The enzymatic technique using 3alpha-hydroxysteroid dehydrogenase for determining bile acids in blood has been modified by measuring the reduced nicotinamide adenine dinucleotide fluorimetrically. The increased sensitivity attained enables the concentration of total bile acids in serum to be estimated using 3 ml for normal subjects and 1 ml for jaundiced patients. The range of normal values in serum was found to be 0-4.7 mumol/litre for males and 1.0-8.2 mumol/litre for females.

Two strains of Pseudomonas putida isolated by enrichment cultures with orcinol as the sole source of carbon were both found to grow with resorcinol. Data are presented which show that one strain (ORC) catabolizes resorcinol by a metabolic pathway, genetically and mechanistically distinct from the orcinol pathway, via hydroxyquinol and ortho oxygenative cleavage to give maleylacetate, but that the other strain (O1) yields mutants that utilize resorcinol. One mutant strain, designated O1OC, was shown to be constitutive for the enzymes of the orcinol pathway. After growth of this strain on resorcinol, two enzymes of the resorcinol pathway are also induced, namely hydroxyquinol 1,2-oxygenase and maleylacetate reductase. Thus hydroxyquniol, formed from resorcinol, undergoes both ortho and meta diol cleavage reactions with the subsequent formation of both pyruvate and maleylacetate. Evidence was not obtained for the expression of resorcinol hydroxylase in strain O1OC; the activity of orcinol hydroxylase appears to be recruited for this hydroxylation reaction. P. putida ORC, on the other hand, possesses individual hydroxylases for orcinol and resorcinol, which are specifically induced by growth on their respective substrates. The spectral changes associated with the enzymic and nonenzymic oxidation of hydroxyquinol are described. Maleylacetate was identified as the product of hydroxyquinol oxidation by partially purified extracts obtained from P. putida ORC grown with resorcinol. Its further metabolism was reduced nicotinamide adenine dinucleotide dependent.

Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is widely expressed in brain tissue including neurons, glia, and endothelia in neurovascular units. It is a major source of oxidants in the post-ischemic brain and significantly contributes to ischemic brain damage. Inflammation occurs after brain ischemia and is known to be associated with post-ischemic oxidative stress. Post-ischemic inflammation also causes progressive brain injury. In this study we investigated the role of NOX2 in post-ischemic cerebral inflammation using a transient middle cerebral artery occlusion model in mice. We demonstrate that mice with NOX2 subunit gp91(phox) knockout (gp91 KO) showed 35-44% less brain infarction at 1 and 3 days of reperfusion compared with wild-type (WT) mice. Minocycline further reduced brain damage in the gp91 KO mice at 3 days of reperfusion. The gp91 KO mice exhibited less severe post-ischemic inflammation in the brain, as evidenced by reduced microglial activation and decreased upregulation of inflammation mediators, including interleukin-1β (IL-1β), tumor necrosis factor-α, inducible nitric oxide synthases, CC-chemokine ligand 2, and CC-chemokine ligand 3. Finally, we demonstrated that an intraventricular injection of IL-1β enhanced ischemia- and reperfusion-mediated brain damage in the WT mice (double the infarction volume), whereas, it failed to aggravate brain infarction in the gp91 KO mice. Taken together, these results demonstrate the involvement of NOX2 in post-ischemic neuroinflammation and that NOX2 inhibition provides neuroprotection against inflammatory cytokine-mediated brain damage.

Redox regulation has been shown to be an important component of malignant cell survival. Tipping the cellular redox balance through pharmacologic regulation in favor of increasing intracellular reactive oxygen species (ROS) and/or depleting protective reducing metabolites (such as glutathione and nicotinamide adenine dinucleotide phosphate) may lead to oxidative stress and resultant induction of apoptosis for the treatment of cancer. We review the biology and importance of ROS with regard to malignant and normal cells. Moreover, we discuss pre-clinical and clinical data regarding novel therapeutic agents that modulate the cellular redox system including buthionine sulfoximine, ascorbic acid, arsenic trioxide, imexon, and motexafin gadolinium as single-agents and in combination. Continued research is needed to better understand the mechanisms and specific apoptotic pathways involved in ROS-induced cell death, as well as, to determine the most rationale and effective combination of redox-active agents.

