The heat-labile enterotoxin of Escherichia coli, like cholera toxin, activates adenylate cyclase by catalyzing the transfer of adenosine diphosphate-ribose from HAD+ (oxidized nicotinamide adenine dinucleotide) to the guanyl nucleotide-dependent regulatory component of the cyclase. A preparation of enterotoxin that had been released from E. coli following exposure to polymyxin B and then partially purified was found to contain two enzymatically active peptides, one of about 29,000 and the other of about 24,000 daltons, which correspond in molecular size to the enzymatically active subunit A and fragment A1 of cholera toxin, respectively. As with cholera toxin, the enzymatic activity of E. coli enterotoxin was elevated by incubation with sodium dodecyl sulfate to release active peptides. Treatment with dithiothreitol, however, had no effect. Dithiothreitol activates subunit A of cholera toxin by reducing an internal disulfide bond, but no corresponding bond appears to be present in the partially purified E. coli enterotoxin.

An l-(+)-lactate dehydrogenase was purified approximately 35-fold from crude extracts of Streptococcus faecalis. The purified enzyme had an absolute and specific requirement for fructose-1,6-diphosphate (FDP) for catalytic activity. The concentration of FDP required for 50% maximal activity was about 0.045 mm. The activator was bound to the enzyme more effectively at pH 5.8 than it was at a neutral or alkaline pH. Activation appeared to involve a conformational change in the enzyme which made the substrate and coenzyme sites more accessible to the respective reactants. Among the evidence supporting this hypothesis was the fact that FDP lowered significantly the apparent K(m) for both pyruvate and reduced nicotinamide adenine dinucleotide. Moreover, the enzyme, which was quite heat stable in the absence of any of the reactants, was rendered heat labile by FDP.

Muscle atrophy (cachexia) is a muscle wasting syndrome associated with several pathological conditions in humans such as congestive heart failure, diabetes, AIDS, cancer and renal failure, and the presence of cachexia worsens outcome. Many of the conditions associated with cachexia are accompanied by stimulation of the renin-angiotensin system and elevation in angiotensin II (ang II) levels. Ang II infusion induces skeletal muscle atrophy in rodents and mechanisms include increased expression of the E3 ligases atrogin-1/MuRF-1, an elevated rate of ubiquitin-proteasome mediated proteolysis and increased reactive oxygen species (ROS) levels, closely mimicking conditions of human cachexia. Ang II-induced oxidative stress contributes to muscle atrophy in a mouse model. Nicotinamide adenine dinucleotide phosphate oxidase- and mitochondria-derived ROS contribute to ang II-induced oxidative stress. Specific targeting of ROS and nicotinamide adenine dinucleotide phosphate oxidase/mitochondria cross-talk could be a beneficial, novel therapy to treat cachexia.

This narrative review summarizes the role of vitamin C in mitigating oxidative injury-induced microcirculatory impairment and associated organ failure in ischemia/reperfusion or sepsis. Preclinical studies show that high-dose vitamin C can prevent or restore microcirculatory flow impairment by inhibiting activation of nicotinamide adenine dinucleotide phosphate-oxidase and inducible nitric oxide synthase, augmenting tetrahydrobiopterin, preventing uncoupling of oxidative phosphorylation, and decreasing the formation of superoxide and peroxynitrite, and by directly scavenging superoxide. Vitamin C can additionally restore vascular responsiveness to vasoconstrictors, preserve endothelial barrier by maintaining cyclic guanylate phosphatase and occludin phosphorylation and preventing apoptosis. Finally, high-dose vitamin C can augment antibacterial defense. These protective effects against overwhelming oxidative stress due to ischemia/reperfusion, sepsis or burn seems to mitigate organ injury and dysfunction, and promote recovery after cardiac revascularization and in critically ill patients, in the latter partially in combination with other antioxidants. Of note, several questions remain to be solved, including optimal dose, timing and combination of vitamin C with other antioxidants. The combination obviously offers a synergistic effect and seems reasonable during sustained critical illness. High-dose vitamin C, however, provides a cheap, strong and multifaceted antioxidant, especially robust for resuscitation of the circulation. Vitamin C given as early as possible after the injurious event, or before if feasible, seems most effective. The latter could be considered at the start of cardiac surgery, organ transplant or major gastrointestinal surgery. Preoperative supplementation should consider the inhibiting effect of vitamin C on ischemic preconditioning. In critically ill patients, future research should focus on the use of short-term high-dose intravenous vitamin C as a resuscitation drug, to intervene as early as possible in the oxidant cascade in order to optimize macrocirculation and microcirculation and limit cellular injury.

