Science.gov

Sample records for acid dihydrate nad

  1. Infrared spectrum of nitric acid dihydrate: Influence of particle shape.

    PubMed

    Wagner, Robert; Möhler, Ottmar; Saathoff, Harald; Stetzer, Olaf; Schurath, Ulrich

    2005-03-24

    In situ Fourier transform infrared (FTIR) extinction spectra of airborne alpha-NAD microparticles generated by two different methods were recorded in the large coolable aerosol chamber AIDA of Forschungszentrum Karlsruhe. The extinction spectrum of alpha-NAD crystals obtained by shock freezing of a HNO3/H2O gas mixture could be accurately reproduced using Mie theory with published refractive indices of alpha-NAD as input. In contrast, Mie theory proved to be inadequate to properly reproduce the infrared extinction spectrum of alpha-NAD crystals which were formed via homogeneous nucleation of supercooled HNO3/H2O solution droplets, evaporating slowly on a time scale of several hours at about 195 K. Much better agreement between measured and calculated extinction spectra was obtained by T-matrix calculations assuming oblate particles with aspect ratios greater than five. This indicates that strongly aspherical alpha-NAD crystals are obtained when supercooled nitric acid solution droplets freeze and grow slowly, a process which has been discussed as a potential pathway to the formation of crystalline polar stratospheric cloud (PSC) particles. PMID:16833561

  2. Precipitation diagrams and solubility of uric acid dihydrate

    NASA Astrophysics Data System (ADS)

    Babić-Ivančić, V.; Füredi-Milhofer, H.; Brown, W. E.; Gregory, T. M.

    1987-07-01

    The solubility of uric acid dihydrate (UA·2H 2O) and the precipitation of UA·2H 2O and anhydrous uric acid (UA) from solutions containing sodium hydroxide and hydrochloric acid have been investigated. For the solubility studies, crystals of pure UA·2H 2O were prepared and equilibrated with water and with solutions of HCl or NaOH for 60 min or 20 h, respectively. The equilibrium pH (pH = 2-6.25) and uric acid concentration were determined. For the precipitation experiments, commercial UA was dissolved in NaOH in a 1:1.1 molar ratio and UA·2H 2O and/or UA were precipitated with hydrochloric acid. The precipitates and/or supernatants were examined 24 h after sample preparation. The results are represented in the form of tables, precipitation diagrams and "chemical potential" diagrams. Solubility measurements with 60 min equilibration times yielded the solubility products of UA·2H 2O, K sp(298 K) = (0.926 ± 0.025) × 10 -9mol2dm-6 and K sp(310 K) = (2.25 ± 0.05) × 10 -9mol2dm-6 and the first dissociation constants of uric acid, K 1(298 K) = (2.45 ± 0.07) × 10 -6moldm-3 and K 1(310 K) = (3.63 ± 0.08) × 10 -6moldm-3. Precipitation diagrams show that under the given experimental conditions, at 298 K, UA·2H 2O is stable for 24 h while at 310 K this was true only for precipitates formed from solutions of high supersaturations. At lower supersaturations, mixtures of UA·2H 2O and UA formed. Consequently, while the Ksp value determined from precipitation data obtained at 298 K (K sp = 1.04 × 10 -9mol2dm-6) was consistent with the respective solubility product, the 310 K precipitation boundary yielded an ion activity product, AP, the value of which fulfills the conditions Ksp(UA) < AP < Ksp (UA·2H 2O). Similar ion activity products were obtained from solubility measurements in pure water at 20 h equilibration time.

  3. NAD

    PubMed Central

    Pétriacq, Pierre; de Bont, Linda; Tcherkez, Guillaume; Gakière, Bertrand

    2013-01-01

    Many metabolic processes that occur in living cells involve oxido-reduction (redox) chemistry underpinned by redox compounds such as glutathione, ascorbate and/or pyridine nucleotides. Among these redox carriers, nicotinamide adenine dinucleotide (NAD) is the cornerstone of cellular oxidations along catabolism and is therefore essential for plant growth and development. In addition to its redox role, there is now compelling evidence that NAD is a signal molecule controlling crucial functions like primary and secondary carbon metabolism. Recent studies using integrative -omics approaches combined with molecular pathology have shown that manipulating NAD biosynthesis and recycling lead to an alteration of metabolites pools and developmental processes, and changes in the resistance to various pathogens. NAD levels should now be viewed as a potential target to improve tolerance to biotic stress and crop improvement. In this paper, we review the current knowledge on the key role of NAD (and its metabolism) in plant responses to pathogen infections. PMID:23104110

  4. Bis(melaminium) succinate succinic acid monosolvate dihydrate

    PubMed Central

    Froschauer, Barbara; Weil, Matthias

    2012-01-01

    The asymmetric unit of the solvated title salt, 2C3H7N6 +·C4H4O4 2−·C4H6O4·2H2O, contains one essentially planar melaminium (2,4,6-triamino-1,3,5-triazin-1-ium) cation (r.m.s. deviation of the non-H atoms = 0.0097 Å), one-half of a succinate anion, one-half of a succinic acid solvent mol­ecule and one water molecule of crystallization; full mol­ecules are generated by inversion symmetry. Supra­molecular layers parallel to (12-1) are formed through extensive inter­molecular hydrogen bonding of the types O—H⋯O, N—H⋯N and N—H⋯O between the components. PMID:22904985

  5. A Neat Trick Using Oxalic Acid Dihydrate and Potassium Permanganate and Other Experiments with Small Organic Amine or Oxygenated Compounds

    ERIC Educational Resources Information Center

    Kelland, Malcolm A.

    2011-01-01

    Solid potassium permanganate (KMnO[subscript 4]) is shown to react in a variety of ways with small organic amines or oxygenated compounds depending on whether they are liquids or solids and whether water is present. In particular, its reaction with solid oxalic acid dihydrate can be initiated by the moisture in one's breath, making an intriguing…

  6. 5-Sulfosalicylic acid dihydrate-based pretreatment for the modification of enzyme-linked immunoassay of fluoroquinolones in fishery products.

    PubMed

    Cui, Mengqi; Lin, Hong; Wang, Xiudan; Cao, Limin; Sui, Jianxin

    2015-01-01

    A simple, rapid sample extraction method for the determination of FQs was developed. Fishery samples were extracted with 2% of 5-sulfosalicylic acid dihydrate and the extracts were analyzed directly without any further purification or clean-up procedures. The FQs were determined with standards of 2% of 5-sulfosalicylic acid dihydrate in the concentration range of 0.1-25.6 μg L(-1), and the limit of detection (LOD) was 0.1 μg L(-1). The matrix interference originated from fishery samples was eliminated by 2% of 5-sulfosalicylic acid dihydrate and did not interact with horseradish peroxidase (HRP) labeled IgG in western blotting. No significant matrix interference was observed as samples extracted with 2% of 5-sulfosalicylic acid dihydrate. Recoveries of FQs in fishery muscle were between 72.37-94.35% in the concentrations range of 10-50 μg kg(-1).This extraction procedure was much rapider and simpler to conventional ELISA extraction procedure and could be used as a time-saving and cost-effective method for FQs monitoring in fishery samples.

  7. Evidence of Polaron Excitations in Low Temperature Raman Spectra of Oxalic Acid Dihydrate.

    PubMed

    Mohaček-Grošev, Vlasta; Grdadolnik, Jože; Hadži, Dušan

    2016-05-12

    Low temperature Raman spectra of oxalic acid dihydrate (8-300 K) for both the polycrystalline and single crystal phase show strong variation with temperature in the interval from 1200 to 2000 cm(-1). Previous low temperature diffraction studies all confirmed the stability of the crystal P21/n phase with no indications of any phase transition, reporting the existence of a strong hydrogen bond between the oxalic acid and a water molecule. A new group of Raman bands in the 1200-1300 cm(-1) interval below 90 K is observed, caused by possible loss of the center of inversion. This in turn could originate either due to disorder in hydroxyl proton positions or due to proton transfer from carboxylic group to water molecule. The hypothesis of proton transfer is further supported by the emergence of new bands centered at 1600 and 1813 cm(-1), which can be explained with vibrations of H3O(+) ions. The broad band at 1600 cm(-1) looses intensity, while the band at 1813 cm(-1) gains intensity on cooling. The agreement between quantum calculations of vibrational spectra and experimentally observed Raman bands of hydronium ions in oxalic acid sesquihydrate crystal corroborates this hypothesis. PMID:27093217

  8. Synthesis and bioactivities of silver nanoparticles capped with 5-Amino-?-resorcylic acid hydrochloride dihydrate

    PubMed Central

    2014-01-01

    Background Conjugated and drug loaded silver nanoparticles are getting an increased attention for various biomedical applications. Nanoconjugates showed significant enhancement in biological activity in comparison to free drug molecules. In this perspective, we report the synthesis of bioactive silver capped with 5-Amino-?-resorcylic acid hydrochloride dihydrate (AR). The in vitro antimicrobial (antibacterial, antifungal), enzyme inhibition (xanthine oxidase, urease, carbonic anhydrase, ?-chymotrypsin, cholinesterase) and antioxidant activities of the developed nanostructures was investigated before and after conjugation to silver metal. Results The conjugation of AR to silver was confirmed through FTIR, UV¿vis and TEM techniques. The amount of AR conjugated with silver was characterized through UV¿vis spectroscopy and found to be 9% by weight. The stability of synthesized nanoconjugates against temperature, high salt concentration and pH was found to be good. Nanoconjugates, showed significant synergic enzyme inhibition effect against xanthine and urease enzymes in comparison to standard drugs, pure ligand and silver. Conclusions Our synthesized nanoconjugate was found be to efficient selective xanthine and urease inhibitors in comparison to Ag and AR. On a per weight basis, our nanoconjugates required less amount of AR (about 11 times) for inhibition of these enzymes. PMID:25201390

  9. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition.

