The last enzyme (LytB) of the methylerythritol phosphate pathway for isoprenoid biosynthesis catalyzes the reduction of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into isopentenyl diphosphate and dimethylallyl diphosphate. This enzyme possesses a dioxygen-sensitive [4Fe-4S] cluster. This prosthetic group was characterized in the Escherichia coli enzyme by UV/visible and electron paramagnetic resonance spectroscopy after reconstitution of the purified protein. Enzymatic activity required the presence of a reducing system such as flavodoxin/flavodoxin reductase/reduced nicotinamide adenine dinucleotide phosphate or the photoreduced deazaflavin radical.

Classical nicotinamide adenine dinucleotide (NAD+) binding proteins contain a beta alpha beta alpha beta unit. By comparing 14 such proteins, it is observed that an additional beta strand associates with this unit to form the "core" topology, the minimum structure necessary to bind cofactor. Although the overall topologies of the cofactor binding domains of nicotinamide binding proteins vary, they all contain at least the core topology. The first 30-35 amino acids of the core topology, called the "fingerprint" region, are diagnostic for the presence of a dinucleotide binding fold. There are four characteristics of this fingerprint region: 1) a phosphate binding consensus sequence, GXGXXG, 2) six positions usually occupied by small hydrophobic amino acids, 3) a conserved, negatively charged residue (Glu or Asp) at the end of the second beta strand of the fingerprint region, and 4) a conserved positively charged residue (Arg or Lys) at the beginning of the first beta strand of the fingerprint region. The core topologies of the classical nicotinamide binding proteins overlap well with root mean squared deviations of main chain atoms ranging from 0.7 to 4.7 A. A conserved interaction (found in 8 of the 12 classical nicotinamide binding proteins studied) between the cofactor and the protein is a hydrogen bond between the pyrophosphate oxygen of NAD(P)+ and the carboxy-terminal glycine of the phosphate binding helix, the first alpha helix of the beta alpha beta alpha beta unit. The classical nicotinamide binding proteins all bind their cofactor in the same location and orientation, with the cofactor itself adopting a similar extended conformation in every structure. Although observed less frequently than the classical fold, numerous nonclassical folding patterns are also used by proteins that bind NAD(P)+.

Human neutrophils have a short half-life and are believed to die by apoptosis or programmed cell death both in vivo and in vitro. We found that caspases are activated in a time-dependent manner in neutrophils undergoing spontaneous apoptosis, concomitant with other characteristic features of apoptotic cell death such as morphologic changes, phosphatidylserine (PS) exposure, and DNA fragmentation. The treatment of neutrophils with agonistic anti-Fas monoclonal antibodies (MoAbs) significantly accelerated this process. However, in cells treated with the potent neutrophil activator phorbol 12-myristate 13-acetate (PMA), caspase activity was only evident after pharmacologic inhibition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Similarily, inhibition of the NADPH oxidase in constitutive and Fas/APO-1-triggered apoptosis resulted in increased rather than suppressed levels of caspase activity, suggesting that reactive oxygen species may prevent caspases from functioning optimally in these cells. Moreover, oxidants generated via the NADPH oxidase were essential for PS exposure during PMA-induced cell death, but not for neutrophils undergoing spontaneous apoptosis. We conclude that caspases are an important component of constitutive and Fas/APO-1-triggered neutrophil apoptosis. However, these redox sensitive enzymes are suppressed in activated neutrophils, and an alternate oxidant-dependent pathway is used to mediate PS exposure and neutrophil clearance under these conditions.

