NAD hydrolysis: Chemical and enzymatic mechanisms

The pyridine nucleotide have important non-redox activities as cellular effectors and metabolic regulators [1–3]. The enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond of NAD⁺ and the attendant delivery of the ADPRibosyl moiety to acceptors is central to these many diverse biological activities. Included are the medically important NAD-dependent toxins associated with cholera, diphtheria, pertussis, and related disease [4]; the reversible ADPRibosylation-mediated biological regulatory systems [5,6]; the synthesis of poly (ADPRibose) in response to DNA damage or cellular, division [7]; and the synthesis of cyclic ADPRibose as part of an independent, calcium-mediated regulatory system[8]. As will be presented in this chapter, all evidence points to both the chemical and enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond being dissociative in character via an oxocarbenium intermediate.     

Effects of nicotinamide on NAD and poly (ADP-ribose) metabolism in DNA-damaged human lymphocytes

The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD⁺ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treat-ment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD⁺ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.     

5-alkylvinyl-1,2,4-triazole nucleosides: Synthesis and biological evaluation

Abstract

Some 5-substituted ribavirin analogues have a high antiviral and anticancer activity, but their mechanisms of action are obviously not the same as their parent compound. The SAR studies performed on 3 (5)-substituted 1,2,4-triazole nucleosides have shown a high dependency between the structure of the 3 (5)-substituent and the level of antiviral/anticancer activity. The most active substances of the row contain coplanar with the 1,2,4-triazole ring aromatic substituent which is connected by a rigid ethynyl bond. However, the compounds with the trans-vinyl linker also had antiviral activity. We decided to study the antitumor activity of ribavirin analogues with alkyl/aryl vinyl substituents in the 5th position of the 1,2,4-triazole ring. Protected nucleoside analogues with various 5-alkylvinyl substituents were obtained by Horner-Wadsworth-Emmons reaction from the common precursor and converted to the nucleosides. Arylvinyl nucleosides were synthesised according the reported procedures. All compounds did not show significant antiproliferative activity on several tumour cell lines. Coplanar aromatic motif in the 5-substituent for the anticancer activity manifestation was confirmed.     

Development of near-infrared firefly luciferin analogue reacted with wild-type and mutant luciferases

Interestingly, only the D-form of firefly luciferin produces light by luciferin–luciferase (L–L) reaction. Certain firefly luciferin analogues with modified structures maintain bioluminescence (BL) activity; however, all L-form luciferin analogues show no BL activity. To this date, our group has developed luciferin analogues with moderate BL activity that produce light of various wavelengths. For in vivo bioluminescence imaging, one of the important factors for detection sensitivity is tissue permeability of the number of photons emitted by L–L reaction, and the wavelengths of light in the near-infrared (NIR) range (700–900 nm) are most appropriate for the purpose. Some NIR luciferin analogues by us had performance for in vivo experiments to make it possible to detect photons from deep target tissues in mice with high sensitivity, whereas only a few of them can produce NIR light by the L–L reactions with wild-type luciferase and/or mutant luciferase. Based on the structure–activity relationships, we designed and synthesized here a luciferin analogue with the 5-allyl-6-dimethylamino-2-naphthylethenyl moiety. This analogue exhibited NIR BL emissions with wild-type luciferase (λ_max = 705 nm) and mutant luciferase AlaLuc (λ_max = 655 nm).     

Pitfalls of using lucigenin in endothelial cells: Implications for NAD(P)H dependent superoxide formation

Since an increased endothelial superoxide formation plays an important role in the pathogenesis of endothelial dysfunction its specific detection is of particular interest. The widely used superoxide probe lucigenin, however, has been reported to induce superoxide under certain conditions, especially in the presence of NADH. This raises questions as to the conclusion of a NAD(P)H oxidase as the major source of endothelial superoxide. Using independent methods, we showed that lucigenin in the presence of NADH leads to the production of substantial amount of superoxide (～ 15-fold of control) in endothelial cell homogenates. On the other hand, these independent methods revealed that endothelial cells without lucigenin still produce superoxide in a NAD(P)H-dependent manner. This was blocked by inhibitors of the neutrophil NADPH oxidase diphenyleniodonium and phenylarsine oxide. Our results demonstrate that a NAD(P)H-dependent oxidase is an important source for endothelial superoxide but the latter, however, cannot be measured reliably by lucigenin.     