    PubMed

    Bogan, Katrina L; Brenner, Charles

    2008-01-01

    Although baseline requirements for nicotinamide adenine dinucleotide (NAD+) synthesis can be met either with dietary tryptophan or with less than 20 mg of daily niacin, which consists of nicotinic acid and/or nicotinamide, there is growing evidence that substantially greater rates of NAD+ synthesis may be beneficial to protect against neurological degeneration, Candida glabrata infection, and possibly to enhance reverse cholesterol transport. The distinct and tissue-specific biosynthetic and/or ligand activities of tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD+ precursor, nicotinamide riboside, reviewed herein, are responsible for vitamin-specific effects and side effects. Because current data suggest that nicotinamide riboside may be the only vitamin precursor that supports neuronal NAD+ synthesis, we present prospects for human nicotinamide riboside supplementation and propose areas for future research. PMID:18429699

  10. Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

    PubMed

    Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

    2015-11-01

    NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors. PMID:26385918

  11. Crystallization of dicalcium phosphate dihydrate with presence of glutamic acid and arginine at 37 °C.

    PubMed

    Li, Chengfeng; Ge, Xiaolu; Li, Guochang; Bai, Jiahai; Ding, Rui

    2014-08-01

    The formations of non-metabolic stones, bones and teeth were seriously related to the morphology, size and surface reactivity of dicalcium phosphate dihydrate (DCPD). Herein, a facile biomimetic mineralization method with presence of glutamic acid and arginine was employed to fabricate DCPD with well-defined morphology and adjustable crystallite size. In reaction solution containing more arginine, crystallization of DCPD occurred with faster rate of nucleation and higher density of stacked layers due to the generation of more OH(-) ions after hydrolysis of arginine at 37 °C. With addition of fluorescein or acetone, the consumption of OH(-) ions or desolvation reaction of Ca(2+) ions was modulated, which resulted in the fabrication of DCPD with adjustable crystallite sizes and densities of stacked layers. In comparison with fluorescein-loading DCPD, dicalcium phosphate anhydrate was prepared with enhanced photoluminescence properties due to the reduction of self-quenching effect and regular arrangement of encapsulated fluorescein molecules. With addition of more acetone, DCPD was prepared with smaller crystallite size via antisolvent crystallization. The simulated process with addition of amino acids under 37 °C would shed light on the dynamic process of biomineralization for calcium phosphate compounds.

  12. OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease.

    PubMed

    Akie, Thomas E; Liu, Lijun; Nam, Minwoo; Lei, Shi; Cooper, Marcus P

    2015-01-01

    OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance--ROS and PKCε activity--were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.

  13. Crystal structure of human aldehyde dehydrogenase 1A3 complexed with NAD+ and retinoic acid

    PubMed Central

    Moretti, Andrea; Li, Jianfeng; Donini, Stefano; Sobol, Robert W.; Rizzi, Menico; Garavaglia, Silvia

    2016-01-01

    The aldehyde dehydrogenase family 1 member A3 (ALDH1A3) catalyzes the oxidation of retinal to the pleiotropic factor retinoic acid using NAD+. The level of ALDHs enzymatic activity has been used as a cancer stem cell marker and seems to correlate with tumour aggressiveness. Elevated ALDH1A3 expression in mesenchymal glioma stem cells highlights the potential of this isozyme as a prognosis marker and drug target. Here we report the first crystal structure of human ALDH1A3 complexed with NAD+ and the product all-trans retinoic acid (REA). The tetrameric ALDH1A3 folds into a three domain-based architecture highly conserved along the ALDHs family. The structural analysis revealed two different and coupled conformations for NAD+ and REA that we propose to represent two snapshots along the catalytic cycle. Indeed, the isoprenic moiety of REA points either toward the active site cysteine, or moves away adopting the product release conformation. Although ALDH1A3 shares high sequence identity with other members of the ALDH1A family, our structural analysis revealed few peculiar residues in the 1A3 isozyme active site. Our data provide information into the ALDH1As catalytic process and can be used for the structure-based design of selective inhibitors of potential medical interest. PMID:27759097

  14. Structure-function studies of human deoxyhypusine synthase: identification of amino acid residues critical for the binding of spermidine and NAD.

    PubMed

    Lee, C H; Um, P Y; Park, M H

    2001-05-01

    Deoxyhypusine synthase catalyses the first step in the biosynthesis of hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. The crystal structure of human deoxyhypusine synthase in complex with NAD revealed four NAD-binding sites per enzyme tetramer, and led to a prediction of the spermidine-binding pocket. We have replaced each of the seven amino acid residues at the predicted spermidine-binding site, and eleven residues that contact NAD, on an individual basis with alanine. Of the amino acid residues at the spermidine site, substitution of Asp-243, Trp-327, His-288, Asp-316 or Glu-323 with alanine caused an almost complete loss of spermidine binding and enzyme activity; only the mutation Tyr-305-->Ala showed partial binding and activity. His-288-->Ala was also deficient in terms of binding NAD. NAD binding was significantly reduced in all of the NAD-site mutant enzymes, except for Glu-137-->Ala, which showed a normal binding of NAD, but was totally lacking in spermidine binding. Of the NAD-site mutant enzymes, Asp-342-->Ala, Asp-313-->Ala and Asp-238-->Ala displayed the lowest binding of NAD. These enzymes and His-288Ala also showed a reduced binding of spermidine, presumably because spermidine binding is dependent on NAD. These findings permit the positive identification of amino acid residues critical for binding of spermidine and NAD, and provide a new insight into the complex molecular interactions involved in the deoxyhypusine synthase reaction. PMID:11311149

  15. Thermochemical Kinetics of H2O and HNO3 on crystalline Nitric Acid Hydrates (alpha-, beta-NAT, NAD) in the range 175-200 K

    NASA Astrophysics Data System (ADS)

    Rossi, Michel J.; Iannarelli, Riccardo

    2015-04-01

    The growth of NAT (Nitric Acid Trihydrate, HNO3x3H2O) and NAD (Nitric Acid Dihydrate, HNO3x2H2O) on an ice substrate, the evaporative lifetime of NAT and NAD as well as the interconversion of alpha- and beta-NAT competing with evaporation and growth under UT/LS conditions depends on the interfacial kinetics of H2O and HNO3 vapor on the condensed phase. Despite the existence of some literature results we have embarked on a systematic investigation of the kinetics using a multidiagnostic experimental approach enabled by the simultaneous observation of both the gas (residual gas mass spectrometry) as well as the condensed phase (FTIR absorption in transmission). We report on thermochemically consistent mass accommodation coefficients alpha and absolute evaporation rates Rev/molecule s-1cm-3 as a function of temperature which yields the corresponding vapor pressures of both H2O and HNO3 in equilibrium with the crystalline phases, hence the term thermochemical kinetics. These results have been obtained using a stirred flow reactor (SFR) using a macroscopic pure ice film of 1 micron or so thickness as a starting substrate mimicking atmospheric ice particles and are reported in a phase diagram specifically addressing UT (Upper Troposphere)/LS (Lower Stratosphere) conditions as far as temperature and partial pressures are concerned. The experiments have been performed either at steady-state flow conditions or in transient supersaturation using a pulsed solenoid valve in order to generate gas pulses whose decay were subsequently monitored in real time. Special attention has been given to the effect of the stainless-steel vessel walls in that Langmuir adsorption isotherms for H2O and HNO3 have been used to correct for wall-adsorption of both probe gases. Typically, the accommodation coefficients of H2O and HNO3 are similar throughout the temperature range whereas the rates of evaporation Rev of H2O are significantly larger than for HNO3 thus leading to the difference in

  16. Studies on the syntheses, structural characterization, antimicrobial-, and DPPH radical scavenging activity of the cocrystals caffeine:cinnamic acid and caffeine:eosin dihydrate

    NASA Astrophysics Data System (ADS)

    Suresh Kumar, G. S.; Seethalakshmi, P. G.; Bhuvanesh, N.; Kumaresan, S.

    2013-10-01

    Two organic cocrystals namely, caffeine:cinnamic acid [(caf)(ca)] (1) and caffeine:eosin dihydrate [(caf)(eos)]·2H2O (2) were synthesized and studied by FT-IR, TGA/DTA, and single crystal XRD. The crystal system of cocrystal 1 is triclinic with space group P-1 and Z = 2 and that of cocrystal 2 is monoclinic with space group P21/C and Z = 4. An imidazole-carboxylic acid synthon is observed in the cocrystal 1. The intermolecular hydrogen bond, O-H⋯N and π-π interactions play a major role in stabilizing 1 whereas the intermolecular hydrogen bonds, O-H⋯O, O-H⋯N, and intramolecular hydrogen bond, O-H⋯Br; along with π-π interactions together play a vital role in stabilizing the structure of 2. The antimicrobial- and DPPH radical scavenging activities of both the cocrystals were studied.

  17. Speciation in experimental C-O-H fluids produced by the thermal dissociation of oxalic acid dihydrate

    USGS Publications Warehouse

    Morgan, G.B.; Chou, I.-Ming; Pasteris, J.D.