Chronic granulomatous disease (CGD) is an immunodeficiency caused by the lack of the superoxide-producing phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. However, CGD patients not only suffer from recurrent infections, but also present with inflammatory, non-infectious conditions. Among the latter, granulomas figure prominently, which gave the name to the disease, and colitis, which is frequent and leads to a substantial morbidity. In this paper, we systematically review the inflammatory lesions in different organs of CGD patients and compare them to observations in CGD mouse models. In addition to the more classical inflammatory lesions, CGD patients and their relatives have increased frequency of autoimmune diseases, and CGD mice are arthritis-prone. Possible mechanisms involved in CGD hyperinflammation include decreased degradation of phagocytosed material, redox-dependent termination of proinflammatory mediators and/or signaling, as well as redox-dependent cross-talk between phagocytes and lymphocytes (e.g. defective tryptophan catabolism). As a conclusion from this review, we propose the existence of ROS high and ROS low inflammatory responses, which are triggered as a function of the level of reactive oxygen species and have specific characteristics in terms of physiology and pathophysiology.

Sirtuin enzymes are a family of highly conserved protein deacetylases that depend on nicotinamide adenine dinucleotide (NAD+) for their activity. There are seven sirtuins in mammals and these proteins have been linked with caloric restriction and aging by modulating energy metabolism, genomic stability and stress resistance. Sirtuin enzymes are potential therapeutic targets in a variety of human diseases including cancer, diabetes, inflammatory disorders and neurodegenerative disease. Modulation of sirtuin activity has been shown to impact the course of several aggregate-forming neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and spinal and bulbar muscular atrophy. Sirtuins can influence the progression of neurodegenerative disorders by modulating transcription factor activity and directly deacetylating proteotoxic species. Here, we describe sirtuin protein targets in several aggregate-forming neurodegenerative diseases and discuss the therapeutic potential of compounds that modulate sirtuin activity in these disorders.

Nicotinamide adenine dinucleotide (NADPH) oxidases have been shown to be involved in various differentiation processes in fungi. We investigated the role of two NADPH oxidases in the necrotrophic phytopathogenic fungus, Botrytis cinerea. The genes bcnoxA and bcnoxB were cloned and characterized; their deduced amino acid sequences show high homology to fungal NADPH oxidases. Analyses of single and double knock-out mutants of both NADPH oxidase genes showed that both bcnoxA and bcnoxB are involved in formation of sclerotia. Both genes have a great impact on pathogenicity: whereas bcnoxB mutants showed a retarded formation of primary lesions, probably due to an impaired formation of penetration structures, bcnoxA mutants were able to penetrate host tissue in the same way as the wild type but were much slower in colonizing the host tissue. Double mutants showed an additive effect: they were aberrant in penetration and colonization of plant tissue and, therefore, almost nonpathogenic. To study the structure of the fungal Nox complex in more detail, bcnoxR (encoding a homolog of the mammalian p67(phox), a regulatory subunit of the Nox complex) was functionally characterized. The phenotype of DeltabcnoxR mutants is identical to that of DeltabcnoxAB double mutants, providing evidence that BcnoxR is involved in activation of both Bcnox enzymes.

Duchenne muscular dystrophy (DMD) is a fatal degenerative muscle disease resulting from mutations in the dystrophin gene. Increased oxidative stress and altered Ca(2+) homeostasis are hallmarks of dystrophic muscle. While impaired autophagy has recently been implicated in the disease process, the mechanisms underlying the impairment have not been elucidated. Here we show that nicotinamide adenine dinucleotide phosphatase (Nox2)-induced oxidative stress impairs both autophagy and lysosome formation in mdx mice. Persistent activation of Src kinase leads to activation of the autophagy repressor mammalian target of rapamycin (mTOR) via PI3K/Akt phosphorylation. Inhibition of Nox2 or Src kinase reduces oxidative stress and partially rescues the defective autophagy and lysosome biogenesis. Genetic downregulation of Nox2 activity in the mdx mouse decreases reactive oxygen species (ROS) production, abrogates defective autophagy and rescues histological abnormalities and contractile impairment. Our data highlight mechanisms underlying the pathogenesis of DMD and identify NADPH oxidase and Src kinase as potential therapeutic targets.

BACKGROUND

Postischemic administration of volatile anesthetics activates reperfusion injury salvage kinases and decreases myocardial damage. However, the mechanisms underlying anesthetic postconditioning are unclear.