Hypoxic pulmonary vasoconstriction (HPV), also known as the von Euler-Liljestrand mechanism, is a physiological response to alveolar hypoxia which distributes pulmonary capillary blood flow to alveolar areas of high oxygen partial pressure. Impairment of this mechanism may result in hypoxaemia. Under conditions of chronic hypoxia generalised vasoconstriction of the pulmonary vasculature in concert with hypoxia-induced vascular remodelling leads to pulmonary hypertension. Although the principle of HPV was recognised decades ago, its exact pathway still remains elusive. Neither the oxygen sensing process nor the exact pathway underlying HPV is fully deciphered yet. The effector pathway is suggested to include L-type calcium channels, nonspecific cation channels and voltage-dependent potassium channels, whereas mitochondria and nicotinamide adenine dinucleotide phosphate oxidases are discussed as oxygen sensors. Reactive oxygen species, redox couples and adenosine monophosphate-activated kinases are under investigation as mediators of hypoxic pulmonary vasoconstriction. Moreover, the role of calcium sensitisation, intracellular calcium stores and direction of change of reactive oxygen species is still under debate. In this context the present article focuses on the basic mechanisms of hypoxic pulmonary vasoconstriction and also outlines differences in current concepts that have been suggested for the regulation of hypoxic pulmonary vasoconstriction.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (T reg cells) play a major role in the control of immune responses but the factors controlling their homeostasis and function remain poorly characterized. Nicotinamide adenine dinucleotide (NAD(+)) released during cell damage or inflammation results in ART2.2-mediated ADP-ribosylation of the cytolytic P2X7 receptor on T cells. We show that T reg cells express the ART2.2 enzyme and high levels of P2X7 and that T reg cells can be depleted by intravenous injection of NAD(+). Moreover, lower T reg cell numbers are found in mice deficient for the NAD-hydrolase CD38 than in wild-type, P2X7-deficient, or ART2-deficient mice, indicating a role for extracellular NAD(+) in T reg cell homeostasis. Even routine cell preparation leads to release of NAD(+) in sufficient quantities to profoundly affect T reg cell viability, phenotype, and function. We demonstrate that T reg cells can be protected from the deleterious effects of NAD(+) by an inhibitory ART2.2-specific single domain antibody. Furthermore, selective depletion of T reg cells by systemic administration of NAD(+) can be used to promote an antitumor response in several mouse tumor models. Collectively, our data demonstrate that NAD(+) influences survival, phenotype, and function of T reg cells and provide proof of principle that acting on the ART2-P2X7 pathway represents a new strategy to manipulate T reg cells in vivo.

Mitochondrial complex I dysfunction is regarded as underlying dopamine neuron death in Parkinson's disease models. However, inactivation of the Ndufs4 gene, which compromises complex I activity, does not affect the survival of dopamine neurons in culture or in the substantia nigra pars compacta of 5-wk-old mice. Treatment with piericidin A, a complex I inhibitor, does not induce selective dopamine neuron death in either Ndufs4(+/+) or Ndufs4(-/-) mesencephalic cultures. In contrast, rotenone, another complex I inhibitor, causes selective toxicity to dopamine neurons, and Ndufs4 inactivation potentiates this toxicity. We identify microtubule depolymerization and the accumulation of cytosolic dopamine and reactive oxygen species as alternative mechanisms underlying rotenone-induced dopamine neuron death. Enhanced rotenone toxicity to dopamine neurons from Ndufs4 knockout mice may involve enhanced dopamine synthesis caused by the accumulation of nicotinamide adenine dinucleotide reduced. Our results suggest that the combination of disrupting microtubule dynamics and inhibiting complex I, either by mutations or exposure to toxicants, may be a risk factor for Parkinson's disease.

DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP-ribose) polymerase-1 (PARP-1) cell death pathway. PARP-1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)+ to form poly(ADP-ribose) on acceptor proteins. The mechanisms by which PARP-1 activation leads to cell death are not understood fully. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD+ depletion by PARP-1 and the role of NAD+ depletion in this cell death program. PARP-1 activation was induced by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), using medium in which glucose was the only exogenous energy substrate. PARP-1 activation led to a rapid but incomplete depletion of astrocyte NAD+, a near-complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD+ restored glycolytic function and prevented cell death. The addition of non-glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP-1 activation. These studies suggest PARP-1 activation leads to rapid depletion of the cytosolic but not the mitochondrial NAD+ pool. Depletion of the cytosolic NAD+ pool renders the cells unable to utilize glucose as a metabolic substrate. Under conditions where glucose is the only available metabolic substrate, this leads to cell death. This cell death pathway is particularly germane to brain because glucose is normally the only metabolic substrate that is transported rapidly across the blood-brain barrier.