    1992-01-01

    Fluid speciations and their related reaction pathways were studied in C-O-H-system fluids produced by the thermal dissociation of oxalic acid dihydrate (OAD: H2C2O4 ?? 2H2O) sealed in silica glass capsules. Experiments were conducted in the temperature range 230-750??C, with bulk fluid densities in the range 0.01-0.53 g/cm3. Pressure was controlled by temperature and density in the isochoric systems. The quenched products of dissociation experiments were an aqueous liquid and one (supercritical fluid) or, rarely, two (vapor plus liquid) carbonic phase (s). In-situ Raman microanalyses were performed on the quenched carbonic phases at room temperature, at which fluid pressures ranged from about 50 to 340 bars. Bulk fluid speciations were reconstructed from the Raman analyses via mass balance constraints, and appear to monitor the true fluid speciations at run conditions. In experiments from the lowtemperature range (230-350??C), fluid speciations record the dissociation of OAD according to the reaction OAD = CO2 + CO + 3H2O. A process of the form CO + H2O = CO2 + H2 is driven to the right with increasing temperature. The hydrogen gas produced tends to escape from the sample systems via diffusion into/through the silica glass capsules, shifting bulk compositions toward equimolar binary H2O-CO2 mixtures. The speciations of fluids in experiments with minimal hydrogen loss show poor agreement with speciations calculated for equilibrium fluids by the corresponding-states model of Saxena and Fei (1988). Such disagreement suggests that the formations of CH4 and graphite are metastably inhibited in the current experiments, which correlates with their absence or trivial abundances in experimental products. Moreover, calculations in which the stabilities of methane and graphite are suppressed suggest that such metastable equilibrium is approached only in experiments at temperatures greater than about 600-650??C. These results have applications to fluid processes in geological

  18. NMR spectra and potentiometry studies of aluminum(III) binding with coenzyme NAD+ in acidic aqueous solutions.

    PubMed

    Yang, Xiaodi; Bi, Shuping; Yang, Xiaoliang; Yang, Li; Hu, Jun; Liu, Jian; Yang, Zhengbiao

    2003-06-01

    Complexation and conformational studies of coenzyme NAD+ with aluminum were conducted in acidic aqueous solutions (pH 2-5) by means of potentiometry as well as multinuclear (1H, 13C, 31P, 27Al) and two-dimensional (1H, 1H-NOESY) NMR spectroscopy. These led to the following results: (1) Al could coordinate with NAD+ through the following binding sites: N7' of adenine and pyrophosphate free oxygen (O(A)1, O(N)1,O(A)2) to form various mononuclear 1:1 (AlLH23+, AlLH2+) and 2:1 (AlL2-) species, and dinuclear 2:2 (Al2L22+) species. (2) The conformations of NAD+ and Al-NAD+ depended on the solvents and different species in the complexes. The results suggest the occurrence of an Al-linked complexation, which causes structural changes at the primary recognition sites and secondary conformational alterations for coenzymes. This finding will help us to understand role of Al in biological enzyme reaction systems.

  19. A Study of the Monohydrate and Dihydrate Complexes of Perfluoropropionic Acid Using Chirped-Pulse Fourier Transform Microwave (CP-FTMW) Spectroscopy.

    PubMed

    Grubbs, G S; Obenchain, Daniel A; Frank, Derek S; Novick, Stewart E; Cooke, S A; Serrato, Agapito; Lin, Wei

    2015-10-22

    This work reports the first known spectroscopic observation of the monohydrate and dihydrate complexes of perfluoropropionic acid (PFPA). The spectra have been observed using a chirped-pulse Fourier transform microwave (CP-FTMW) spectrometer in the 7750 to 14,250 MHz region. The structures of the species have been confirmed with the aid of ab initio quantum chemical calculations. Rotational constants A, B, and C have been determined and reported for both species along with centrifugal distortion constants ΔJ, ΔJK, ΔK, δJ, δK for H2O-PFPA and ΔJ, ΔJK, and δJ for (H2O)2-PFPA. Effects due to large amplitude motions were not observable in these experiments. Structures of the complexes have been determined using a combination of experimental second moment values and ab initio results. The complexation of the -OH of one or two water molecules has been found to occur in the plane of the carboxylic acid group forming a six- or eight-member ring.

  20. Dehydration behavior of eprosartan mesylate dihydrate.

    PubMed

    Sheng, J; Venkatesh, G M; Duddu, S P; Grant, D J

    1999-10-01

    Eprosartan mesylate (SKF 108566-J; EM) is an antihypertensive agent approved for marketing in the USA. EM dihydrate was prepared by three methods, one of which included suspending the anhydrous drug in an aqueous solution of 1.0 M methanesulfonic acid to form a slurry, followed by filtration. The dehydration kinetics of EM dihydrate were derived by analyzing the fit of the isothermal thermogravimetric analytical (TGA) data to numerous kinetic models. EM dihydrate undergoes dehydration in two distinct steps, each involving the loss of 1 mol of water at 25-70 degrees C and 70-120 degrees C, respectively. Recrystallization of EM occurs at approximately 120-140 degrees C after dehydration to the anhydrous phase. This explanation is supported by variable temperature powder X-ray diffractometry. The mechanism of the dehydration reaction is complex, the dependence of the reaction rate on temperature varying as a function of the particles size. For the dihydrate of sieve fraction <125 microm, the kinetics of the first and second dehydration steps are consistent with the Avrami-Erofeev equation (A3, n = 1/3) over the temperature range studied, corresponding to three-dimensional growth of nuclei. In contrast, for the 125-180-microm and 180-250-microm sieve fractions, the kinetics are best described by the two-dimensional phase boundary reaction (R2) at a lower dehydration temperature (i.e., 28.3 degrees C), and by the Avrami-Erofeev equation (A3, n = 1/3) at a higher dehydration temperature (i.e., 93.7 degrees C). The activation energies (15-40 kcal/mol) and frequency factors of the dehydration of EM dihydrate were determined both by Arrhenius plots of the isothermal rates determined by TGA and by Kissinger plots of the nonisothermal differential scanning calorimetric data. Hot stage microscopy of single crystals of EM dihydrate showed random nucleation at the surface and dehydration with the growth of microcrystals along the needle a axis. Cerius(2) molecular modeling

  1. Fabrication of Poly-l-lactic Acid/Dicalcium Phosphate Dihydrate Composite Scaffolds with High Mechanical Strength-Implications for Bone Tissue Engineering.

    PubMed

    Tanataweethum, Nida; Liu, Wai Ching; Goebel, W Scott; Li, Ding; Chu, Tien Min

    2015-11-04

    Scaffolds were fabricated from poly-l-lactic acid (PLLA)/dicalcium phosphate dihydrate (DCPD) composite by indirect casting. Sodium citrate and PLLA were used to improve the mechanical properties of the DCPD scaffolds. The resulting PLLA/DCPD composite scaffold had increased diametral tensile strength and fracture energy when compared to DCPD only scaffolds (1.05 vs. 2.70 MPa and 2.53 vs. 12.67 N-mm, respectively). Sodium citrate alone accelerated the degradation rate by 1.5 times independent of PLLA. Cytocompatibility of all samples were evaluated using proliferation and differentiation parameters of dog-bone marrow stromal cells (dog-BMSCs). The results showed that viable dog-BMSCs attached well on both DCPD and PLLA/DCPD composite surfaces. In both DCPD and PLLA/DCPD conditioned medium, dog-BMSCs proliferated well and expressed alkaline phosphatase (ALP) activity indicating cell differentiation. These findings indicate that incorporating both sodium citrate and PLLA could effectively improve mechanical strength and biocompatibility without increasing the degradation time of calcium phosphate cement scaffolds for bone tissue engineering purposes.

  2. Fabrication of Poly-l-lactic Acid/Dicalcium Phosphate Dihydrate Composite Scaffolds with High Mechanical Strength—Implications for Bone Tissue Engineering

    PubMed Central

    Tanataweethum, Nida; Liu, Wai Ching; Scott Goebel, W.; Li, Ding; Chu, Tien Min

    2015-01-01

    Scaffolds were fabricated from poly-l-lactic acid (PLLA)/dicalcium phosphate dihydrate (DCPD) composite by indirect casting. Sodium citrate and PLLA were used to improve the mechanical properties of the DCPD scaffolds. The resulting PLLA/DCPD composite scaffold had increased diametral tensile strength and fracture energy when compared to DCPD only scaffolds (1.05 vs. 2.70 MPa and 2.53 vs. 12.67 N-mm, respectively). Sodium citrate alone accelerated the degradation rate by 1.5 times independent of PLLA. Cytocompatibility of all samples were evaluated using proliferation and differentiation parameters of dog-bone marrow stromal cells (dog-BMSCs). The results showed that viable dog-BMSCs attached well on both DCPD and PLLA/DCPD composite surfaces. In both DCPD and PLLA/DCPD conditioned medium, dog-BMSCs proliferated well and expressed alkaline phosphatase (ALP) activity indicating cell differentiation. These findings indicate that incorporating both sodium citrate and PLLA could effectively improve mechanical strength and biocompatibility without increasing the degradation time of calcium phosphate cement scaffolds for bone tissue engineering purposes. PMID:26556380

  3. Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant.

    PubMed Central

    Stols, L; Donnelly, M I

    1997-01-01

    NAD(+)-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene. The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions. The Km values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction). When malic enzyme was induced under appropriate culture conditions in a strain of E. coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism of glucose was restored. Succinic acid was the major fermentation product formed. When this fermentation was performed in the presence of hydrogen, the yield of succinic acid increased. The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks. PMID:9212416

  4. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  5. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor. PMID:26802007

  6. Metastable nitric acid hydrates--possible constituents of polar stratospheric clouds?

    PubMed

    Grothe, Hinrich; Tizek, Heinz; Ortega, Ismael K

    2008-01-01

    Crystallization kinetics of the metastable modifications of Nitric Acid Dihydrate (NAD) was investigated by time-dependent X-Ray Diffraction (XRD) measurements. Kinetic conversion curves were evaluated adopting the Avrami model. The growth and morphology of the respective crystallites and particles were monitored in situ on the cryo-stage of an Environmental Scanning Electron Microscope (ESEM) under a partial pressure of nitrogen gas (0.5 Torr, 67 Pa). The morphologies were used to adapt the InfraRed (IR) extinction spectra by T-matrix calculation using respective optical indices of NAD. The results show a significant dependence of the band shapes on different morphologies.