METHODS

Isolated perfused rat hearts were exposed to 40 min of ischemia followed by 1 h of reperfusion. Anesthetic postconditioning was induced by 15 min of 2.1 vol% isoflurane (1.5 minimum alveolar concentration) administered at the onset of reperfusion. In some experiments, atractyloside (10 microm), a mitochondrial permeability transition pore (mPTP) opener, and LY294002 (15 microm), a phosphatidylinositol 3-kinase inhibitor, were coadministered with isoflurane. Western blot analysis was used to determine phosphorylation of protein kinase B/Akt and its downstream target glycogen synthase kinase 3beta after 15 min of reperfusion. Myocardial tissue content of nicotinamide adenine dinucleotide served as a marker for mPTP opening. Accumulation of MitoTracker Red 580 (Molecular Probes, Invitrogen, Basel, Switzerland) was used to visualize mitochondrial function.

RESULTS

Anesthetic postconditioning significantly improved functional recovery and decreased infarct size (36 +/- 1% in unprotected hearts vs. 3 +/- 2% in anesthetic postconditioning; P < 0.05). Isoflurane-mediated protection was abolished by atractyloside and LY294002. LY294002 inhibited isoflurane-induced phosphorylation of protein kinase B/Akt and glycogen synthase kinase 3beta and opened mPTP as determined by nicotinamide adenine dinucleotide measurements. Atractyloside, a direct opener of the mPTP, did not inhibit phosphorylation of protein kinase B/Akt and glycogen synthase kinase 3beta by isoflurane but reversed isoflurane-mediated cytoprotection. Microscopy showed accumulation of the mitochondrial tracker in isoflurane-protected functional mitochondria but no staining in mitochondria of unprotected hearts.

CONCLUSIONS

Anesthetic postconditioning by isoflurane effectively protects against reperfusion damage by preventing opening of the mPTP through inhibition of glycogen synthase kinase 3beta.

The nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1 is a major metabolic regulator activated by energy stresses such as fasting or calorie restriction. SIRT1 activation during fasting not only relies on the increase in the NAD(+)/NADH ratio caused by energy deprivation but also involves an upregulation of SIRT1 mRNA and protein levels in various metabolic tissues. We demonstrate that SIRT1 expression is controlled systemically by the activation of the cyclic AMP response-element-binding protein upon low nutrient availability. Conversely, in the absence of energetic stress, the carbohydrate response-element-binding protein represses the expression of SIRT1. Altogether, these results demonstrate that SIRT1 expression is tightly controlled at the transcriptional level by nutrient availability and further underscore that SIRT1 is a crucial metabolic checkpoint connecting the energetic status with transcriptional programmes.

Nicotinamide adenine dinucleotide (NAD) is a central metabolic cofactor by virtue of its redox capacity, and as such regulates a wealth of metabolic transformations. However, the identification of the longevity protein silent regulator 2 (Sir2), the founding member of the sirtuin protein family, as being NAD⁺-dependent reignited interest in this metabolite. The sirtuins (SIRT1-7 in mammals) utilize NAD⁺ to deacetylate proteins in different subcellular compartments with a variety of functions, but with a strong convergence on optimizing mitochondrial function. Since cellular NAD⁺ levels are limiting for sirtuin activity, boosting its levels is a powerful means to activate sirtuins as a potential therapy for mitochondrial, often age-related, diseases. Indeed, supplying excess precursors, or blocking its utilization by poly(ADP-ribose) polymerase (PARP) enzymes or CD38/CD157, boosts NAD⁺ levels, activates sirtuins and promotes healthy aging. Here, we discuss the current state of knowledge of NAD⁺ metabolism, primarily in relation to sirtuin function. We highlight how NAD⁺ levels change in diverse physiological conditions, and how this can be employed as a pharmacological strategy.