Vitamin K, 2,3-Epoxide And Quinone Reduction: Mechanism And Inhibition

The chemical and enzymatic pathways of vitamin K₁ epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase. E.C. I.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes. but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K₁ epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K, 10^?5 as rapidly as it reduced dichlorophenylindophenol(DCPIP). Reduction of 10 μM vitamin K, by200 μM NADH was not inhibited by 10μM dicoumarol. whereas DCPIP reduction was fully inhibited. In contrast to vitamin K, (menadione). vitamin K₁ (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions. suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K₁ epoxide and quinone. and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin. lapachol. and sulphaquinoxaline     

Anti-hypertensive effects of Rosuvastatin are associated with decreased inflammation and oxidative stress markers in hypertensive rats

Among their pleiotropic effects, statins exert antioxidant and anti-inflammatory properties. The aim of this study was to evaluate in normotensive (WKY) and in spontaneously hypertensive rats (SHR) the effect of rosuvastatin (ROSU) treatment on (1) plasma inflammation markers and endogenous NO synthase inhibitor (ADMA) levels, (2) reactive oxygen species (ROS) generated by circulating leukocytes and (3) vascular oxidative stress and tissue inflammation markers. Plasma cytokines were higher in SHR than in WKY, except for IL-4, which was lower in SHR than in WKY. SHR monocytes exhibited higher production of ROS than did WKY monocytes. In the experimental conditions, ROSU did not modify plasma cholesterol levels in SHR but attenuated the increase in systolic blood pressure. In SHR only, ROSU lessened pro-inflammatory cytokines and ADMA levels, increased IL-4 and reduced ROS production in circulating monocytes. These results demonstrate the beneficial effects of ROSU in SHR, independently of any lowering of cholesterol levels.     

Interactions of vasopressin and oxytocin receptors with vasopressin analogues substituted in position 2 with 3,3′‐diphenylalanine – a molecular docking study

Vasopressin and oxytocin receptors belong to the superfamily of G protein‐coupled receptors and play an important role in many physiological functions. They are also involved in a number of pathological conditions being important drug targets. In this work, four vasopressin analogues substituted at position 2 with 3,3′‐diphenylalanine have been docked into partially flexible vasopressin and oxytocin receptors. The bulky residue at position 2 acts as a structural restraint much stronger in the oxytocin receptor (OTR) than in the vasopressin V2 receptor (V2R), resulting in a different location of the analogues in these receptors. This explains the different, either agonistic or antagonistic, activities of the analogues in V2R and OTR, respectively. In all complexes, the conserved polar residues serve as anchor points for the ligand both in OTR and V2R. Strong interactions of the C‐terminus of analogue II ([Mpa¹,d ‐Dpa²,Val⁴,d ‐Arg⁸]VP) with extracellular loop 3 may be responsible for its highest activity at V2R. It also appears that V2R adapts more readily to the docking analogues by conformational changes in the aromatic side chains triggering receptor activation. A weak activity at V1a vasopressin receptor appears to be caused by weak receptor–ligand interactions. Results of this study may facilitate a rational design of new analogues with the highest activity/selectivity at vasopressin and OTRs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Purification and Characterization op a 31-Kilodalton Iron-Regulated Periplasmic Protein from Pasteurella Haenolytica A1

ABSTRACT

A prominent iron-regulated periplasmic protein was purified from Pasteurella haemolytica grown in an iron-deficient chemically defined medium. The protein was purified by anion exchange chromatography and appeared as a single band by SDS-PAGE with a molecular weight of 32,000. A yield of five mg was obtained from 91 mg of protein extract. The iron-regulated protein existed as a monomer in the native state with an average molecular weight of 29,877 as determined by analytical ultracentrifugation. The protein had a molecular weight of 30,880 as determined by matrix-assisted laser desorption mass spectrometry, hence the protein is referred to as the 31 kDa protein. Isoelectric focusing showed four bands with pIs of 7.15, 6.8, 6.6, and 5.9, The secondary structure of the protein was determined by circular dichroism and contained 16% α-helical structure. The N-terminal sequence, EPFKVVTTFTVIQDIAQNVAGDKAT, showed a 95% identity with the 31 kDa iron-binding protein from Haemophilus influenzae. Isolation and characterization of iron-regulated proteins are of particular interest because of their potential roles in iron assimilation and microbial virulence.