  7. l-Nebiviololinium chloride dihydrate

    PubMed Central

    Tuchalski, Gisbert; Hänsicke, Andre; Reck, Günther; Emmerling, Franziska

    2008-01-01

    The hydro­chloride salt of chiral l-nebivolol {systematic name: (+)−(R,S,S,S)-bis­[2-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl)-2-hydroxy­ethyl]ammonium chloride dihydrate}, C22H26F2NO4 +·Cl−·2H2O, was obtained by chiral liquid chromatography as a dihydrate. The pyran rings adopt half-chair conformations. Hydrogen bonds between the cation, anions and water mol­ecules contribute to the formation of layers parallel to the ac plane. PMID:21200930

  8. Metabolic bypass of the tricarboxylic acid cycle during lipid mobilization in germinating oilseeds. Regulation Of nad+-dependent isocitrate dehydrogenase versus fumarase

    PubMed

    Falk; Behal; Xiang; Oliver

    1998-06-01

    Biosynthesis of sucrose from triacylglycerol requires the bypass of the CO2-evolving reactions of the tricarboxylic acid (TCA) cycle. The regulation of the TCA cycle bypass during lipid mobilization was examined. Lipid mobilization in Brassica napus was initiated shortly after imbibition of the seed and proceeded until 2 d postimbibition, as measured by in vivo [1-14C]acetate feeding to whole seedlings. The activity of NAD+-isocitrate dehydrogenase (a decarboxylative enzyme) was not detected until 2 d postimbibition. RNA-blot analysis of B. napus seedlings demonstrated that the mRNA for NAD+-isocitrate dehydrogenase was present in dry seeds and that its level increased through the 4 d of the experiment. This suggested that NAD+-isocitrate dehydrogenase activity was regulated by posttranscriptional mechanisms during early seedling development but was controlled by mRNA level after the 2nd or 3rd d. The activity of fumarase (a component of the nonbypassed section of the TCA cycle) was low but detectable in B. napus seedlings at 12 h postimbibition, coincident with germination, and increased for the next 4 d. RNA-blot analysis suggested that fumarase activity was regulated primarily by the level of its mRNA during germination and early seedling development. It is concluded that posttranscriptional regulation of NAD+-isocitrate dehydrogenase activity is one mechanism of restricting carbon flux through the decarboxylative section of the TCA cycle during lipid mobilization in germinating oilseeds.

  9. Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism.

    PubMed

    Belenky, Peter; Christensen, Kathryn C; Gazzaniga, Francesca; Pletnev, Alexandre A; Brenner, Charles

    2009-01-01

    NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification. PMID:19001417

  10. Kinetic analysis of bifidobacterial metabolism reveals a minor role for succinic acid in the regeneration of NAD+ through its growth-associated production.

    PubMed

    Van der Meulen, Roel; Adriany, Tom; Verbrugghe, Kristof; De Vuyst, Luc

    2006-08-01

    Several strains belonging to the genus Bifidobacterium were tested to determine their abilities to produce succinic acid. Bifidobacterium longum strain BB536 and Bifidobacterium animalis subsp. lactis strain Bb 12 were kinetically analyzed in detail using in vitro fermentations to obtain more insight into the metabolism and production of succinic acid by bifidobacteria. Changes in end product formation in strains of Bifidobacterium could be related to the specific rate of sugar consumption. When the specific sugar consumption rate increased, relatively more lactic acid and less acetic acid, formic acid, and ethanol were produced, and vice versa. All Bifidobacterium strains tested produced small amounts of succinic acid; the concentrations were not more than a few millimolar. Succinic acid production was found to be associated with growth and stopped when the energy source was depleted. The production of succinic acid contributed to regeneration of a small part of the NAD+, in addition to the regeneration through the production of lactic acid and ethanol.

  11. Microbial NAD metabolism: lessons from comparative genomics.

    PubMed

    Gazzaniga, Francesca; Stebbins, Rebecca; Chang, Sheila Z; McPeek, Mark A; Brenner, Charles

    2009-09-01

    NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life via horizontal gene transfer. Biochemical, genetic, and genomic analyses have advanced to the point at which the precursors and pathways utilized by a microorganism can be predicted. Challenges remain in dissecting regulation of pathways. PMID:19721089

  12. A Dual Function α-Dioxygenase-Peroxidase and NAD+ Oxidoreductase Active Enzyme from Germinating Pea Rationalizing α-Oxidation of Fatty Acids in Plants12

    PubMed Central

    Saffert, Alexander; Hartmann-Schreier, Jenny; Schön, Astrid; Schreier, Peter

    2000-01-01

    An enzyme with fatty acid α-oxidation activity (49 nkat mg−1; substrate: lauric acid) was purified from germinating pea (Pisum sativum) by a five-step procedure to apparent homogeneity. The purified protein was found to be a 230-kD oligomer with two dominant subunits, i.e. a 50-kD subunit with NAD+ oxidoreductase activity and a 70-kD subunit, homolog to a pathogen-induced oxygenase, which in turn shows significant homology to animal cyclooxygenase. On-line liquid chromatography-electrospray ionization-tandem mass spectrometry revealed rapid α-oxidation of palmitic acid incubated at 0°C with the purified α-oxidation enzyme, leading to (R)-2-hydroperoxypalmitic acid as the major product together with (R)-2-hydroxypalmitic acid, 1-pentadecanal, and pentadecanoic acid. Inherent peroxidase activity of the 70-kD fraction decreased the amount of the (R)-2-hydroperoxy product rapidly and increased the level of (R)-2-hydroxypalmitic acid. Incubations at room temperature accelerated the decline toward the chain-shortened aldehyde. With the identification of the dual function α-dioxygenase-peroxidase (70-kD unit) and the related NAD+ oxidoreductase (50-kD unit) we provided novel data to rationalize all steps of the classical scheme of α-oxidation in plants. PMID:10938370

  13. Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans

    PubMed Central

    Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian

    2014-01-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production—NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)—were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6. PMID:25217009

  14. Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans.

    PubMed

    Wang, Limin; Cai, Yumeng; Zhu, Lingfeng; Guo, Honglian; Yu, Bo

    2014-12-01

    Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production-NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)-were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6.

  15. Cytosinium orotate dihydrate.

    PubMed

    Portalone, Gustavo

    2013-01-01

    The title compound, C4H6N3O(+)·C5H3N2O4(-)·2H2O or Cyt(+)·Or(-)·2H2O, was synthesized by a reaction between cytosine (4-amino-2-hy-droxy-pyrimidine, Cyt) and orotic acid (2,4-dihy-droxy-6-carb-oxy-pyrimidine, Or) in aqueous solution. The two ions are joined by two N(+)-H⋯O(-) (±)-(CAHB) hydrogen bonds, forming a dimer with graph-set motif R2(2)(8). In the crystal, the ion pairs of the asymmetric unit are joined by four N-H⋯O inter-actions to adjacent dimers, forming hydrogen-bonded rings with R2(2)(8) graph-set motif in a two-dimensional network. The formation of the three-dimensional array is facilitated by water mol-ecules, which act as bridges between structural sub-units linked in R3(2)(8) and R3(2)(7) hydrogen-bonded rings. The orotate anion is essentially planar, as the dihedral angle between the planes defined by the carboxylate group and the uracil fragment is 4.0 (4)°. PMID:23476396

  16. Cytosinium orotate dihydrate

    PubMed Central

    Portalone, Gustavo

    2013-01-01

    The title compound, C4H6N3O+·C5H3N2O4 −·2H2O or Cyt+·Or−·2H2O, was synthesized by a reaction between cytosine (4-amino-2-hy­droxy­pyrimidine, Cyt) and orotic acid (2,4-dihy­droxy-6-carb­oxy­pyrimidine, Or) in aqueous solution. The two ions are joined by two N+—H⋯O− (±)-(CAHB) hydrogen bonds, forming a dimer with graph-set motif R 2 2(8). In the crystal, the ion pairs of the asymmetric unit are joined by four N—H⋯O inter­actions to adjacent dimers, forming hydrogen-bonded rings with R 2 2(8) graph-set motif in a two-dimensional network. The formation of the three-dimensional array is facilitated by water mol­ecules, which act as bridges between structural sub-units linked in R 3 2(8) and R 3 2(7) hydrogen-bonded rings. The orotate anion is essentially planar, as the dihedral angle between the planes defined by the carboxylate group and the uracil fragment is 4.0 (4)°. PMID:23476396