Beyond its antidiabetic activity justifying its use in the treatment of the type 2 diabetes, metformin (MET [dimethylguanidine, Glucophage]) has been shown to exhibit antioxidant properties in vitro, which could contribute to limit the deleterious vascular complications of diabetes. We investigated whether MET, at the pharmacological level of 10 -5 mol/L, was able to modulate intracellular production of reactive oxygen species (ROS) both in quiescent bovine aortic endothelial cells (BAECs) and in BAECs stimulated by a short incubation with high levels of glucose (30 mmol/L, 2 hours) or angiotensin II (10 -7 mol/L, 1 hour). Intracellular ROS production was measured by fluorescence of the DCF (2,7-dichlorodihydrofluorescein) probe. Our results showed that MET was able to reduce the intracellular production of ROS in both nonstimulated BAECs (-20%, P < .05) and BAEC stimulated by high levels of glucose or angiotensin II (-28% and -72%, respectively, P < .01). Experiments performed in the presence of the nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase inhibitor apocynin or the respiratory mitochondrial chain inhibitor rotenone indicated that MET exerted its effect partly through an inhibition of the formation of ROS produced mainly by NAD(P)H oxidase and also, to a lesser extent, by the respiratory mitochondrial chain.

A neurodegenerative tauopathy endemic to the Caribbean island of Guadeloupe has been associated with the consumption of anonaceous plants that contain acetogenins, potent lipophilic inhibitors of complex I of the mitochondrial respiratory chain. To test the hypothesis that annonacin, a prototypical acetogenin, contributes to the etiology of the disease, we investigated whether annonacin affects the cellular distribution of the protein tau. In primary cultures of rat striatal neurons treated for 48 h with annonacin, there was a concentration-dependent decrease in ATP levels, a redistribution of tau from the axons to the cell body, and cell death. Annonacin induced the retrograde transport of mitochondria, some of which had tau attached to their outer membrane. Taxol, a drug that displaces tau from microtubules, prevented the somatic redistribution of both mitochondria and tau but not cell death. Antioxidants, which scavenged the reactive oxygen species produced by complex I inhibition, did not affect either the redistribution of tau or cell death. Both were prevented, however, by forced expression of the NDI1 nicotinamide adenine dinucleotide (NADH)-quinone-oxidoreductase of Saccharomyces cerevisiae, which can restore NADH oxidation in complex I-deficient mammalian cells and stimulation of energy production via anaerobic glycolysis. Consistently, other ATP-depleting neurotoxins (1-methyl-4-phenylpyridinium, 3-nitropropionic, and carbonyl cyanide m-chlorophenylhydrazone) reproduced the somatic redistribution of tau, whereas toxins that did not decrease ATP levels did not cause the redistribution of tau. Therefore, the annonacin-induced ATP depletion causes the retrograde transport of mitochondria to the cell soma and induces changes in the intracellular distribution of tau in a way that shares characteristics with some neurodegenerative diseases.

Numerous studies indicate that Sirtuin 1 (SIRT1), a mammalian nicotinamide adenine dinucleotide (NAD(+))-dependent histone deacetylase (HDAC), plays a crucial role in p53-mediated stress responses by deacetylating p53. Nevertheless, the acetylation levels of p53 are dramatically increased upon DNA damage, and it is not well understood how the SIRT1-p53 interaction is regulated during the stress responses. Here, we identified Set7/9 as a unique regulator of SIRT1. SIRT1 interacts with Set7/9 both in vitro and in vivo. In response to DNA damage in human cells, the interaction between Set7/9 and SIRT1 is significantly enhanced and coincident with an increase in p53 acetylation levels. Importantly, the interaction of SIRT1 and p53 is strongly suppressed in the presence of Set7/9. Consequently, SIRT1-mediated deacetylation of p53 is abrogated by Set7/9, and p53-mediated transactivation is increased during the DNA damage response. Of note, whereas SIRT1 can be methylated at multiple sites within its N terminus by Set7/9, a methylation-defective mutant of SIRT1 still retains its ability to inhibit p53 activity. Taken together, our results reveal that Set7/9 is a critical regulator of the SIRT1-p53 interaction and suggest that Set7/9 can modulate p53 function indirectly in addition to acting through a methylation-dependent mechanism.

