Expression profiles of fetal membrane nicotinamide adenine dinucleotide phosphate oxidases (NOX) 2 and 3 differentiates spontaneous preterm birth and pPROM pathophysiologies

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.

The anti-lymphoma activity of APO866, an inhibitor of nicotinamide adenine dinucleotide biosynthesis, is potentialized when used in combination with anti-CD20 antibody.

Abstract APO866 is an inhibitor of nicotinamide adenine dinucleotide (NAD) biosynthesis that exhibits potent anti-lymphoma activity. Rituximab (RTX), an anti-CD20 antibody, kills lymphoma cells by direct apoptosis and antibody- and complement-dependent cell-mediated cytotoxicities, and has clinical efficacy in non-Hodgkin cell lymphomas. In the present study, we evaluated whether RTX could potentiate APO866-induced human B-lymphoma cell death and shed light on death-mediated mechanisms associated with this drug combination. We found that RTX significantly increases APO866-induced death in lymphoma cells from patients and lines. Mechanisms include enhancement of autophagy-mediated cell death, activation of caspase 3 and exacerbation of mitochondrial depolarization, but not increase of reactive oxygen species (ROS) production, when compared with those induced by each drug alone. In vivo, combined administration of APO866 with RTX in a laboratory model of human aggressive lymphoma significantly decreased tumor burden and prolonged survival over single-agent treatment. Our study demonstrates that the combination of RTX and APO866 optimizes B-cell lymphoma apoptosis and therapeutic efficacy over both compounds administered separately.

Assessment of a quantitative 5' nuclease real-time polymerase chain reaction using the nicotinamide adenine dinucleotide dehydrogenase gamma subunit (nuoG) for Bartonella species in domiciled and stray cats in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tissue specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding

Funding: The Scottish Government's Rural and Environment Science and Analytical Services Division.

Nicotinamide nucleotide and adenylate concentrations in mante, siphon and gill tissue of old and young Laternula elliptica individuals under control and experimental conditions

Future oceans are predicted to contain less oxygen than at present. This is because oxygen is less soluble in warmer water and predicted stratification will reduce mixing. Hypoxia in marine environments is thus likely to become more widespread in marine environments and understanding species-responses is important to predicting future impacts on biodiversity. This study used a tractable model, the Antarctic clam, Laternula elliptica, which can live for 36 years, and has a well-characterized ecology and physiology to understand responses to hypoxia and how the effect varied with age. Younger animals had a higher condition index, higher adenylate energy charge and transcriptional profiling indicated that they were physically active in their response to hypoxia, whereas older animals were more sedentary, with higher levels of oxidative damage and apoptosis in the gills. These effects could be attributed, in part, to age-related tissue scaling; older animals had proportionally less contractile muscle mass and smaller gills and foot compared with younger animals, with consequential effects on the whole-animal physiological response. The data here emphasize the importance of including age effects, as large mature individuals appear to be less able to resist hypoxic conditions and this is the size range that is the major contributor to future generations. Thus, the increased prevalence of hypoxia in future oceans may have marked effects on benthic organisms' abilities to persist and this is especially so for long-lived species when predicting responses to environmental perturbation.

Effect of the effluent released from the canine internal mammary artery after intraluminal and extraluminal perfusion of acetylcholine and adenosine diphosphate

Segments of the canine internal mammary artery (35 mm in length) were suspended in vitro in an organ chamber containing physiological salt solution (95% O(2)/5% CO(2), pH = 7.4, 37 degrees C). Segments were individually cannulated and perfused at 5 ml/minute using a roller pump. Vasorelaxant activity of the effluent from the perfused internal mammary arteries was bioassayed by measuring the decrease in tension induced by the effluent of the coronary artery endothelium-free ring which had been contracted with prostaglandin F(2 alpha) (2 x 10(-6) M). Intraluminal perfusion of adenosine diphosphate (10(-5) M) induced significant increase in relaxant activity in the effluent from the perfused blood vessel. However, when adenosine diphosphate (10(-5) M) was added extraluminally to the internal mammary artery, no change in relaxant activity in the effluent was noted. In contrast, acetylcholine produced significant increase in the relaxant activity on the effluent of the perfused internal mammary artery with both intraluminal and extraluminal perfusion. The intraluminal and extraluminal release of endothelium-derived relaxing factor (EDRF) by acetylcholine (10(-5) M) can be inhibited by site-specific administration of atropine (10(-5) M). These experiments indicate that certain agonists can induce the release of EDRF only by binding to intravascular receptors while other agonists can induce endothelium-dependent vasodilatation by acting on neural side receptors.

Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors as Therapeutics: Rationales, Controversies, Clinical Experience.

Nicotinamide adenine dinucleotide (NAD+) biosynthesis from nicotinamide is used by mammalian cells to replenish their NAD+ stores and to avoid unwanted nicotinamide accumulation. Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in this biosynthetic pathway, almost invariably leads to intracellular NAD+ depletion and, when protracted, to ATP shortage and cell demise. Cancer cells and activated immune cells express high levels of NAMPT and are highly susceptible to NAMPT inhibitors, as shown by the activity of these agents in models of malignant and inflammatory disorders. As the spectrum of conditions which could benefit from pharmacological NAMPT inhibition becomes broader, the mechanisms accounting for their activity are also eventually becoming apparent, including the induction of autophagy and the impairment of Ca(2+) - and NF-κB-dependent signaling. Here, we discuss the rationales for exploiting NAMPT inhibitors in cancer and inflammatory diseases and provide an overview of the preclinical and clinical studies in which these agents have been evaluated.

Identification of the nicotinamide mononucleotide adenylyltransferase of Trypanosoma cruzi

The intracellular parasite Trypanosoma cruzi is the aetiological agent of Chagas disease, a public health concern with an increasing incidence rate. This increase is due, among other reasons, to the parasite217;s drug resistance mechanisms, which require nicotinamide adenine dinucleotide (NAD+). Furthermore, this molecule is involved in metabolic and intracellular signalling processes necessary for the survival of T. cruzi throughout its life cycle. NAD+ biosynthesis is performed by de novo and salvage pathways, which converge on the step that is catalysed by the enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (enzyme commission number: 2.7.7.1). The identification of the NMNAT of T. cruzi is important for the development of future therapeutic strategies to treat Chagas disease. In this study, a hypothetical open reading frame (ORF) for NMNAT was identified in the genome of T. cruzi. The corresponding putative protein was analysed by simulating structural models. The ORF was amplified from genomic DNA by polymerase chain reaction and was further used for the construction of a corresponding recombinant expression vector. The expressed recombinant protein was partially purified and its activity was evaluated using enzymatic assays. These results comprise the first identification of an NMNAT in T. cruzi using bioinformatics and experimental tools and hence represent the first step to understanding NAD+ metabolism in these parasites.

Impact de facteurs sanguins et d'agents thérapeutiques sur la survie de fibroblastes de sujets atteints de la forme canadienne-française du syndrome de Leigh (LSFC)

La forme canadienne-française du syndrome de Leigh (LSFC) est une maladie métabolique associée à une déficience en cytochrome oxydase (COX) et caractérisée par des crises d’acidose lactique, menant à une mort prématurée. Les mécanismes qui sous-tendent l’induction des crises restent inconnus et il n’existe aucune thérapie efficace pour les prévenir. Cette étude vise à caractériser l'effet de facteurs métaboliques périphériques potentiellement altérés chez les patients LSFC sur la mort de lignées cellulaires issues de ces patients et de témoins puis, à identifier des agents thérapeutiques pouvant la prévenir. Nous postulons que (i) ces facteurs métaboliques induiront une mort prématurée des cellules de patients et que (ii) les interventions susceptibles de la prévenir pallieront les conséquences de la déficience en COX, soit la diminution des taux d’adénosine triphosphate (ATP) et l’augmentation du stress oxydant, du nicotinamide adénine dinucléotide (NADH) et des lipides toxiques. Un criblage de 8 facteurs sanguins et 10 agents thérapeutiques a été réalisé. Les paramètres mesurés incluent la nécrose, l’apoptose, l’ATP et l’activité de la COX. Les fibroblastes LSFC sont plus susceptibles à la mort par nécrose (39±6%) induite par du palmitate plus lactate, un effet associé à des niveaux d’ATP diminués (53±8%). La mort cellulaire est réduite de moitié par l’ajout combiné d’agents ciblant le NADH, l’ATP et les lipides toxiques, alors que l’ajout d’antioxydants l’augmente. Ainsi, un excès de nutriments pourrait induire la mort prématurée des cellules LSFC et, pour atténuer cette mort, il serait important de combiner plusieurs interventions ciblant différents mécanismes.