  17. A catalytic triad is responsible for acid-base chemistry in the Ascaris suum NAD-malic enzyme.

    PubMed

    Karsten, William E; Liu, Dali; Rao, G S Jagannatha; Harris, Ben G; Cook, Paul F

    2005-03-01

    The pH dependence of kinetic parameters of several active site mutants of the Ascaris suum NAD-malic enzyme was investigated to determine the role of amino acid residues likely involved in catalysis on the basis of three-dimensional structures of malic enzyme. Lysine 199 is positioned to act as the general base that accepts a proton from the 2-hydroxyl of malate during the hydride transfer step. The pH dependence of V/K(malate) for the K199R mutant enzyme reveals a pK of 5.3 for an enzymatic group required to be unprotonated for activity and a second pK of 6.3 that leads to a 10-fold loss in activity above the pK of 6.3 to a new constant value up to pH 10. The V profile for K199R is pH independent from pH 5.5 to pH 10 and decreases below a pK of 4.9. Tyrosine 126 is positioned to act as the general acid that donates a proton to the enolpyruvate intermediate to form pyruvate. The pH dependence of V/K(malate) for the Y126F mutant is qualitatively similar to K199R, with a requirement for a group to be unprotonated for activity with a pK of 5.6 and a partial activity loss of about 3-fold above a pK of 6.7 to a new constant value. The Y126F mutant enzyme is about 60000-fold less active than the wild-type enzyme. In contrast to K199R, the V rate profile for Y126F also shows a partial activity loss above pH 6.6. The wild-type pH profiles were reinvestigated in light of the discovery of the partial activity change for the mutant enzymes. The wild-type V/K(malate) pH-rate profile exhibits the requirement for a group to be unprotonated for catalysis with a pK of 5.6 and also shows the partial activity loss above a pK of 6.4. The wild-type V pH-rate profile decreases below a pK of 5.2 and is pH independent from pH 5.5 to pH 10. Aspartate 294 is within hydrogen-bonding distance to K199 in the open and closed forms of malic enzyme. D294A is about 13000-fold less active than the wild-type enzyme, and the pH-rate profile for V/K(malate) indicates the mutant is only active above p

  18. Large supplements of nicotinic acid and nicotinamide increase tissue NAD+ and poly(ADP-ribose) levels but do not affect diethylnitrosamine-induced altered hepatic foci in Fischer-344 rats.

    PubMed

    Jackson, T M; Rawling, J M; Roebuck, B D; Kirkland, J B

    1995-06-01

    Poly(ADP-ribose) is a homopolymer of ADP-ribose units synthesized from NAD+ on nuclear acceptor proteins and is known to be involved in DNA repair. It is not known whether large oral doses of the clinically utilized NAD precursors nicotinic acid or nicotinamide affect poly(ADP-ribose) metabolism or the cellular response to DNA damage. In our first study, using Fischer-344 rats, 2 wk of dietary nicotinic acid supplementation (500 and 1000 mg/kg diet) caused elevated levels of NAD+ in the blood, liver, heart and kidney, while nicotinamide caused elevated levels only in the blood and liver, compared with controls fed a diet containing 30 mg/kg nicotinic acid. Both nicotinic acid and nicotinamide, at 1000 mg/kg diet, caused elevations in liver NAD+, by 44 and 43%, respectively. Only nicotinamide, however, elevated liver poly(ADP-ribose) (63% higher than control group). Following treatment with the hepatocarcinogen diethylnitrosamine, higher levels of hepatic NAD+ were observed in rats fed both nicotinic acid and nicotinamide at 1000 mg/kg diet, but only nicotinic acid supplementation caused a greater accumulation of hepatic poly(ADP-ribose) (61% higher than control group). Neither of the dietary treatments significantly affected the proportion of the liver occupied by placental glutathione-S-transferase positive foci. These results show that poly(ADP-ribose) synthesis is not directly responsive to hepatic NAD+ levels during niacin supplementation, and that the mechanisms of action of nicotinic acid and nicotinamide are different. The observed changes in poly(ADP-ribose) metabolism do not appear to cause any change in susceptibility to chemically induced carcinogenesis in this organ.

  19. Genetic linkage in Pseudomonas aeruginosa of algT and nadB: mutation in nadB does not affect NAD biosynthesis or alginate production.

    PubMed

    DeVries, C A; Hassett, D J; Flynn, J L; Ohman, D E

    1995-04-14

    The 68-min region of the chromosome of Pseudomonas aeruginosa (Pa) contains the gene algT, encoding a putative alternate sigma factor similar to sigma E in Escherichia coli, that is required for the expression of several genes in the alginate biosynthetic regulon. Sequences immediately upstream from algT were found to contain a divergently expressed open reading frame encoding a 60-kDa protein with 64 and 36% identity to the nadB gene products of E. coli and Bacillus subtilis, respectively. The nadB gene encodes L-aspartate oxidase and has been shown in several bacteria to be essential for de novo nicotinamide-adenine dinucleotide (NAD) biosynthesis. Pa nadB complemented the growth requirement for nicotinic acid in a nadB mutant strain of E. coli, suggesting that this gene encodes a functional homologue of L-aspartate oxidase. A nadB::Tn501 mutant was constructed by gene replacement in the alginate-producing strain, Pa FRD. This NadB- mutant still produced alginate and appeared normal with respect to the regulation of alginate synthesis. Interestingly, the NadB- mutant did not have an auxotrophic phenotype for nicotinic acid, indicating that this nadB was not essential for NAD biosynthesis in Pa. These results suggest the possibility that Pa has an alternate mechanism for de novo NAD biosynthesis.

  20. Genetic structure of the nadA and nadB antivirulence loci in Shigella spp.

    PubMed

    Prunier, Anne-Laure; Schuch, Raymond; Fernández, Reinaldo E; Maurelli, Anthony T

    2007-09-01

    Comparison of nadA and nadB in 14 Shigella strains and enteroinvasive Escherichia coli versus E. coli showed that at least one locus is altered in all strains. These observations explain the characteristic nicotinic acid auxotrophy of Shigella organisms and are consistent with the previously identified antivirulence nature of these genes for these pathogens.

  1. Rationally re-designed mutation of NAD-independent l-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli

    PubMed Central

    2012-01-01

    Background NAD-independent l-lactate dehydrogenase (l-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. Results Val-108 of l-iLDH was changed to Ala by rationally site-directed mutagenesis. The l-iLDH mutant exhibited much higher activity than wide-type l-iLDH towards l-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant l-iLDH as a biocatalyst, 40 g·L-1 of dl-mandelic acid was converted to 20.1 g·L-1 of d-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L-1 of benzoylformic acid. Conclusions A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure d-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system. PMID:23176608

  2. Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production.

    PubMed

    Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L; Evans, Charles R; Brenner, Charles

    2011-05-11

    NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.

  3. Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production.

    PubMed

    Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L; Evans, Charles R; Brenner, Charles

    2011-01-01

    NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins. PMID:21589930

  4. Enrofloxacin hydro-chloride dihydrate.

    PubMed

    Miranda-Calderón, Jorge E; Gutiérrez, Lilia; Flores-Alamo, Marcos; García-Gutiérrez, Ponciano; Sumano, Héctor

    2014-04-01

    The asymmetric unit of the title compound, C19H23FN3O3 (+)·Cl(-)·2H2O [systematic name: 4-(3-carb-oxy-1-cyclo-propyl-6-fluoro-4-oxo-1,4-di-hydro-quin-o-lin-7-yl)-1-ethyl-piperazin-1-ium chloride dihydrate], consists of two independent monocations of the protonated enrofloxacin, two chloride anions and four water mol-ecules. In the cations, the piperazinium rings adopt chair conformations and the dihedral angles between the cyclo-propyl ring and the 10-membered quinoline ring system are 56.55 (2) and 51.11 (2)°. An intra-molecular O-H⋯O hydrogen bond is observed in each cation. In the crystal, the components are connected via O-H⋯Cl, N-H⋯Cl and O-H⋯O hydrogen bonds, and a π-π inter-action between the benzene rings [centroid-centroid distance = 3.6726 (13) Å], resulting in a three-dimensional array.

  5. Studies on the de novo biosynthesis of NAD in Escherichia coli. The separation of the nadB gene product from the nadA gene product and its purification.

    PubMed

    Griffith, G R; Chandler, J L; Gholson, R K

    1975-05-01

    Quinolinic acid (pyridine 2,3-dicarboxylic acid) which is an immediate precursor of the pyridine nucleotides, is synthesised from L-asparate and dihydroxyacetone phosphate in Escherichia coli. Extracts from certain nadB mutants complement the extracts prepared from all nadA mutants for the enzymic synthesis of quinolinate. Using the complementation assay, the quinolinate synthetase B protein has been purified more than 300-fold. The quinolinate synthetase B protein exists in all nadA and nadC mutants examined. The quinolinate synthetase A protein was present in all nadC mutants and most (but not all) nadB mutants. The facile separation of the wild-type quinolinate synthetase A and B proteins out of a nadC mutant suggests that quinolinate synthetase does not exists as a tightly bound complex. The partially purified quinolinate synthetase is inhibited by physiological concetrations of NAD and NADH but not by NADP or NADPH.

  6. Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum.

    PubMed

    Pérez-Pomares, F; Ferrer, J; Camacho, M; Pire, C; LLorca, F; Bonete, M J

    1999-02-01

    The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.

  7. Analysis of NAD 2D-NMR spectra of saturated fatty acids in polypeptide aligning media by experimental and modeling approaches.

    PubMed

    Serhan, Zeinab; Borgogno, Andrea; Billault, Isabelle; Ferrarini, Alberta; Lesot, Philippe

    2012-01-01

    The overall and detailed elucidation (including the stereochemical aspects) of enzymatic mechanisms requires the access to all reliable information related to the natural isotopic fractionation of both precursors and products. Natural abundance deuterium (NAD) 2D-NMR experiments in polypeptide liquid-crystalline solutions are a new, suitable tool for analyzing site-specific deuterium isotopic distribution profiles. Here this method is utilized for analyzing saturated C14 to C18 fatty acid methyl esters (FAMEs), which are challenging because of the crowding of signals in a narrow spectral region. Experiments in achiral and chiral oriented solutions were performed. The spectral analysis is supplemented by the theoretical prediction of quadrupolar splittings as a function of the geometry and flexibility of FAMEs, based on a novel computational methodology. This allows us to confirm the spectral assignments, while providing insights into the mechanism of solute ordering in liquid-crystalline polypeptide solutions. This is found to be dominated by steric repulsions between FAMEs and polypeptides.