Peroxisomes are subcellular respiratory organelles which contain catalase and H2O2-producing flavin oxidases as basic enzymatic constituents. These organelles have an essentially oxidative type of metabolism and have the potential to carry out different important metabolic pathways. In recent years the presence of different types of superoxide dismutase (SOD) have been demonstrated in peroxisomes from several plant species, and more recently the occurrence of SOD has been extended to peroxisomes from human and transformed yeast cells. A copper,zinc-containing SOD from plant peroxisomes has been purified and partially characterized. The production of hydroxyl and superoxide radicals has been studied in peroxisomes. There are two sites of O2- production in peroxisomes: (1) in the matrix, the generating system being xanthine oxidase; and (2) in peroxisomal membranes, dependent on reduced nicotinamide adenine dinucleotide (NADH), and the electron transport components of the peroxisomal membrane are possibly responsible. The generation of oxygen radicals in peroxisomes could have important effects on cellular metabolism. Diverse cellular implications of oxyradical metabolism in peroxisomes are discussed in relation to phenomena such as cell injury, peroxisomal genetic diseases, peroxisome proliferation and oxidative stress, metal and salt stress, catabolism of nucleic acids, senescence, and plant pathogenic processes.

The renin-angiotensin-aldosterone system contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. Angiotensin II and aldosterone (corticosterone in rodents) together generate reactive oxygen species (ROS) via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which likely facilitate this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo mineralocorticoid receptor (MR) blockade in a rodent model of the chronically elevated tissue renin-angiotensin-aldosterone system, the transgenic TG (mRen2) 27 rat (Ren2). The Ren2 overexpresses the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6- to 7-wk-old) male Ren2 and age-matched Sprague-Dawley rats were treated with spironolactone or placebo for 3 wk. Heart tissue ROS, immunohistochemical analysis of 3-nitrotyrosine, and NADPH oxidase (NOX) subunits (gp91(phox) recently renamed NOX2, p22(phox), Rac1, NOX1, and NOX4) were measured. Structural changes were assessed with cine-magnetic resonance imaging, transmission electron microscopy, and light microscopy. Significant increases in Ren2 septal wall thickness (cine-magnetic resonance imaging) were accompanied by perivascular fibrosis, increased mitochondria, and other ultrastructural changes visible by light microscopy and transmission electron microscopy. Although there was no significant reduction in systolic blood pressure, significant improvements were seen with MR blockade on ROS formation and NOX subunits (each P < 0.05). Collectively, these data suggest that MR blockade, independent of systolic blood pressure reduction, improves cardiac oxidative stress-induced structural and functional changes, which are driven, in part, by angiotensin type 1 receptor-mediated increases in NOX.

CONCLUSIONS

Reactive oxygen species (ROS) are signaling molecules that are important in physiological processes, including host defense, aging, and cellular homeostasis. Increased ROS bioavailability and altered redox signaling (oxidative stress) have been implicated in the onset and/or progression of chronic diseases, including hypertension.

BACKGROUND

Although oxidative stress may not be the only cause of hypertension, it amplifies blood pressure elevation in the presence of other pro-hypertensive factors, such as salt loading, activation of the renin-angiotensin-aldosterone system, and sympathetic hyperactivity, at least in experimental models. A major source for ROS in the cardiovascular-renal system is a family of nicotinamide adenine dinucleotide phosphate oxidases (Noxs), including the prototypic Nox2-based Nox, and Nox family members: Nox1, Nox4, and Nox5.

RESULTS

Although extensive experimental data support a role for increased ROS levels and altered redox signaling in the pathogenesis of hypertension, the role in clinical hypertension is unclear, as a direct causative role of ROS in blood pressure elevation has yet to be demonstrated in humans. Nevertheless, what is becoming increasingly evident is that abnormal ROS regulation and aberrant signaling through redox-sensitive pathways are important in the pathophysiological processes which is associated with vascular injury and target-organ damage in hypertension.