Recent studies suggest that increased mitochondrial metabolism and the concomitant decrease in NADH levels mediate calorie restriction (CR)-induced life span extension. The mitochondrial inner membrane is impermeable to NAD (nicotinamide adenine dinucleotide, oxidized form) and NADH, and it is unclear how CR relays increased mitochondrial metabolism to multiple cellular pathways that reside in spatially distinct compartments. Here we show that the mitochondrial components of the malate-aspartate NADH shuttle (Mdh1 [malate dehydrogenase] and Aat1 [aspartate amino transferase]) and the glycerol-3-phosphate shuttle (Gut2, glycerol-3-phosphate dehydrogenase) are novel longevity factors in the CR pathway in yeast. Overexpressing Mdh1, Aat1, and Gut2 extend life span and do not synergize with CR. Mdh1 and Aat1 overexpressions require both respiration and the Sir2 family to extend life span. The mdh1Deltaaat1Delta double mutation blocks CR-mediated life span extension and also prevents the characteristic decrease in the NADH levels in the cytosolic/nuclear pool, suggesting that the malate-aspartate shuttle plays a major role in the activation of the downstream targets of CR such as Sir2. Overexpression of the NADH shuttles may also extend life span by increasing the metabolic fitness of the cells. Together, these data suggest that CR may extend life span and ameliorate age-associated metabolic diseases by activating components of the NADH shuttles.

The influence of a H(2)-utilizing organism, Vibrio succinogenes, on the fermentation of limiting amounts of glucose by a carbohydrate-fermenting, H(2)-producing organism, Ruminococcus albus, was studied in continuous cultures. Growth of V. succinogenes depended on the production of H(2) from glucose by R. albus. V. succinogenes used the H(2) produced by R. albus to obtain energy for growth by reducing fumarate in the medium. Fumarate was not metabolized by R. albus alone. The only products detected in continuous cultures of R. albus alone were acetate, ethanol, and H(2). CO(2) was not measured. The only products detected in the mixed cultures were acetate and succinate. No free H(2) was produced. No formate or any other volatile fatty acid, no succinate or other dicarboxylic acids, lactate, alcohols other than ethanol, pyruvate, or other keto-acids, acetoin, or diacetyl were detected in cultures of R. albus alone or in mixed cultures. The moles of product per 100 mol of glucose fermented were approximately 69 for ethanol, 74 for acetate, 237 for H(2) for R. albus alone and 147 for acetate and 384 for succinate for the mixed culture. Each mole of succinate is equivalent to the production of 1 mol of H(2) by R. albus. Thus, in the mixed cultures, ethanol production by R. albus is eliminated with a corresponding increase in acetate and H(2) formation. The mixed-culture pattern is consistent with the hypothesis that nicotinamide adenine dinucleotide (reduced form), formed during glycolysis by R. albus, is reoxidized during ethanol formation when R. albus is grown alone and is reoxidized by conversion to nicotinamide adenine dinucleotide and H(2) when R. albus is grown with V. succinogenes. The ecological significance of this interspecies transfer of H(2) gas and the theoretical basis for its causing changes in fermentation patterns of R. albus are discussed.

OBJECTIVE

Oxidative stress is implicated in the pathogenesis of pancreatitis, but clinical trials of antioxidants have produced conflicting results. We examined the role of intracellular reactive oxygen species (ROS) in pancreatic acinar cell injury.

METHODS

Freshly isolated murine and human pancreatic acinar cells were studied using confocal microscopy to measure changes in intracellular and mitochondrial ROS concentrations ([ROS]I and [ROS]M), cytosolic and mitochondrial calcium concentrations ([Ca2+]C and [Ca2+]M), reduced nicotinamide adenine dinucleotide phosphate levels, and death pathways in response to taurolithocholate acid sulfate (TLC-S) or the oxidant menadione. Ca2+-activated Cl- currents were measured using whole-cell patch clamp, with or without adenosine triphosphate (ATP).