  8. Isothermal Decomposition of Hydrogen Peroxide Dihydrate

    NASA Technical Reports Server (NTRS)

    Loeffler, M. J.; Baragiola, R. A.

    2011-01-01

    We present a new method of growing pure solid hydrogen peroxide in an ultra high vacuum environment and apply it to determine thermal stability of the dihydrate compound that forms when water and hydrogen peroxide are mixed at low temperatures. Using infrared spectroscopy and thermogravimetric analysis, we quantified the isothermal decomposition of the metastable dihydrate at 151.6 K. This decomposition occurs by fractional distillation through the preferential sublimation of water, which leads to the formation of pure hydrogen peroxide. The results imply that in an astronomical environment where condensed mixtures of H2O2 and H2O are shielded from radiolytic decomposition and warmed to temperatures where sublimation is significant, highly concentrated or even pure hydrogen peroxide may form.

  9. Assimilation of NAD(+) precursors in Candida glabrata.

    PubMed

    Ma, Biao; Pan, Shih-Jung; Zupancic, Margaret L; Cormack, Brendan P

    2007-10-01

    The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection. PMID:17725566

  10. NAD+ and vitamin B3: from metabolism to therapies.

    PubMed

    Sauve, Anthony A

    2008-03-01

    The role of NAD(+) metabolism in health and disease is of increased interest as the use of niacin (nicotinic acid) has emerged as a major therapy for treatment of hyperlipidemias and with the recognition that nicotinamide can protect tissues and NAD(+) metabolism in a variety of disease states, including ischemia/reperfusion. In addition, a growing body of evidence supports the view that NAD(+) metabolism regulates important biological effects, including lifespan. NAD(+) exerts potent effects through the poly(ADP-ribose) polymerases, mono-ADP-ribosyltransferases, and the recently characterized sirtuin enzymes. These enzymes catalyze protein modifications, such as ADP-ribosylation and deacetylation, leading to changes in protein function. These enzymes regulate apoptosis, DNA repair, stress resistance, metabolism, and endocrine signaling, suggesting that these enzymes and/or NAD(+) metabolism could be targeted for therapeutic benefit. This review considers current knowledge of NAD(+) metabolism in humans and microbes, including new insights into mechanisms that regulate NAD(+) biosynthetic pathways, current use of nicotinamide and nicotinic acid as pharmacological agents, and opportunities for drug design that are directed at modulation of NAD(+) biosynthesis for treatment of human disorders and infections. PMID:18165311

  11. Altered behavioral and metabolic circadian rhythms in mice with disrupted NAD+ oscillation

    PubMed Central

    Sahar, Saurabh; Nin, Veronica; Barbosa, Maria Thereza; Chini, Eduardo Nunes; Sassone-Corsi, Paolo

    2011-01-01

    The Intracellular levels of nicotinamide adenine dinucleotide (NAD+) are rhythmic and controlled by the circadian clock. However, whether NAD+ oscillation in turn contributes to circadian physiology is not fully understood. To address this question we analyzed mice mutated for the NAD+ hydrolase CD38. We found that rhythmicity of NAD+ was altered in the CD38-deficient mice. The high, chronic levels of NAD+ results in several anomalies in circadian behavior and metabolism. CD38-null mice display a shortened period length of locomotor activity and alteration in the rest-activity rhythm. Several clock genes and, interestingly, genes involved in amino acid metabolism were deregulated in CD38-null livers. Metabolomic analysis identified alterations in the circadian levels of several amino acids, specifically tryptophan levels were reduced in the CD38-null mice at a circadian time paralleling with elevated NAD+ levels. Thus, CD38 contributes to behavioral and metabolic circadian rhythms and altered NAD+ levels influence the circadian clock. PMID:21937766

  12. NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I

    SciTech Connect

    Levova, Katerina; Moserova, Michaela; Nebert, Daniel W.; Phillips, David H.; Frei, Eva; Schmeiser, Heinz H.; Arlt, Volker M.; Stiborova, Marie

    2012-12-15

    Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)—the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1(−/−), Cyp1a2(−/−) and Cyp1a1/1a2(−/−) knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential. Highlights: ► NAD(P)H:quinone oxidoreductase expression in Cyp1a knockout and humanized CYP1A mice ► Reductive activation of the nephrotoxic and carcinogenic aristolochic acid I (AAI) ► NAD(P)H:quinone oxidoreductase is induced in mice treated with AAI. ► Induced hepatic enzyme activity resulted in elevated AAI-DNA adduct levels.

  13. Caffeic acid phenethyl ester stimulates human antioxidant response element-mediated expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene.

    PubMed

    Jaiswal, A K; Venugopal, R; Mucha, J; Carothers, A M; Grunberger, D

    1997-02-01

    Caffeic acid phenethyl ester (CAPE) is a phenolic antioxidant derived from the propolis of honeybee hives. CAPE was shown to inhibit the formation of intracellular hydrogen peroxide and oxidized bases in DNA of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated HeLa cells and was also found to induce a redox change that correlated with differential growth effects in transformed cells but not the nontumorigenic parental ones. Mediated via the electrophile or human antioxidant response element (hARE), induction of the expression of NAD(P)H quinone oxidoreductase (NQO1) and glutathione S-transferase Ya subunit genes by certain phenolic antioxidants has been correlated with the chemopreventive properties of these agents. Here, we determined by Northern analysis that CAPE treatment of hepatoma cells stimulates NQO1 gene expression in cultured human hepatoma cells (HepG2), and we characterized the effects of CAPE treatment on the expression of a reporter gene either containing or lacking the hARE or carrying a mutant version of this element in rodent hepatoma (Hepa-1) transfectants. A dose-dependent transactivation of human hARE-mediated chloramphenicol acetyltransferase (cat) gene expression was observed upon treatments of the Hepa-1 transfectants with TPA, a known inducer, as well as with CAPE. The combined treatments resulted in an apparent additive stimulation of the reporter expression. To learn whether this activation of cat gene expression was effected by protein kinase C in CAPE-treated cells, a comparison was made of cat gene activity after addition of calphostin, a protein kinase C inhibitor. Calphostin reduced the cat gene induction by TPA but not by CAPE, suggesting that stimulation of gene expression in this system by these agents proceeds via distinct mechanisms. Band-shift experiments to examine binding of transactivator proteins from nuclear extracts of treated and untreated cells to a hARE DNA probe showed that TPA exposure increased the binding level

  14. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

    PubMed

    Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

    2015-01-01

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

  15. Tri-calcium phosphate (ß-TCP) can be artificially synthesized by recycling dihydrate gypsum hardened.

    PubMed

    Han-Cheol, Cho; Hori, Masaharu; Yoshida, Takakazu; Yamada, Naoko; Komada, Yuko; Tamaki, Yukimichi; Miyazaki, Takashi

    2014-01-01

    Calcium phosphate is known as a major component of biological hard tissues. This study aimed to produce calcium phosphate by recycling kneaded surplus gypsum. β-dihydrate gypsum was derived from commercial dental β-hemihydrate gypsum, which was mechanically powdered and mixed with the liquid component of a commercial zinc phosphate cement. This mixture was fired at 1,200°C and evaluated by XRD analysis, thermal analysis and scanning electron microscopy (SEM). An acceptable ratio of mixing was 4 g of β-dihydrate gypsum powder to 1.5 mL of phosphoric acid liquid. XRD peaks were monotonic below 800°C, but new ß-TCP was formed by firing at 900°C or more, although TG-DTA analysis of synthetic ß-TCP suggested that some residual dihydrate gypsum remained in the sample. SEM images indicated a fused-block bone-like structure covered with phosphorus and calcium. These results suggest that production of synthetic β-TCP is possible through ecological techniques using recycled materials. PMID:25483384

  16. NAD+ Auxotrophy is Bacteriocidal for the Tubercle Bacilli

    PubMed Central

    Vilchèze, Catherine; Weinrick, Brian; Wong, Ka-Wing; Chen, Bing; Jacobs, William R.

    2010-01-01

    SUMMARY The human tubercle bacillus Mycobacterium tuberculosis can synthesize NAD+ using the de novo biosynthesis pathway or the salvage pathway. The salvage pathway of the bovine tubercle bacillus M. bovis was reported defective due to a mutation in the nicotinamidase PncA. This defect prevents nicotinic acid secretion, which is the basis for the niacin test that clinically distinguishes M. bovis from M. tuberculosis. Surprisingly, we found that the NAD+ de novo biosynthesis pathway (nadABC) can be deleted from M. bovis, demonstrating a functioning salvage pathway. M. bovis ΔnadABC fails to grow in mice, whereas M. tuberculosis ΔnadABC grows normally in mice, suggesting that M. tuberculosis can acquire nicotinamide from its host. The introduction of M. tuberculosis pncA into M. bovis ΔnadABC is sufficient to fully restore growth in a mouse, proving that the functional salvage pathway enables nicotinamide acquisition by the tubercle bacilli. This study demonstrates that NAD+ starvation is a cidal event in the tubercle bacilli and confirms that enzymes common to the de novo and salvage pathways may be good drug targets. PMID:20199601

  17. Calcium chloride rhenate(VII) dihydrate.

    PubMed

    Jarek, Urszula; Hołyńska, Małgorzata; Rlepokura, Katarzyna; Lis, Tadeusz

    2007-09-01

    The crystal structure of calcium chloride rhenate(VII) dihydrate, CaCl(ReO4).2H2O, investigated at 85 K, consists of calcium cations, chloride anions, rhenate(VII) anions and water molecules. In the nearly tetrahedral rhenate(VII) anion, all constituent atoms lie on special positions of m2m (Re) and m (O) site symmetries. The Cl- anion and water O atom lie on special positions of m2m and 2 site symmetries, respectively. The Ca2+ ion, also on a special position (m2m), is eight-coordinated in a distorted square-antiprismatic coordination mode. The crystal has a layered structure stabilized by Ca-O coordination bonds and O-H...Cl hydrogen bonds.