CONCLUSIONS

There is a paucity of clinical information related to the mechanisms of oxidative stress and blood pressure elevation, and a few assays accurately measure ROS directly in patients. Such further ROS research is needed in humans and in the development of adequately validated analytical methods to accurately assess oxidative stress in the clinic.

OBJECTIVE

Nicotinamide adenine dinucleotides (NAD+ and NADH) play a crucial role in cellular energy metabolism, and a dysregulated NAD+-to-NADH ratio is implicated in metabolic syndrome. However, it is still unknown whether a modulating intracellular NAD+-to-NADH ratio is beneficial in treating metabolic syndrome. We tried to determine whether pharmacological stimulation of NADH oxidation provides therapeutic effects in rodent models of metabolic syndrome.

METHODS

We used beta-lapachone (betaL), a natural substrate of NADH:quinone oxidoreductase 1 (NQO1), to stimulate NADH oxidation. The betaL-induced pharmacological effect on cellular energy metabolism was evaluated in cells derived from NQO1-deficient mice. In vivo therapeutic effects of betaL on metabolic syndrome were examined in diet-induced obesity (DIO) and ob/ob mice.

RESULTS

NQO1-dependent NADH oxidation by betaL strongly provoked mitochondrial fatty acid oxidation in vitro and in vivo. These effects were accompanied by activation of AMP-activated protein kinase and carnitine palmitoyltransferase and suppression of acetyl-coenzyme A (CoA) carboxylase activity. Consistently, systemic betaL administration in rodent models of metabolic syndrome dramatically ameliorated their key symptoms such as increased adiposity, glucose intolerance, dyslipidemia, and fatty liver. The treated mice also showed higher expressions of the genes related to mitochondrial energy metabolism (PPARgamma coactivator-1alpha, nuclear respiratory factor-1) and caloric restriction (Sirt1) consistent with the increased mitochondrial biogenesis and energy expenditure.

CONCLUSIONS

Pharmacological activation of NADH oxidation by NQO1 resolves obesity and related phenotypes in mice, opening the possibility that it may provide the basis for a new therapy for the treatment of metabolic syndrome.

Insulin resistance (IR) underlines aging and aging-associated medical (diabetes, obesity, dyslipidemia, hypertension) and psychiatric (depression, cognitive decline) disorders. Molecular mechanisms of IR in genetically or metabolically predisposed individuals remain uncertain. Current review of the literature and our data presents the evidences that dysregulation of tryptophan (TRP)-kynurenine (KYN) and KYN-nicotinamide adenine dinucleotide (NAD) metabolic pathways is one of the mechanisms of IR. The first and rate-limiting step of TRP-KYN pathway is regulated by enzymes inducible by pro-inflammatory factors and/or stress hormones. The key enzymes of KYN-NAD pathway require pyridoxal-5-phosphate (P5P), an active form of vitamin B6, as a cofactor. Deficiency of P5P diverts KYN-NAD metabolism from production of NAD to the excessive formation of xanthurenic acid (XA). Human and experimental studies suggested that XA and some other KYN metabolites might impair production, release, and biological activity of insulin. We propose that one of the mechanisms of IR is inflammation- and/or stress-induced upregulation of TRP-KYN metabolism in combination with P5P deficiency-induced diversion of KYN-NAD metabolism towards formation of XA and other KYN derivatives affecting insulin activity. Monitoring of KYN/P5P status and formation of XA might help to identify subjects at risk for IR. Pharmacological regulation of the TRP-KYN and KYN-NAD pathways and maintaining of adequate vitamin B6 status might contribute to prevention and treatment of IR in conditions associated with inflammation/stress-induced excessive production of KYN and deficiency of vitamin B6, e.g., type 2 diabetes, obesity, cardiovascular diseases, aging, menopause, pregnancy, and hepatitis C virus infection.