Cardiovascular disease (CVD) remains the leading cause of morbidity and premature mortality in both women and men in most industrialized countries, and has for some time also established a prominent role in developing nations. In fact, obesity, diabetes mellitus and hypertension are now commonplace even in children and youths. Regular exercise is rapidly gaining widespread advocacy as a preventative measure in schools, medical circles and in the popular media. There is overwhelming evidence garnered from a number of sources, including epidemiological, prospective cohort and intervention studies, suggesting that CVD is largely a disease associated with physical inactivity. A rapidly advancing body of human and animal data confirms an important beneficial role for exercise in the prevention and treatment of CVD. In Part 1 of this review we discuss the impact of exercise on CVD, and we highlight the effects of exercise on (i) endothelial function by regulation of endothelial genes mediating oxidative metabolism, inflammation, apoptosis, cellular growth and proliferation, increased superoxide dismutase (SOD)-1, down-regulation of p67phox, changes in intracellular calcium level, increased vascular endothelial nitric oxide synthase (eNOS), expression and eNOS Ser-1177 phosphorylation; (ii) vascular smooth muscle function by either an increased affinity of the Ca2+ extrusion mechanism or an augmented Ca2+ buffering system by the superficial sarcoplasmic reticulum to increase Ca2+ sequestration, increase in K+ channel activity and/or expression, and increase in L-type Ca2+ current density; (iii) antioxidant systems by elevation of Mn-SOD, Cu/Zn-SOD and catalase, increases in glutathione peroxidase activity and activation of vascular nicotinamide adenine dinucleotide phosphate [(NAD(P)H] oxidase and p22phox expression; (iv) heat shock protein (HSP) expression by stimulating HSP70 expression in myocardium, skeletal muscle and even in human leucocytes, probably through heat shock transcription factor 1 activity; (v) inflammation by reducing serum inflammatory cytokines such as high-sensitivity C-reactive protein (hCRP), interleukin (IL)-6, IL-18 and tumour necrosis factor-alpha and by regulating Toll-like receptor 4 pathway. Exercise also alters vascular remodelling, which involves two forms of vessel growth including angiogenesis and arteriogenesis. Angiogenesis refers to the formation of new capillary networks. Arteriogenesis refers to the growth of pre-existent collateral arterioles leading to formation of large conductance arteries that are well capable to compensate for the loss of function of occluded arteries. Another aim of this review is to focus on exercise-related cardiovascular protection against CVD and associated risk factors such as aging, coronary heart disease, hypertension, heart failure, diabetes mellitus and peripheral arterial diseases mediated by vascular remodelling. Lastly, this review examines the benefits of exercise in mitigating pre-eclampsia during pregnancy by mechanisms that include improved blood flow, reduced blood pressure, enhanced placental growth and vascularity, increased activity of antioxidant enzymes, reduced oxidative stress and restored vascular endothelial dysfunction.

CD38 is a membrane-associated ecto-nicotinamide adenine dinucleotide (NAD+) glycohydrolase that is expressed on multiple hematopoietic cells. The extracellular domain of CD38 can mediate the catalysis of NAD+ to cyclic adenosine diphosphoribose (cADPR), a Ca2+-mobilizing second messenger, adenosine diphosphoribose (ADPR), and nicotinamide. In addition to its enzymatic properties, murine CD38 has been shown to act as a B-cell coreceptor capable of modulating signals through the B-cell antigen receptor. To investigate the in vivo physiological function(s) of this novel class of ectoenzyme we generated mice carrying a null mutation in the CD38 gene. CD38-/- mice showed a complete loss of tissue-associated NAD+ glycohydrolase activity, showing that the classical NAD+ glycohydrolases and CD38 are likely identical. Although murine CD38 is expressed on hematopoietic stem cells as well as on committed progenitors, we show that CD38 is not required for hematopoiesis or lymphopoiesis. However, CD38-/- mice did exhibit marked deficiencies in antibody responses to T-cell-dependent protein antigens and augmented antibody responses to at least one T-cell-independent type 2 polysaccharide antigen. These data suggest that CD38 may play an important role in vivo in regulating humoral immune responses.