Therapeutic benefit of low-dose clopidogrel in patients undergoing carotid surgery is linked to variability in the platelet adenosine diphosphate response and patients' weight

BACKGROUND AND PURPOSE: We have previously shown that a single 75-mg tablet of clopidogrel, taken before carotid endarterectomy, significantly reduces postoperative embolization, a marker of thromboembolic stroke. This study explores the antiplatelet effect of this submaximal dose. METHODS: Fifty-six patients on long-term aspirin (150 mg) were randomized to 75 mg clopidogrel or placebo before carotid endarterectomy. Blood samples were taken pre- and postdrug administration and at the end of surgery to measure platelet activation and adenosine diphosphate (ADP) response by flow cytometry and aggregometry. RESULTS: Surgery produced a significant rise in platelet activation in vivo as evidenced by a rise in the percentage of monocyte-platelet aggregates in patients given placebo, but this was not seen in patients receiving clopidogrel. Before surgery, clopidogrel produced a significant reduction in the platelet response to ADP; for example, with 10(-6)M ADP, 77.32+/-2.3% bound fibrinogen in placebo group compared with 67.16+/-3.1% after clopidogrel (P=0.01). This was accentuated after surgery when the percentage of platelets binding fibrinogen in response to ADP was 76.53+/-2.2% in patients given placebo and 62.84+/-3.3% in the clopidogrel group (P=0.002). Similar differences were seen over a range of ADP concentrations and by aggregometry. Platelet responsiveness before treatment was highly variable and was positively correlated with the inhibitory effect of clopidogrel; patients with the highest baseline response to ADP showed the greatest response to clopidogrel. A negative correlation was seen between the effect of clopidogrel and patients' weight (r=0.57; P=0.002). CONCLUSIONS: These results explain how a single 75-mg dose of clopidogrel produces a significant clinical impact on embolization.

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 Å resolution

Background: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldose-ketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. Results: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 Å resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. Conclusions: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Morphological and histochemical analysis of the human vestibular fold

A morphological and histochemical study of the human vestibular fold was carried out using routine histological techniques. Seven μm-thick histological sections stained with hematoxylin-eosin (HE) and Calleja showed the presence of elastic collagen fibers and seromucous glands in the vestibular fold. Muscle fibers forming the ventricular muscle were also identified. Ultrastructural analyses of the epithelial layer by scanning electron microscopy (SEM) revealed ciliated cells and gland ducts opening on the epithelial surface. Histochemical analyses were performed on ventricular muscles submitted to nicotinamide-adenine-dinucleotide tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), and myofibrillar adenosine triphosphatase (mATPase) reactions. Based on these reactions, it was observed that the muscle is formed by three types of muscle fibers: slow-twitch oxidative (SO), fast-twitch oxydative glycolytic (FOG) and fast-twitch glycolytic (FG) fibers distributed in a mosaic pattern. The fiber frequency was 22.7%, 69.9% and 7.4%, respectively. The higher frequency of SO and FOG fibers characterized the muscle as having aerobic metabolism and resistance to fatigue. The ventricular muscle was considered fast. The study of the neuromuscular junctions performed after nonspecific esterase reaction showed that they are of the en-plaque type and have multiple occurrences in the ventricular muscle.