CONCLUSIONS

Stroke, a leading cause of death and disability, poses a substantial burden for patients, relatives, and our healthcare systems. Only one drug is approved for treating stroke, and more than 30 contraindications exclude its use in 90% of all patients. Thus, new treatments are urgently needed. In this review, we discuss oxidative stress as a pathomechanism of poststroke neurodegeneration and the inhibition of its source, type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4), as a conceptual breakthrough in stroke therapy.

BACKGROUND

Among potential sources of reactive oxygen species (ROS), the NOXes stand out as the only enzyme family that is solely dedicated to forming ROS. In rodents, three cerebrovascular NOXes exist: the superoxide-forming NOX1 and 2 and the hydrogen peroxide-forming NOX4. Studies using NOX1 knockout mice gave conflicting results, which overall do not point to a role for this isoform. Several reports find NOX2 to be relevant in stroke, albeit to variable and moderate degrees. In our hands, NOX4 is, by far, the major source of oxidative stress and neurodegeneration on ischemic stroke.

RESULTS

We critically discuss the tools that have been used to validate the roles of NOX in stroke. We also highlight the relevance of different animal models and the need for advanced quality control in preclinical stroke research.

CONCLUSIONS

The development of isoform-specific NOX inhibitors presents a precious tool for further clarifying the role and drugability of NOX homologues. This could pave the avenue for the first clinically effective neuroprotectant applied poststroke, and even beyond this, stroke could provide a proof of principle for antioxidative stress therapy.

Synaptic ribbons are large, dynamic structures in the active zone complex of ribbon synapses and important for the physiological properties of these tonically active synapses. RIBEYE is a unique and major protein component of synaptic ribbons. The aim of the present study was to understand how the synaptic ribbon is built and how the construction of the ribbon could contribute to its ultrastructural plasticity. In the present study, we demonstrate that RIBEYE self-associates using different independent approaches (yeast two-hybrid analyses, protein pull downs, synaptic ribbon-RIBEYE interaction assays, coaggregation experiments, transmission electron microscopy and immunogold electron microscopy). The A-domain [RIBEYE(A)] and B-domain [RIBEYE(B)] of RIBEYE contain five distinct sites for RIBEYE-RIBEYE interactions. Three interaction sites are present in the A-domain of RIBEYE and mediate RIBEYE(A)-RIBEYE(A) homodimerization and heterodimerization with the B-domain. The docking site for RIBEYE(A) on RIBEYE(B) is topographically and functionally different from the RIBEYE(B) homodimerization interface and is negatively regulated by nicotinamide adenine dinucleotide. The identified multiple RIBEYE-RIBEYE interactions have the potential to build the synaptic ribbon: heterologously expressed RIBEYE forms large electron-dense aggregates that are in part physically associated with surrounding vesicles and membrane compartments. These structures resemble spherical synaptic ribbons. These ribbon-like structures coassemble with the active zone protein bassoon, an interaction partner of RIBEYE at the active zone of ribbon synapses, emphasizing the physiological relevance of these RIBEYE-containing aggregates. Based on the identified multiple RIBEYE-RIBEYE interactions, we provide a molecular mechanism for the dynamic assembly of synaptic ribbons from individual RIBEYE subunits.

CONCLUSIONS

The nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) and NADP+/reduced NADP+ (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD+-consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics.

RESULTS

The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism.

CONCLUSIONS

Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD+ precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.

Both DNA double- and single-strand break repair are highly coordinated processes utilizing signal transduction cascades and post-translational modifications such as phosphorylation, acetylation and ADP ribosylation. 'Drugable' targets within these networks have been identified that could potentially lead to novel therapeutic approaches within the oncology arena. Key regulators within these signalling cascades, such as DNA-dependent protein kinase, ataxia-telangiectasia mutated, checkpoint kinase 1 (CHK1), checkpoint kinase 2 (CHK2) and poly(ADP-ribose) polymerase, use either ATP or nicotinamide adenine dinucleotide for their enzymatic functions and are therefore readily accessible to small molecule inhibition at their catalytic sites. A range of highly potent and selective inhibitors of these DNA damage response pathways has now been identified through drug discovery efforts, with candidate molecules either approaching or already in clinical trials. This review will describe the small molecule inhibitors and drug discovery activities that focus on DNA break repair, along with the therapeutic rationale behind chemosensitization and the concept of synthetic lethality. We will also describe the emerging clinical data coming from this exciting new approach to targeted cancer therapy.