RESULTS

TLC-S induced prolonged increases in [Ca2+]C and [Ca2+]M, which led to dose-dependent increases in [ROS]I and [ROS]M, impaired production of ATP, apoptosis, and necrosis. Inhibition of the antioxidant reduced nicotinamide adenine dinucleotide phosphate quinine oxidoreductase by 2,4-dimethoxy-2-methylnaphthalene potentiated the increases in [ROS]I and apoptosis but reduced necrosis, whereas the antioxidant N-acetyl-L-cysteine reduced [ROS]I and apoptosis but increased necrosis. Inhibition of mitochondrial ROS production prevented apoptosis but did not alter necrosis; autophagy had no detectable role. Patched ATP prevented sustained increases in [Ca2+]C and necrosis.

CONCLUSIONS

Increases in [ROS]M and [ROS]I during bile acid injury of pancreatic acinar cells promote apoptosis but not necrosis. These results indicate that alternative strategies to antioxidants are required for oxidative stress in acute pancreatitis.

Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP(+)) dose-dependently stimulated intracellular Ca(2+)-signaling. At a concentration of 10 nM NAADP(+) evoked repetitive and long-lasting Ca(2+)-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca(2+)-peak followed by trains of smaller Ca(2+)-oscillations was observed. Higher concentrations of NAADP(+) (1 and 10 microM) gradually reduced the initial Ca(2+)-peak, and a complete self-inactivation of Ca(2+)-signals was seen at 100 microM. The effect of NAADP(+) was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4, 5-trisphosphate- and cyclic adenosine diphosphoribose-mediated Ca(2+)-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP(+). Most importantly, microinjection of a self-inactivating concentration of NAADP(+) completely abolished subsequent stimulation of Ca(2+)-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP(+) Ca(2+)-release system is essential for T-lymphocyte Ca(2+)-signaling.

The rate of hydrolysis of adenosine triphosphate (ATP) by chemically skinned rabbit muscle fibres was measured as a function of Mg ATP concentration in the range 5 microM to 5 mM. Pyruvate kinase and lactate dehydrogenase were used to link adenosine diphosphate formation to oxidation of nicotinamide adenine dinucleotide which was followed by the change in absorption at 340 nm. The ATPase rate of a fully activated fibre (pCa = 4.5) increased monotonically with Mg ATP concentration in a manner that could be readily fitted by a hyperbola. At 15 degrees C, pH 7 and an ionic strength of 0.2 M the rate at saturating Mg ATP (Vm) was 1.78 +/- 0.2 s-1 per myosin head (mean +/- S.D.; n = 6) and the Mg ATP concentration needed for half the maximal rate (Km) was 16.6 +/- 2 microM. The ATPase of fibres that had been stabilized by cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) was also investigated. Cross-linking did not significantly affect the Vm or Km and these fibres proved useful for investigating the adequacy of the pyruvate kinase activity for regenerating hydrolysed ATP. Myofibrils were cross-linked with EDC or glutaraldehyde to prevent shortening. Their ATPase properties were investigated: the values of Vm were 0.85 +/- 0.18 (mean +/- S.D.; n = 14) and 0.82 +/- 0.05 s-1 (n = 6) and of Km were 18.0 +/- 2.8 and 12.4 +/- 2.4 microM respectively. The values of Vm and Km for EDC cross-linked myofibrils were fairly insensitive to ionic strength, the Km decreasing 40% and the Vm increasing 50% for a change from 0.2 to 0.3 M. This slight dependence on ionic strength is considered in relation to the ionic strength dependence of the elementary rate constants of the actomyosin subfragment-1 ATPase cycle.

The dorsomedial portion of the nucleus tractus solitarius (dmNTS) is the site of termination of baroreceptor and cardiorespiratory vagal afferents and plays a critical role in cardiovascular regulation. Angiotensin II (Ang II) is a powerful signaling molecule in dmNTS neurons and exerts some of its biological effects by modulating Ca(2+) currents via reactive oxygen species (ROS) derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. We investigated whether a Nox2-containing NADPH oxidase is the source of the Ang II-induced ROS production and whether the signaling mechanisms of its activation require intracellular Ca(2+) or protein kinase C (PKC). Second-order dmNTS neurons were anterogradely labeled with 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide transported from the vagus and isolated from the brain stem. ROS production was assessed in 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide-positive dmNTS neurons using the fluorescent dye 6-carboxy-2',7'-dichlorodihydro-fluorescein di(acetoxymethyl ester). Ang II (3 to 2000 nmol/L) increased ROS production in dmNTS neurons (EC(50)=38.3 nmol/L). The effect was abolished by the ROS scavenger Mn (III) porphyrin 5,10,20-tetrakis (benzoic acid) porphyrin manganese (III), the Ang II type 1 receptor antagonist losartan, or the NADPH oxidase inhibitors apocynin or gp91ds-tat. Ang II failed to increase ROS production or to potentiate L-type Ca(2+) currents in dmNTS neurons of mice lacking Nox2. The PKC inhibitor GF109203X or depletion of intracellular Ca(2+) attenuated Ang II-elicited ROS production. We conclude that the powerful effects of Ang II on Ca(2+) currents in dmNTS neurons are mediated by PKC activation leading to ROS production via Nox2. Thus, a Nox2-containing NADPH oxidase is the critical link between Ang II and the enhancement of Ca(2+) currents that underlie the actions of Ang II on central autonomic regulation.