Platelets play a crucial role in the physiology of primary hemostasis and pathophysiologic processes such as arterial thrombosis. Accumulating evidence suggests a role of reactive oxygen species (ROSs) in platelet activation. Here we show that platelets activated with different agonists produced intracellular ROSs, which were reduced by reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase inhibitors and superoxide scavengers. In addition, we demonstrate that ROSs produced in platelets significantly affected alphaIIbbeta3 integrin activation but not alpha and dense granule secretion and platelet shape change. Thrombin-induced integrin alphaIIbbeta3 activation was significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors (diphenylene iodonium [DPI] [45% +/- 9%] and apocynin [43% +/- 11%]) and superoxide scavengers (tiron [60% +/- 9%] and Mn(III)tetrakis (1-methyl-4-pyridyl)porphyrin [MnTMPyP] [70% +/- 6%]). These inhibitors also reduced platelet aggregation and thrombus formation on collagen under high shear and achieved their effects independent of the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway.

Clinical studies have shown that in vivo fluorescence spectroscopy can improve the diagnosis of cervical precancer. Recent work suggests that epithelial fluorescence increases, whereas stromal fluorescence decreases, with precancer. However, the microanatomic and biochemical sources of fluorescence in living cervical tissue have not yet been established. This study aims to characterize the origins of living normal and precancerous cervical fluorescence at microscopic levels using laser-scanning fluorescence confocal microscopy. Ten pairs of colposcopically normal and abnormal biopsies were obtained; transverse, 200 microm thick, short-term tissue cultures were prepared and imaged when viable with UV (351-364 nm) and 488 nm excitation before and after addition of the vital dye, Mitotracker Orange. In normal epithelium basal epithelial cells showed cytoplasmic fluorescence; parabasal, intermediate and superficial cells showed fluorescence only at the periphery of the cell. In low-grade precancers cytoplasmic fluorescence was visible in the bottom one-third of the epithelium; in high-grade precancers cytoplasmic fluorescence was visible throughout the lower two-thirds of the epithelium. Cytoplasmic fluorescence was colocalized with the MitoTracker probe and is attributed to mitochondrial reduced form of nicotinamide adenine dinucleotide at UV excitation and mitochondrial flavin adenine dinucleotide at 488 nm excitation. Stromal fluorescence originated from matrix fibers; with the development of precancer the density and fluorescence intensity of matrix fibers decrease. Autofluorescence properties of precancerous cervix reflect an increased number of metabolically active mitochondria in epithelial cells and a reduced stromal fluorescence, which can be an indicator for altered communication between precancerous epithelium and stroma. These changes can explain differences in in vivo fluorescence spectra of normal and precancerous cervical tissue.

Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II-dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22(phox)) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria.

Increasing evidence shows that reactive oxygen species (ROS) may be critical mediators of liver damage during the relative hypoxia of ischemia/reperfusion injury (IRI) associated with transplant surgery or of the tissue microenvironment created as a result of chronic hepatic inflammation or infection. Much work has been focused on Kupffer cells or liver resident macrophages with respect to the generation of ROS during IRI. However, little is known about the contribution of endogenous hepatocyte ROS production or its potential impact on the parenchymal cell death associated with IRI and chronic hepatic inflammation. For the first time, we show that human hepatocytes isolated from nondiseased liver tissue and human hepatocytes isolated from diseased liver tissue exhibit marked differences in ROS production in response to hypoxia/reoxygenation (H-R). Furthermore, several different antioxidants are able to abrogate hepatocyte ROS-induced cell death during hypoxia and H-R. These data provide clear evidence that endogenous ROS production by mitochondria and nicotinamide adenine dinucleotide phosphate oxidase drives human hepatocyte apoptosis and necrosis during hypoxia and H-R and may therefore play an important role in any hepatic diseases characterized by a relatively hypoxic liver microenvironment. In conclusion, these data strongly suggest that hepatocytes and hepatocyte-derived ROS are active participants driving hepatic inflammation. These novel findings highlight important functional/metabolic differences between hepatocytes isolated from normal donor livers, hepatocytes isolated from normal resected tissue obtained during surgery for malignant neoplasms, and hepatocytes isolated from livers with end-stage disease. Furthermore, the targeting of hepatocyte ROS generation with antioxidants may offer therapeutic potential for the adjunctive treatment of IRI and chronic inflammatory liver diseases.