OBJECTIVE

Laser activated gold nanoshell thermal ablation represents a new, minimally invasive technology that offers benign tissue sparing thermal ablation of malignant tumors. We evaluated the efficacy of this technology for eradicating prostate cancer in a subcutaneous tumor model.

METHODS

The 110 nm gold nanoshells with a 10 nm gold shell are designed to act as intense near infrared absorbers. PC-3 cells were injected on the dorsum of nude mice in 3 groups, including 1-gold nanoshell plus near infrared laser, 2-saline alone and 3-near infrared laser alone. Animals received 7.0 ml/gm body weight (low dose) or 8.5 ml/gm body weight (high dose) nanoshells via tail vein injection. Control animals received saline. A 810 nm near infrared laser with a 200 mu laser fiber and an energy setting of 4 W/cm(2) was aimed at the tumor bed for 3 minutes. Tumors were measured at days 0, 7, 14 and 21. Tissue temperature was monitored during laser activation. Tumors were harvested at day 21 and stained with hematoxylin and eosin, and for nicotinamide adenine dinucleotide diaphorase activity.

RESULTS

We observed 93% tumor necrosis and regression in the high dose treated group. Nicotinamide adenine dinucleotide staining corroborated this finding. The ablation zone was sharply limited to the laser spot size. There was no difference in the size or tumor histology in control groups, indicating a benign course for near infrared laser treatment alone. Temperatures up to 65.4C were attained in the treated group.

CONCLUSIONS

Laser activated gold nanoshell ablation is an effective and selective technique for prostate cancer ablation in an ectopic murine tumor model.

BACKGROUND

Angiotensin II infusion has been shown to cause hypertension and endothelial dysfunction and to increase superoxide (O-.2) production in vascular tissue, mainly via an activation of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-dependent oxidase, the most significant O-.2 source in endothelial and/or smooth muscle cells. With these studies, we sought to determine whether endothelial dysfunction in renovascular hypertension is secondary to an activation of these oxidases.

METHODS

Endothelial function in aortas from rats with two kidney-one clip (2K-1C) hypertension and age-matched controls was assessed using isometric tension studies in organ chambers. Changes in vascular O-.2 production were measured using lucigenin-enhanced chemiluminescence and electron spin resonance spectroscopy.

RESULTS

In hypertensive animals, relaxation to endothelium-dependent (acetylcholine) and endothelium-independent nitrovasodilators (nitroglycerin) was impaired. Constriction to a direct activator of protein kinase C (PKC) phorbol ester 12,13 dibutyrate (PDBu) was enhanced, and vascular O-.2 was significantly increased compared with controls. Vascular O-.2 was normalized by the PKC inhibitor calphostin C, by the inhibitor of flavin-dependent oxidases, diphenylene iodonium, and recombinant heparin-binding superoxide dismutase, whereas inhibitors of the xanthine oxidase (oxypurinol), nitric oxide synthase (NG-nitro-l-arginine) and mitochondrial NADH dehydrogenase (rotenone) were ineffective. Studies of vascular homogenates demonstrated that the major source of O-.2 was a NAD(P)H-dependent oxidase. Incubation of intact tissue with PDBu markedly increased O-. 2, the increase being significantly stronger in vessels from hypertensive animals as compared with vessels from controls. Endothelial dysfunction was improved by preincubation of vascular tissue with superoxide dismutase and calphostin C.

CONCLUSIONS

We therefore conclude that renovascular hypertension in 2K-1C rats is associated with increased vascular O-.2 leading to impaired vasodilator responses to endogenous and exogenous nitrovasodilators. Increased vascular O-.2 is likely secondary to a PKC-mediated activation of a membrane-associated NAD(P)H-dependent oxidase.