  18. Pseudopolymorphism of levodopa: A novel “disappearing” dihydrate

    NASA Astrophysics Data System (ADS)

    André, Vânia; Duarte, M. Teresa

    2014-11-01

    We report herein the crystal structure of a novel L-dopa dihydrate, an unstable pseudopolymorph detected in some co-crystallization studies. This form is obtained by traditional solution techniques and tends to convert to the anhydrous form under ambient conditions. Even though pseudopolymorphism has generally been subject of large conceptual discussions, it is indeed of importance in the pharmaceutical industry and, in this particularly case, the knowledge of the formation of this novel dihydrate may be very relevant for processing issues.

  19. Boosting NAD to spare hearing.

    PubMed

    Brenner, Charles

    2014-12-01

    Ex vivo experiments have strangely shown that inhibition or stimulation of NAD metabolism can be neuroprotective. In this issue of Cell Metabolism, Brown et al. (2014) demonstrate that cochlear NAD is diminished by deafening noise but protected by nicotinamide riboside or WldS mutation. Hearing protection by nicotinamide riboside depends on Sirt3. PMID:25470539

  20. Emerging therapeutic roles for NAD(+) metabolism in mitochondrial and age-related disorders.

    PubMed

    Srivastava, Sarika

    2016-12-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a central metabolic cofactor in eukaryotic cells that plays a critical role in regulating cellular metabolism and energy homeostasis. NAD(+) in its reduced form (i.e. NADH) serves as the primary electron donor in mitochondrial respiratory chain, which involves adenosine triphosphate production by oxidative phosphorylation. The NAD(+)/NADH ratio also regulates the activity of various metabolic pathway enzymes such as those involved in glycolysis, Kreb's cycle, and fatty acid oxidation. Intracellular NAD(+) is synthesized de novo from L-tryptophan, although its main source of synthesis is through salvage pathways from dietary niacin as precursors. NAD(+) is utilized by various proteins including sirtuins, poly ADP-ribose polymerases (PARPs) and cyclic ADP-ribose synthases. The NAD(+) pool is thus set by a critical balance between NAD(+) biosynthetic and NAD(+) consuming pathways. Raising cellular NAD(+) content by inducing its biosynthesis or inhibiting the activity of PARP and cADP-ribose synthases via genetic or pharmacological means lead to sirtuins activation. Sirtuins modulate distinct metabolic, energetic and stress response pathways, and through their activation, NAD(+) directly links the cellular redox state with signaling and transcriptional events. NAD(+) levels decline with mitochondrial dysfunction and reduced NAD(+)/NADH ratio is implicated in mitochondrial disorders, various age-related pathologies as well as during aging. Here, I will provide an overview of the current knowledge on NAD(+) metabolism including its biosynthesis, utilization, compartmentalization and role in the regulation of metabolic homoeostasis. I will further discuss how augmenting intracellular NAD(+) content increases oxidative metabolism to prevent bioenergetic and functional decline in multiple models of mitochondrial diseases and age-related disorders, and how this knowledge could be translated to the clinic for human relevance. PMID

  1. Enrofloxacin hydro­chloride dihydrate

    PubMed Central

    Miranda-Calderón, Jorge E.; Gutiérrez, Lilia; Flores-Alamo, Marcos; García-Gutiérrez, Ponciano; Sumano, Héctor

    2014-01-01

    The asymmetric unit of the title compound, C19H23FN3O3 +·Cl−·2H2O [systematic name: 4-(3-carb­oxy-1-cyclo­propyl-6-fluoro-4-oxo-1,4-di­hydro­quin­o­lin-7-yl)-1-ethyl­piperazin-1-ium chloride dihydrate], consists of two independent monocations of the protonated enrofloxacin, two chloride anions and four water mol­ecules. In the cations, the piperazinium rings adopt chair conformations and the dihedral angles between the cyclo­propyl ring and the 10-membered quinoline ring system are 56.55 (2) and 51.11 (2)°. An intra­molecular O—H⋯O hydrogen bond is observed in each cation. In the crystal, the components are connected via O—H⋯Cl, N—H⋯Cl and O—H⋯O hydrogen bonds, and a π–π inter­action between the benzene rings [centroid–centroid distance = 3.6726 (13) Å], resulting in a three-dimensional array. PMID:24826167

  2. Hydrolysis of dicalcium phosphate dihydrate to hydroxyapatite.

    PubMed

    Fulmer, M T; Brown, P W

    1998-04-01

    Dicalcium phosphate dihydrate (DCPD) was hydrolysed in water and in 1 M Na2HPO4 solution at temperatures from 25-60 degrees C. Hydrolysis was incomplete in water. At 25 degrees C, DCPD partially hydrolysed to hydroxyapatite (HAp). Formation of HAp is indicative of incongruent DCPD dissolution. At the higher temperatures, hydrolysis to HAp was more extensive and was accompanied by the formation of anhydrous dicalcium phosphate (DCP). Both of these processes are endothermic. When hydrolysis was carried out in 1 M Na2HPO4 solution, heat absorption was greater at any given temperature than for hydrolysis in water. Complete hydrolysis to HAp occurred in this solution. The hydrolysis of DCPD to HAp in sodium phosphate solution was also endothermic. The complete conversion of DCPD to HAp in sodium phosphate solution would not be expected if the only effect of this solution was to cause DCPD dissolution to become congruent. Because of the buffering capacity of a dibasic sodium phosphate solution, DCPD hydrolysed completely to HAp. Complete conversion to HAp was accompanied by the conversion of dibasic sodium phosphate to monobasic sodium phosphate. The formation of DCP was not observed indicating that the sodium phosphate solution precluded the DCPD-to-DCP dehydration reaction. In addition to affecting the extent of hydrolysis, reaction in the sodium phosphate solution also caused a morphological change in the HAp which formed. HAp formed by hydrolysis in water was needle-like to globular while that formed in the sodium phosphate solution exhibited a florette-like morphology.

  3. Permeability of Rickettsia prowazekii to NAD

    SciTech Connect

    Atkinson, W.H.; Winkler, H.H.

    1989-02-01

    Rickettsia prowazekii accumulated radioactivity from (adenine-2,8-3H)NAD but not from (nicotinamide-4-3H)NAD, which demonstrated that NAD was not taken up intact. Extracellular NAD was hydrolyzed by rickettsiae with the products of hydrolysis, nicotinamide mononucleotide and AMP, appearing in the incubation medium in a time- and temperature-dependent manner. The particulate (membrane) fraction contained 90% of this NAD pyrophosphatase activity. Rickettsiae which had accumulated radiolabel after incubation with (adenine-2,8-3H)NAD were extracted, and the intracellular composition was analyzed by chromatography. The cells contained labeled AMP, ADP, ATP, and NAD. The NAD-derived intracellular AMP was transported via a pathway distinct from and in addition to the previously described AMP translocase. Exogenous AMP (1 mM) inhibited uptake of radioactivity from (adenine-2,8-3H)NAD and hydrolysis of extracellular NAD. AMP increased the percentage of intracellular radiolabel present as NAD. Nicotinamide mononucleotide was not taken up by the rickettsiae, did not inhibit hydrolysis of extracellular NAD, and was not a good inhibitor of the uptake of radiolabel from (adenine-2,8-3H)NAD. Neither AMP nor ATP (both of which are transported) could support the synthesis of intracellular NAD. The presence of intracellular (adenine-2,8-3H)NAD within an organism in which intact NAD could not be transported suggested the resynthesis from AMP of (adenine-2,8-3H)NAD at the locus of NAD hydrolysis and translocation.

  4. Transcriptional regulation of the nadA gene in Neisseria meningitidis impacts the prediction of coverage of a multicomponent meningococcal serogroup B vaccine.

    PubMed

    Fagnocchi, Luca; Biolchi, Alessia; Ferlicca, Francesca; Boccadifuoco, Giuseppe; Brunelli, Brunella; Brier, Sébastien; Norais, Nathalie; Chiarot, Emiliano; Bensi, Giuliano; Kroll, J Simon; Pizza, Mariagrazia; Donnelly, John; Giuliani, Marzia Monica; Delany, Isabel

    2013-02-01

    The NadA adhesin is a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B (MenB) infection. Under in vitro growth conditions, nadA is repressed by the regulator NadR and poorly expressed, resulting in inefficient killing of MenB strains by anti-NadA antibodies. Interestingly, sera from children infected with strains that express low levels of NadA in laboratory growth nevertheless recognize the NadA antigen, suggesting that NadA expression during infection may be different from that observed in vitro. In a strain panel covering a range of NadA levels, repression was relieved through deleting nadR. All nadR knockout strains expressed high levels of NadA and were efficiently killed by sera from subjects immunized with 4CMenB. A selected MenB strain, NGP165, mismatched for other vaccine antigens, is not killed by sera from immunized infants when the strain is grown in vitro. However, in an in vivo passive protection model, the same sera effectively protected infant rats from bacteremia with NGP165. Furthermore, we identify a novel hydroxyphenylacetic acid (HPA) derivative, reported by others to be produced during inflammation, which induces expression of NadA in vitro, leading to efficient antibody-mediated killing. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h postinfection. Our results suggest that during infectious disease, NadR repression is alleviated due to niche-specific signals, resulting in high levels of NadA expression from any nadA-positive (nadA(+)) strain and therefore efficient killing by anti-NadA antibodies elicited by the 4CMenB vaccine.