OBJECTIVE

Resveratrol activates Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent deacetylase which modulates metabolic homeostasis and improves several pathophysiological features present in diseases of ageing. In particular, it has been shown that SIRT1 activation improves endothelial dysfunction and suppresses vascular inflammation, two central pathophysiological processes involved in the initiation and progression of cardiovascular disease. The downstream targets of SIRT1 activation in this context, however, remain poorly defined. Therefore, in this study, we aimed to characterize mechanistically how SIRT1 activation regulates the endothelial vasoprotective phenotype.

RESULTS

We demonstrate that SIRT1 activation by resveratrol increases the expression of the transcription factor Krüppel-like factor 2 (KLF2) in human vascular endothelial cells, resulting in the orchestrated regulation of transcriptional programs critical for conferring an endothelial vasoprotective phenotype. Moreover, we show that KLF2 upregulation by resveratrol occurs via a mitogen-activated protein kinase 5/myocyte enhancing factor 2-dependent signalling pathway.

CONCLUSIONS

Collectively, these observations provide a new mechanistic framework to understand the vascular protective effects mediated by SIRT1 activators and define KLF2 as a critical mediator of these effects.

Transcription is regulated by acetylation/deacetylation reactions of histone and nonhistone proteins mediated by enzymes called KATs and HDACs, respectively. As a major mechanism of transcriptional regulation, protein acetylation is a key controller of physiological processes such as cell cycle, DNA damage response, metabolism, apoptosis, and autophagy. The deacetylase activity of class III histone deacetylases or sirtuins depends on the presence of NAD(+) (nicotinamide adenine dinucleotide), and therefore, their function is closely linked to cellular energy consumption. This activity of sirtuins connects the modulation of chromatin dynamics and transcriptional regulation under oxidative stress to cellular lifespan, glucose homeostasis, inflammation, and multiple aging-related diseases including cancer. Here we provide an overview of the recent developments in relation to the diverse biological activities associated with sirtuin enzymes and stress responsive transcription factors, DNA damage, and oxidative stress and relate the involvement of sirtuins in the regulation of these processes to oncogenesis. Since the majority of the molecular mechanisms implicated in these pathways have been described for Sirt1, this sirtuin family member is more extensively presented in this paper.

Signalling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalysed by the E1, E2 and E3 three-enzyme cascade, which links the carboxy terminus of ubiquitin to the ε-amino group of, in most cases, a lysine of the substrate via an isopeptide bond. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents. For example, many bacterial pathogens exploit ubiquitin signalling using virulence factors that function as E3 ligases, deubiquitinases or as enzymes that directly attack ubiquitin. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a permissive niche for its replication in phagocytes. Here we demonstrate that members of the SidE effector family of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum. Moreover, we show that these proteins are capable of catalysing ubiquitination without the need for the E1 and E2 enzymes. A putative mono-ADP-ribosyltransferase motif critical for the ubiquitination activity is also essential for the role of the SidE family in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalysed by these enzymes is energized by nicotinamide adenine dinucleotide, which activates ubiquitin by the formation of ADP-ribosylated ubiquitin. These results establish that ubiquitination can be catalysed by a single enzyme, the activity of which does not require ATP.

SIRT1 is the closest mammalian homologue of enzymes that extend life in lower organisms. Its role in mammals is incompletely understood, but includes modulation of at least 34 distinct targets through its nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase activity. Recent experiments using small molecule activators and genetically engineered mice have provided new insight into the role of this enzyme in mammalian biology and helped to highlight some of the potentially relevant targets. The most widely employed activator is resveratrol, a small polyphenol that improves insulin sensitivity and vascular function, boosts endurance, inhibits tumor formation, and ameliorates the early mortality associated with obesity in mice. Many of these effects are consistent with modulation of SIRT1 targets, such as PGC1alpha and NFkappaB, however, resveratrol can also activate AMPK, inhibit cyclooxygenases, and influence a variety of other enzymes. A novel activator, SRT1720, as well as various methods to manipulate NAD(+) metabolism, are emerging as alternative methods to increase SIRT1 activity, and in many cases recapitulate effects of resveratrol. At present, further studies are needed to more directly test the role of SIRT1 in mediating beneficial effects of resveratrol, to evaluate other strategies for SIRT1 activation, and to confirm the specific targets of SIRT1 that are relevant in vivo. These efforts are especially important in light of the fact that SIRT1 activators are entering clinical trials in humans, and "nutraceutical" formulations containing resveratrol are already widely available.