The nicotinamide adenine dinucleotide (NAD)-binding domains of dehydrogenases, containing a conserved double beta-alpha-beta-alpha-beta motif, are common structural feature of many enzymes that bind NAD, nicotinamide adenine dinucleotide phosphate (NADP) and related cofactors. Features of this folding pattern that create a natural binding site for such molecules have been described. The domain continues to appear in many structures, in the form of a common core with different peripheral additions or variations. Other structures that bind NAD and related molecules use entirely different topologies, although, in many, a phosphate group appears at the N terminus of an alpha helix. Ferredoxin reductase seems to show convergent evolution, containing a single beta-alpha-beta motif that is similar both in its structure and in its interactions with the ligand to a region in dehydrogenases.

Protein ADP-ribosyltransferases catalyze the transfer of adenosine diphosphate ribose (ADP-ribose) from nicotinamide adenine dinucleotide (NAD) onto specific target proteins. Sirtuins, a class of enzymes with NAD-dependent deacetylase activity, have been reported to possess ADP-ribosyltransferase activity, too. Here we used NAD analogues and 32P-NAD to study the ADP-ribosyltransferase activity of several different sirtuins, including yeast Sir2, human SirT1, mouse SirT4, and mouse SirT6. The results showed that an alkyne-tagged NAD is the substrate for deacetylation reactions but cannot detect the ADP-ribosylation activity. Furthermore, comparing with a bacterial ADP-ribosyltransferase diphtheria toxin, the observed rate constant of sirtuin-dependent ADP-ribosylation is >5000-fold lower. Compared with the kcat/Km values of the deacetylation activity of sirtuins, the observed rate constant of sirtuin-dependent ADP-ribosyltion is 500 times weaker. The weak ADP-ribosylation events can be explained by both enzymatic and nonenzymatic reaction mechanisms. Combined with recent reports on several other sirtuins, we propose that the reported ADP-ribosyltransferase activity of sirtuins is likely some inefficient side reactions of the deacetylase activity and may not be physiologically relevant.

OBJECTIVE

PARP inhibitors are being developed as therapeutic agents for cancer. More than six compounds have entered clinical trials. The majority of these compounds are β-nicotinamide adenine dinucleotide (NAD(+))-competitive inhibitors. One exception is iniparib, which has been proposed to be a noncompetitive PARP inhibitor. In this study, we compare the biologic activities of two different structural classes of NAD(+)-competitive compounds with iniparib and its C-nitroso metabolite.

METHODS

Two chemical series of NAD(+)-competitive PARP inhibitors, iniparib and its C-nitroso metabolite, were analyzed in enzymatic and cellular assays. Viability assays were carried out in MDA-MB-436 (BRCA1-deficient) and DLD1(-/-) (BRCA2-deficient) cells together with BRCA-proficient MDA-MB-231 and DLD1(+/+) cells. Capan-1 and B16F10 xenograft models were used to compare iniparib and veliparib in vivo. Mass spectrometry and the (3)H-labeling method were used to monitor the covalent modification of proteins.

RESULTS

All NAD(+)-competitive inhibitors show robust activity in a PARP cellular assay, strongly potentiate the activity of temozolomide, and elicit robust cell killing in BRCA-deficient tumor cells in vitro and in vivo. Cell killing was associated with an induction of DNA damage. In contrast, neither iniparib nor its C-nitroso metabolite inhibited PARP enzymatic or cellular activity, potentiated temozolomide, or showed activity in a BRCA-deficient setting. We find that the nitroso metabolite of iniparib forms adducts with many cysteine-containing proteins. Furthermore, both iniparib and its nitroso metabolite form protein adducts nonspecifically in tumor cells.

