 reduces neuronal vulnerability to forebrain ischemia

Brief forebrain ischemia is a model of the delayed hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation. Previous studies demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes occurs hours to days before delayed neuronal death. In this study we tested the strategy of directing protection to astrocytes to protect neighboring neurons from forebrain ischemia. Two well-studied protective proteins, heat shock protein 72 (Hsp72) or superoxide dismutase 2 (SOD2), were genetically targeted for expression in astrocytes using the astrocyte-specific human glial fibrillary acidic protein (GFAP) promoter. The expression constructs were injected stereotacticly immediately above the hippocampal CA1 region on one side of the rat brain two days prior to forebrain ischemia. Cell type specific expression was confirmed by double label immunohistochemistry. When the expression constructs were injected two days before transient forebrain ischemia, the loss of CA1 hippocampal neurons observed seven days later was significantly reduced on the injected side compared with controls. This neuroprotection was associated with significantly better preservation of astrocyte glutamate transporter-1 immunoreactivity at 5-h reperfusion and reduced oxidative stress. Improving the resistance of astrocytes to ischemic stress by targeting either the cytosolic or mitochondrial compartment was thus associated with preservation of CA1 neurons following forebrain ischemia. Targeting astrocytes is a promising strategy for neuronal preservation following cardiac arrest and resuscitation.

Cold tolerance of UCP1-ablated mice: A skeletal muscle mitochondria switch toward lipid oxidation with marked UCP3 up-regulation not associated with increased basal, fatty acid- or ROS-induced uncoupling or enhanced GDP effects

Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1) - and thus all heat production from brown adipose tissue - can still adapt to a cold environment (4°C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(–/–) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(–/–) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(–/–) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(–/–), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.

Clinical utility of antioxidant gene expression levels in circulating cancer cell clusters for the detection of prostate cancer in patients with prostate‐specific antigen levels of 4–10 ng/mL and disease prognostication after radical prostatectomy

To test antioxidant genes (AOX) expression in circulating cancer cell clusters (CCC). A novel method using molecular, polymerase chain reaction (PCR)-based detection of CCC was applied for predicting prostate cancer and to assess the effect of radical prostatectomy (RP) on reducing CCC and for prognostication of relapse-free survival (RFS), as serum total prostate-specific antigen (tPSA) has limited specificity at 4-10 ng/mL. In all, 240 patients were enrolled in the study, 129 for tumour diagnosis and 111 after RP for disease prognostication. Filtration assay in previously fractionated mononuclear cells (MNC) was used to enrich the CCC and large cells, which were retained in a mesh of 20 microm width. To establish the malignant nature of these cells they were analysed for genomic imbalances detected via PCR-assays of loss of heterozygosity in tumour suppressor loci and of DNA amplification in protooncogen loci. As a screening test in daily practice, real-time reverse transcription (RT)-PCR of AOX was introduced to overcome the laborious and expensive DNA tests. The AOX chosen were glutathione peroxidase (GPX1), Mn-dependent superoxide dismutase 2 (SOD2) and thioredoxine reductase (TXNRD1); selected from 67 marker candidate genes according to sensitivity and specificity data. AOX overexpression in CCC serves as a general marker for solid tumours needing, however, organ markers to relate to the organ of origin. Androgen receptor (AR), PSA and prostate-specific membrane antigen mRNAs served as organ markers for the prostate. Signals were detected in patients' MNC and to a minor level in CCC, rendering to CCC a substantial loss in epithelial features equivalent to a lower grade of epithelial differentiation. Organ markers in the MNC fraction were positive in <85% of AOX testing. The AOX test was tumour predicting (P < 0.001) with a sensitivity of 86%, specificity 82%, positive predictive value 69%, negative predictive value 92%, accuracy 83% and odds ratio (OR) of 28. SOD2 and TXNRD1 expression correlated to tumour size and Gleason score. Objective assessment for the evaluation of the molecular cell markers was achieved by receiver operating characteristic (ROC) curves. The areas under the ROC curve values of the AOXs were 0.7-0.9. RP was followed by a complete clearance of AOX-expressing cells. After RP, a subgroup of patients had residual CCC over-expressing only SOD2 and GPX1 indicating incomplete clearance by RP. Sustained overexpression of SOD2 and GPX1 accounted as risk factors for distant tumour recurrence (P = 0.003) mainly for bone metastases (97% M1b) as evaluated by Kaplan-Meier curves. In univariate analysis the tumour size had a limited effect on the probability of RFS (P = 0.05). In multivariate analysis tumour size, nodal status and Gleason score had no effect. This can partially be attributed to the higher risk level of pathological variables in the AOX over-expressing group but also to ineffective endocrine therapy resulting in marked overexpression of ARs and GPX1, the lead prognosticator gene. The AOX expression level allowed the identification of patients with high progression risk, who have more favourable pathological variables. The AOX testing of CCC is a novel method with excellent prognostic and predictive properties enabling the monitoring of therapies, e.g. effects of RP and endocrine therapy. We speculate that the continuing elevated expression of AOX with special emphasis on GPX1 acts as survival and defence mechanism in CCC required in an atypical environment prone to escape from immune surveillance.

Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes

Aggresomes are dynamic structures formed when the ubiquitin–proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6 h. However, at longer times (18 h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with γ-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.