Glaucomas are neurodegenerative diseases that cause vision loss, especially in the elderly. The mechanisms initiating glaucoma and driving neuronal vulnerability during normal aging are unknown. Studying glaucoma-prone mice, we show that mitochondrial abnormalities are an early driver of neuronal dysfunction, occurring before detectable degeneration. Retinal levels of nicotinamide adenine dinucleotide (NAD+, a key molecule in energy and redox metabolism) decrease with age and render aging neurons vulnerable to disease-related insults. Oral administration of the NAD+ precursor nicotinamide (vitamin B3), and/or gene therapy (driving expression of Nmnat1, a key NAD+-producing enzyme), was protective both prophylactically and as an intervention. At the highest dose tested, 93% of eyes did not develop glaucoma. This supports therapeutic use of vitamin B3 in glaucoma and potentially other age-related neurodegenerations.

Poly[adenosine diphosphate (ADP)-ribose] polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from the oxidized form of nicotinamide adenine dinucleotide (NAD(+)) onto substrate proteins. Here we report a robust NAD(+) analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1-mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits negative elongation factor (NELF), a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1 or mutation of the ADP-ribosylation sites on NELF-E promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals.

The objective of this study is to induce experimental diabetes mellitus by Streptozotocin in normal adult Wistar rats via comparison of changes in body weight, consumption of food and water, volume of urine and levels of glucose, insulin and C-peptide in serum, between normal and diabetic rats. Intra-venous injection of 60mg/kg dose of Streptozotocin in adult wistar rats, makes pancreas swell and at last causes degeneration in Langerhans islet beta cells and induces experimental diabetes mellitus in the 2-4 days. Induction of experimental diabetes mellitus is indeed the first step in the plan of purification of pancreatic Langerhans islet cells of normal rats for transplanting under the testis subcutaneous of experimentally induced diabetic rats. Streptozotocin induces one type of diabetes which is similar to diabetes mellitus with non-ketosis hyperglycemia in some animal species. For induction of experimental diabetes in male adult rats weighted 250-300 grams (75-90 days), 60mg/kg of Streptozotocin was injected intravenously. Three days after degeneration of beta cells, diabetes was induced in all animals. The diabetic and normal animals were kept in the metabolic cages separately and their body weight, consumption of food and water, urine volume, the levels of serum glucose, insulin and C-peptide quantities in all animals were measured and then these quantities were compared. For a microscopic study of degeneration of Langerhans islet beta cells of diabetic rats, sampling from pancreas tissue of diabetic and normal rats, staining and comparison between them, were done. Induction of diabetes with Streptozotocin decreases Nicotinamide-adenine dinucleotide (NAD) in pancreas islet beta cells and causes histopathological effects in beta cells which probably intermediates induction of diabetes. In this study, we used Streptozotocin for our experiments in induction of experimental diabetes mellitus. After Induction of diabetes, consumption of food and water, volume of urine and glucose increased in the diabetic animals in comparison with normal animals, but the weight of body and the volume of insulin and C-peptide decreased in the diabetic animals. Sampling and staining of pancreas tissue of diabetic and normal rats showed that the Langerhans islet beta cells of diabetic rats have been clearly degenerated. In three days, Streptozotocin makes pancreas swell and at last causes degeneration in Langerhans islet beta cells and induces experimental diabetes. It also changes normal metabolism in diabetic rats in comparison with normal rats. Consumption of water and food, volume of urine, serum glucose increases in diabetic animals in comparison with normal rats but the levels of serum insulin, C-peptide and body weight decreases.

Reactive oxygen species (ROS) are products of normal cellular metabolism and are known to act as second messengers. Under physiological conditions, ROS participate in maintenance of cellular 'redox homeostasis' in order to protect cells against oxidative stress through various redox-regulatory mechanisms. Overproduction of ROS, most frequently due to excessive stimulation of either reduced nicotinamide adenine dinucleotide phosphate by cytokines or the mitochondrial electron transport chain and xanthine oxidase, results in oxidative stress. Oxidative stress is a deleterious process that leads to lung damage and consequently to various disease states. Knowledge of the mechanisms of ROS regulation could lead to the pharmacological manipulation of antioxidants in lung inflammation and injury.