CONCLUSIONS

Iniparib nonselectively modifies cysteine-containing proteins in tumor cells, and the primary mechanism of action for iniparib is likely not via inhibition of PARP activity.

OBJECTIVE

Several studies have found that chronic treatment with the dietary flavonoid quercetin lowers blood pressure and restores endothelial dysfunction in hypertensive animal models. We hypothesized that increased endothelial nitric oxide synthase (eNOS) and/or decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase protein expression and activity, and reduced reactive oxygen species might be involved in the improvement of endothelial function induced by quercetin in spontaneously hypertensive rats (SHR).

METHODS

Male SHR and Wistar-Kyoto (WKY) rats (5 weeks old) were treated with quercetin (10 mg/kg) or vehicle for 13 weeks. Changes in vascular expression of eNOS, caveolin-1 and p47 were analysed by Western blot, eNOS activity by conversion of [H]arginine to L-[H]citrulline, and NADPH oxidase activity by NADPH-enhanced chemoluminescence of lucigenin.

RESULTS

In SHR, quercetin reduced the increase in blood pressure and heart rate and enhanced the endothelium-dependent aortic vasodilation induced by acetylcholine, but had no effect on the endothelium-independent response induced by nitroprusside. However, quercetin had no effect on endothelium-dependent vasoconstriction and aortic thromboxane B2 production. Compared to WKY, SHR showed upregulated eNOS and p47 protein expression, downregulated caveolin-1 expression, increased NADPH-induced superoxide production but, paradoxically, eNOS activity was reduced. Chronic quercetin treatment prevented all these changes in SHR. In WKY, quercetin had no effect on blood pressure, endothelial function or the expression or activity of the proteins analysed.

CONCLUSIONS

Enhanced eNOS activity and decreased NADPH oxidase-mediated superoxide anion (O2) generation associated with reduced p47 expression appear to be essential mechanisms for the improvement of endothelial function and the antihypertensive effects of chronic quercetin.

The slow Wallerian degeneration (Wld(S)) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70-amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide-synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of Wld(S)-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.

OBJECTIVE

Subpopulations of interstitial cells of Cajal are regarded as the source of spontaneous slow waves of the gut musculature (pacemaker cells). Their ontogeny remains unclear, but a role of the tyrosine kinase receptor c-kit in their development has recently been recognized. This study examined the interstitial cells in the human colon and in Hirschsprung's disease (aganglionosis).

METHODS

The distribution of the c-kit receptor was studied using specific antibodies in 5 normal patients, 10 patients with Hirschsprung's disease, and 3 patients with diversion loop enterostomies. c-kit immunohistochemistry was also combined with reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry or with c-kit ligand (stem cell factor) immunohistochemistry. Transmission electron microscopy was performed in 1 patient with Hirschsprung's disease.

RESULTS

c-kit immunoreactivity labeled a network of interstitial cells at the outer edge of the submucosa, in the muscular layers, and around the myenteric plexus. In aganglionic segments, interstitial cells were scarce and its network appeared disrupted. Interstitial cells of Cajal were identified in aganglionic regions by electron microscopy. Interstitial cells of Cajal are identifiable in newborns and exhibit similar distribution in diversion loops independent of contact with luminal nutrients.

CONCLUSIONS

Our morphological data may explain the abnormal spontaneous electrical activity in aganglionic segments of Hirschsprung's disease and may give new insight into the ontogeny of interstitial cells.

This article reviews current concepts on the pathogenesis and treatment of alcoholic liver disease. It has been known that the hepatotoxicity of ethanol results from alcohol dehydrogenase-mediated excessive generation of hepatic nicotinamide adenine dinucleotide, reduced form, and acetaldehyde. It is now recognized that acetaldehyde is also produced by an accessory (but inducible) microsomal pathway that additionally generates oxygen radicals and activates many xenobiotics to toxic metabolites, thereby explaining the increased vulnerability of heavy drinkers to industrial solvents, anesthetics, commonly used drugs, over-the-counter medications, and carcinogens. The contribution of gastric alcohol dehydrogenase to the first-pass metabolism of ethanol and alcohol-drug interactions is discussed. Roles for hepatitis C, cytokines, sex, genetics, and age are now emerging. Alcohol also alters the degradation of key nutrients, thereby promoting deficiencies as well as toxic interactions with vitamin A and beta carotene. Conversely, nutritional deficits may affect the toxicity of ethanol and acetaldehyde, as illustrated by the depletion in glutathione, ameliorated by S-adenosyl-L-methionine. Other "supernutrients" include polyunsaturated lecithin, shown to correct the alcohol-induced hepatic phosphatidylcholine depletion and to prevent alcoholic cirrhosis in nonhuman primates. Thus, a better understanding of the pathology induced by ethanol is now generating improved prospects for therapy.