Glioblastoma multiforme (GBM) is a devastating brain tumor with poor prognosis and low median survival time. Standard treatment includes radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). However, a large percentage of tumors are resistant to the cytotoxic effects of the TMZ-induced DNA lesion O(6)-methylguanine due to elevated expression of the repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) or a defect in the mismatch repair (MMR) pathway. Although a majority of the TMZ-induced lesions (N7-methylguanine and N3-methyladenine) are base excision repair (BER) substrates, these DNA lesions are also readily repaired. However, blocking BER can enhance response to TMZ and therefore the BER pathway has emerged as an attractive target for reversing TMZ resistance. Our lab has recently reported that inhibition of BER leads to the accumulation of repair intermediates that induce energy depletion-mediated cell death via hyperactivation of poly(ADP-ribose) polymerase. On the basis of our observation that TMZ-induced cell death via BER inhibition is dependent on the availability of nicotinamide adenine dinucleotide (NAD(+)), we have hypothesized that combined BER and NAD(+) biosynthesis inhibition will increase TMZ efficacy in glioblastoma cell lines greater than BER inhibition alone. Importantly, we find that the combination of BER and NAD(+) biosynthesis inhibition significantly sensitizes glioma cells with elevated expression of MGMT and those deficient in MMR, two genotypes normally associated with TMZ resistance. Dual targeting of these two interacting pathways (DNA repair and NAD(+) biosynthesis) may prove to be an effective treatment combination for patients with resistant and recurrent GBM.

Nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high-throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is transferred to alpha-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)+, which is measured spectrophotometrically at 340 nm. Using this continuous assay with sirtuin-1 (Sirt1) and the ADP-ribosyl cyclase CD38, the resulting steady-state kinetic parameters are in excellent agreement with values obtained by other published methods. Importantly, this assay permitted determination of k(cat) and K(m) values with the native acetylated substrate acetyl-CoA synthetase-1; measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts; and determination of IC(50) values of various Sirt1 inhibitors. This assay is applicable to any nicotinamide-forming enzyme and will be an important tool to address many outstanding questions surrounding their regulation.

We have determined the amounts of a number of small molecules and enzymes in the mother cell compartment and the developing forespore during sporulation of Bacillus megaterium. Significant amounts of adenosine 5'-triphosphate and reduced nicotinamide adenine dinucleotide were present in the forespore compartment before accumulation of dipicolinic acid (DPA), but these compounds disappeared as DPA was accumulated. 3-Phosphoglyceric acid (3-PGA) accumulated only within the developing forespore, beginning 1 to 2 h before DPA accumulation. Throughout its development the forespore contained constant levels of enzymes of both 3-PGA synthesis (phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase) and 3-PGA utilization (phosphoglycerate mutase, enolase, and pyruvate kinase) at levels similar to those in the mother cell and the dormant spore. Despite the presence of enzymes for 3-PGA utilization, this compound was stable within isolated forespores. Two acid-soluble proteins (A and B proteins) also accumulated only in the forespore, beginning 1 to 2 h before DPA accumulation. At this time the specific protease involved in degradation of the A and B proteins during germination also appeared, but only in the forespore compartment. Nevertheless, the A and B proteins were stable within isolated forespores. Arginine and glutamic acid accumulated within the forespore in parallel with DPA accumulation. The forespore also contained the enzyme arginase at a level similar to that in the mother cell and a level of glutamic acid decarboxylase 2- to 25-fold higher than that in the mother cell, depending on when in sporulation the forespores were isolated. The specific activities of several other enzymes (protease active on hemoglobin, ornithine transcarbamylase, malate dehydrogenase, aconitase, and isocitrate dehydrogenase) in forespores were about 10% or less of the values in the mother cell. Aminopeptidase was present at similar levels in both compartments; threonine deaminase was not found in either compartment.

The effect of different nitrogen compounds on the induction of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase was examined in Neurospora crassa. Whereas in the wild-type strain several amino acids and ammonia inhibit the formation of nitrate reductase, only glutamine, cysteine, and histidine are shown to inhibit the synthesis of nitrate reductase in a glutamine-requiring auxotroph. None of the amino acids inhibited nitrate reductase activity in vitro. The effects of cysteine and histidine are nonspecific, these amino acids being inhibitory of the growth of the organism. The effect of glutamine on the induction of nitrate reductase is not due to an inhibition of the uptake of the inducer nitrate. By the use of histidine-, pyrimidine-, and arginine-requiring auxotrophs, it was shown that glutamine appears to act per se and does not seem to be converted to another product in order to be effective in repression. The repression of nitrate reductase by ammonia appears, from the results described herein, to be indirect; ammonia has to be converted first to glutamine in order to be effective in repression.

The brain renin-angiotensin system (RAS) contributes to increased sympathetic drive in heart failure (HF). The factors upregulating the brain RAS in HF remain unknown. We hypothesized that aldosterone (ALDO), a downstream product of the systemic RAS that crosses the blood-brain barrier, signals the brain to increase RAS activity in HF. We examined the relationship between circulating and brain ALDO in normal intact rats, in adrenalectomized rats receiving subcutaneous infusions of ALDO, and in rats with ischemia-induced HF and sham-operated controls. Brain ALDO levels were proportional to plasma ALDO levels across the spectrum of rats studied. Compared with sham-operated controls rats, HF rats had higher plasma and hypothalamic tissue levels of ALDO. HF rats also had higher expression of mRNA and protein for angiotensin-converting enzyme and angiotensin type 1 receptors in the hypothalamus, increased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity and superoxide generation in the paraventricular nucleus of the hypothalamus, increased excitation of paraventricular nucleus neurons, and increased plasma norepinephrine. HF rats treated for 4 weeks with intracerebroventricular RU28318 (1 microg/h), a selective mineralocorticoid receptor antagonist, had less hypothalamic angiotensin-converting enzyme and angiotensin type 1 receptor mRNA and protein, less reduced nicotinamide-adenine dinucleotide phosphate-induced superoxide in the paraventricular nucleus, fewer excited paraventricular nucleus neurons, and lower plasma norepinephrine. RU28318 had no effect on plasma ALDO or on angiotensin-converting enzyme or angiotensin type 1 receptor expression in brain cortex. The data demonstrate that ALDO of adrenal origin enters the hypothalamus in direct proportion to plasma levels and suggest that ALDO contributes to the upregulation of hypothalamic RAS activity and sympathetic drive in heart failure.

The low rate of endogenous respiration exhibited by the blue-green algae Anacystis nidulans and Phormidium luridum was not increased by the addition of respiratory substrates. However, endogenous respiration was inhibited by low concentrations of cyanide and by high carbon monoxide tensions. In addition, the uncouplers dinitrophenol and carbonyl cyanide p-trifluoromethoxyphenylhydrazone both stimulated the respiratory rate. The transition of cells from the aerobic steady state to anaerobiosis was accompanied by a decrease in the concentration of cellular nicotinamide adenine dinucleotide phosphate (NADP(+)) and adenosine triphosphate (ATP), whereas the concentration of nicotinamide adenine dinucleotide (NAD(+)) was unchanged. Concomitant with the metabolite decreases were stoichiometric increases io reduced NADP(+) (NADPH), adenosine diphosphate, and adenosine monophosphate. A decrease in ATP was also observed after the addition of uncouplers. These data are interpreted as evidence for the association of oxidative phosphorylation with the oxidation of NADP(+)-linked substrates in these algae. Membrane fragments isolated from the algal cells oxidized succinate, malate, ferrocytochrome c, ascorbate-tetramethyl-p-phenylenediamine, and reduced 2,6-dichlorophenol indophenol but did not oxidize NADPH or reduced NAD(+) in a cyanide-sensitive system. Oxidative phosphorylation has not yet been demonstrated in these fragments, but a dark ATP-P(i) exchange, distinct from the lighttriggered exchange associated with photosynthesis, is readily observed. This exchange was inhibited by phloridzin, Atabrine, and uncouplers in concentrations which suggest that the mechanism of oxidative phosphorylation in blue-green algae is different from that found in other bacteria and in mitochondria. These results led to the conclusion that the biochemical basis for obligate autotrophy in these organisms does not lie in the metabolic events associated with terminal electron transport and energy conservation.