Sir2 is a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase involved in gene silencing and longevity. Cellular stresses affect Sir2 activity, but the mechanisms of Sir2 regulation are debated. Nicotinamide has been proposed as a physiological regulator that inhibits Sir2 deacetylase activity by chemical reversal of a covalent reaction intermediate. We demonstrate a chemical strategy to activate Sir2-dependent transcriptional silencing and present evidence that the endogenous level of nicotinamide limits Sir2 activity in wild-type (wt) yeast cells. Nicotinamide inhibition of Sir2 is antagonized in vitro by isonicotinamide, which causes an increase in Sir2 deacetylation activity. Isonicotinamide also substantially increases transcriptional silencing at Sir2-regulated loci in wt strains and in strains lacking key NAD+ salvage pathway enzymes (PNC1 and NPT1). Thus, a nicotinamide antagonist is a Sir2 agonist in vitro and in vivo.

Nicotine adenine dinucleotide (NAD(+)) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD(+)-dependent protein deacetylases. In the latter two processes, NAD(+) is consumed and converted to ADP-ribose and nicotinamide. NAD(+) levels can be maintained by regeneration of NAD(+) from nicotinamide via a salvage pathway or by de novo synthesis of NAD(+) from tryptophan. Both pathways are conserved from yeast to humans. We describe a critical role of the NAD(+)-dependent deacetylase Hst1p as a sensor of NAD(+) levels and regulator of NAD(+) biosynthesis. Using transcript arrays, we show that low NAD(+) states specifically induce the de novo NAD(+) biosynthesis genes while the genes in the salvage pathway remain unaffected. The NAD(+)-dependent deacetylase activity of Hst1p represses de novo NAD(+) biosynthesis genes in the absence of new protein synthesis, suggesting a direct effect. The known Hst1p binding partner, Sum1p, is present at promoters of highly inducible NAD(+) biosynthesis genes. The removal of HST1-mediated repression of the NAD(+) de novo biosynthesis pathway leads to increased cellular NAD(+) levels. Transcript array analysis shows that reduction in cellular NAD(+) levels preferentially affects Hst1p-regulated genes in comparison to genes regulated with other NAD(+)-dependent deacetylases (Sir2p, Hst2p, Hst3p, and Hst4p). In vitro experiments demonstrate that Hst1p has relatively low affinity toward NAD(+) in comparison to other NAD(+)-dependent enzymes. These findings suggest that Hst1p serves as a cellular NAD(+) sensor that monitors and regulates cellular NAD(+) levels.

There is growing evidence implicating the kynurenine pathway (KP) and particularly one of its metabolites, quinolinic acid (QUIN), as important contributors to neuroinflammation in several brain diseases. While QUIN has been shown to induce neuronal and astrocytic apoptosis, the exact mechanisms leading to cell death remain unclear. To determine the mechanism of QUIN-mediated excitotoxicity in human brain cells, we measured intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) and poly(ADP-ribose) polymerase (PARP) and extracellular lactate dehydrogenase (LDH) activities in primary cultures of human neurons and astrocytes treated with QUIN. We found that QUIN acts as a substrate for NAD(+) synthesis at very low concentrations (<50 nM) in both neurons and astrocytes, but is cytotoxic at sub-physiological concentrations (>150 nM) in both the cell types. We have shown that the NMDA ion channel blockers, MK801 and memantine, and the nitric oxide synthase (NOS) inhibitor, L-NAME, significantly attenuate QUIN-mediated PARP activation, NAD(+) depletion, and LDH release in both neurons and astrocytes. An increased mRNA and protein expression of the inducible (iNOS) and neuronal (nNOS) forms of nitric oxide synthase was also observed following exposure of both cell types to QUIN. Taken together these results suggests that QUIN-induced cytotoxic effects on neurons and astrocytes are likely to be mediated by an over activation of an NMDA-like receptor with subsequent induction of NOS and excessive nitric oxide (NO(*))-mediated free radical damage. These results contribute significantly to our understanding of the pathophysiological mechanisms involved in QUIN neuro- and gliotoxicity and are relevant for the development of therapies for neuroinflammatory diseases.