  5. Enzymatic assay for calmodulins based on plant NAD kinase activity

    SciTech Connect

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  6. NAD+-dependent deacetylase Hst1p controls biosynthesis and cellular NAD+ levels in Saccharomyces cerevisiae.

    PubMed

    Bedalov, Antonio; Hirao, Maki; Posakony, Jeffrey; Nelson, Melisa; Simon, Julian A

    2003-10-01

    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. PMID:12972620

  7. 21 CFR 184.1845 - Stannous chloride (anhydrous and dihydrated).

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... Anhydrous stannous chloride (SnCl2, CAS Reg. No. 7772-99-8) is the chloride salt of metallic tin. It is prepared by reacting molten tin with either chlorine or gaseous tin tetrachloride. Dihydrated stannous chloride (SnCl2·2H2O, CAS Reg. No. 10025-69-1) is the chloride salt of metallic tin that contains...

  8. 21 CFR 184.1845 - Stannous chloride (anhydrous and dihydrated).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... Anhydrous stannous chloride (SnCl2, CAS Reg. No. 7772-99-8) is the chloride salt of metallic tin. It is prepared by reacting molten tin with either chlorine or gaseous tin tetrachloride. Dihydrated stannous chloride (SnCl2·2H2O, CAS Reg. No. 10025-0969-091) is the chloride salt of metallic tin that contains...

  9. 21 CFR 184.1845 - Stannous chloride (anhydrous and dihydrated).

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... Anhydrous stannous chloride (SnCl2, CAS Reg. No. 7772-99-8) is the chloride salt of metallic tin. It is prepared by reacting molten tin with either chlorine or gaseous tin tetrachloride. Dihydrated stannous chloride (SnCl2·2H2O, CAS Reg. No. 10025-69-1) is the chloride salt of metallic tin that contains...

  10. 21 CFR 184.1845 - Stannous chloride (anhydrous and dihydrated).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... Anhydrous stannous chloride (SnCl2, CAS Reg. No. 7772-99-8) is the chloride salt of metallic tin. It is prepared by reacting molten tin with either chlorine or gaseous tin tetrachloride. Dihydrated stannous chloride (SnCl2·2H2O, CAS Reg. No. 10025-0969-091) is the chloride salt of metallic tin that contains...

  11. The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation.

    PubMed

    Gazanion, E; Garcia, D; Silvestre, R; Gérard, C; Guichou, J F; Labesse, G; Seveno, M; Cordeiro-Da-Silva, A; Ouaissi, A; Sereno, D; Vergnes, B

    2011-10-01

    NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target. PMID:21819459

  12. NAD+ and sirtuins in aging and disease.

    PubMed

    Imai, Shin-ichiro; Guarente, Leonard

    2014-08-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a classical coenzyme mediating many redox reactions. NAD(+) also plays an important role in the regulation of NAD(+)-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD(+) biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD(+) levels decline during the aging process and may be an Achilles' heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD(+) by supplementing NAD(+) intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD(+) intermediate supplementation may be an effective antiaging intervention, providing hope to aging societies worldwide. PMID:24786309

  13. Structure of a NAD kinase from Thermotoga maritima at 2.3 Å resolution

    SciTech Connect

    Oganesyan, Vaheh; Huang, Candice; Adams, Paul D.; Jancarik, Jaru; Yokota, Hisao A.; Kim, Rosalind; Kim, Sung-Hou

    2005-07-01

    The expression, purification, crystallization, and structure determination of NAD-kinase from T. maritima are reported. Similarity to other NAD-kinases as well as homo-oligomrization state of the enzyme from T. maritima are discussed. NAD kinase is the only known enzyme that catalyzes the formation of NADP, a coenzyme involved in most anabolic reactions and in the antioxidant defense system. Despite its importance, very little is known regarding the mechanism of catalysis and only recently have several NAD kinase structures been deposited in the PDB. Here, an independent investigation of the crystal structure of inorganic polyphosphate/ATP-NAD kinase, PPNK-THEMA, a protein from Thermotoga maritima, is reported at a resolution of 2.3 Å. The crystal structure was solved using single-wavelength anomalous diffraction (SAD) data collected at the Se absorption-peak wavelength in a state in which no cofactors or substrates were bound. It revealed that the 258-amino-acid protein is folded into two distinct domains, similar to recently reported NAD kinases. The N-terminal α/β-domain spans the first 100 amino acids and the last 30 amino acids of the polypeptide and has several topological matches in the PDB, whereas the other domain, which spans the middle 130 residues, adopts a unique β-sandwich architecture and only appreciably matches the recently deposited PDB structures of NAD kinases.

  14. Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

    PubMed

    Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

    2015-11-01

    Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

  15. Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

    SciTech Connect

    Sporty, J; Lin, S; Kato, M; Ognibene, T; Stewart, B; Turteltaub, K; Bench, G

    2009-02-18

    Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses on BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD

  16. Topography, purification and characterization of thyroidal NAD+ glycohydrolase

    PubMed Central

    De Wolf, Marc J. S.; Van Dessel, Guido A. F.; Lagrou, Albert R.; Hilderson, Herwig J. J.; Dierick, Wilfried S. H.

    1985-01-01

    Subcellular fractionation of bovine thyroid tissue by differential pelleting and isopycnic gradient centrifugation in a zonal rotor indicated that NAD+ glycohydrolase is predominantly located and rather uniformly distributed in the plasma membrane. Comparison of NAD+ glycohydrolase activities of intact thyroid tissue slices, functional rat thyroid cells in culture (FRTl) and their respective homogenates indicated that most if not all of the enzyme (catalytic site) is accessible to extracellular NAD+. The reaction product nicotinamide was predominantly recovered from the extracellular medium. The diazonium salt of sulphanilic acid, not penetrating into intact cells, was able to decrease the activity of intact thyroid tissue slices to the same extent as in the homogenate. Under the same conditions this reagent almost completely abolished NAD+ glycohydrolase activity associated with intact thyroid cells in culture. The triazine dye Cibacron Blue F3GA and its high-Mr derivative Blue Dextran respectively completely eliminated or caused a severe depression in the NAD+ glycohydrolase activity of FRTl cells. The enzyme could be readily solubilized from bovine thyroid membranes by detergent extraction, and was further purified by gel filtration and affinity chromatography on Blue Sepharose CL-6B. The overall procedure resulted in a 1940-fold purification (specific activity 77.6μmol of nicotinamide released/h per mg). The purified enzyme displays a Km of 0.40mm for β-NAD+, a broad pH optimum around pH7.2 (0.1 m-potassium phosphate buffer) and an apparent Mr of 120000. Nicotinamide is an inhibitor (Ki 1.9mm) of the non-competitive type. The second reaction product ADP-ribose acts as a competitive inhibitor (Ki 2.7mm). The purified enzyme splits β-NAD+, β-NADP+, β-NADH and α-NAD+ at rates in the relative proportions 1:0.75:<0.02:<0.02 and exhibits transglycosidase (pyridine-base exchange) activity. Anionic phospholipids such as phosphatidylinositol and phosphatidylserine

  17. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

    PubMed Central

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A.; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E.; Nikolaev, Evgeni V.; Magni, Giulio; Zhang, Hong; Osterman, Andrei L.

    2009-01-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-Å resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN → NMN → NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN → NaAD → NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis. PMID:19204287

  18. Effects of gamma radiation on solid trisodium citrate dihydrate: radical kinetics, radiosensitivity and dosimetry.

    PubMed

    Tuner, H; Korkmaz, M

    2010-11-01

    In the present work, radiosensitivity and dosimetric potential of solid trisodium citrate dihydrate (SC) were explored through a detailed electron spin resonance (ESR) study performed at various temperatures. Irradiated SC was observed to exhibit an ESR spectrum consisting of many intense and weak resonance lines spread over a magnetic field range of 7 mT and centered at g = 2.0039. An evaluation technique based on the variations of the characteristic resonance line intensities and the spectrum area under different experimental conditions was adopted, to determine the spectroscopic, kinetic and dosimetric features of radical species responsible for the observed experimental ESR spectrum. Radicals exhibiting similar ESR characteristics to those reported in the literature for irradiated tricarboxilic acids and their organic compounds were shown to be also produced in gamma-irradiated SC.

  19. N-(L-2-aminopentanoyl)-L-phenylalanine dihydrate, a hydrophobic dipeptide with a nonproteinogenic residue.

    PubMed

    Görbitz, Carl Henrik; Yadav, Vitthal N

    2013-09-01

    The title dipeptide, better known as L-norvalyl-L-phenylalanine {systematic name: (S)-2-[(S)-2-aminopentanamido]-3-phenylpropanoic acid dihydrate}, C14H20N2O3·2H2O, has a nonproteinogenic N-terminal residue. In the solid state, it takes on a molecular conformation typical for one of the three classes of nanoporous dipeptides, but like two related compounds with a hydrophobic N-terminal residue and a C-terminal L-phenylalanine, it fails to form channels or pores. Instead, the crystal structure is divided into distinct hydrophobic and hydrophilic layers, the latter encompassing cocrystallized water molecules connecting the charged N- and C-terminal groups.

  20. Analysis of NAD(P)+/NAD(P)H cofactors by imprinted polymer membranes associated with ion-sensitive field-effect transistor devices and Au-quartz crystals.

    PubMed

    Pogorelova, Svetlana P; Zayats, Maya; Bourenko, Tatyana; Kharitonov, Andrei B; Lioubashevski, Oleg; Katz, Eugenii; Willner, Itamar

    2003-02-01

    Specific recognition sites for the NAD(P)+ and NAD(P)H cofactors are imprinted in a cross-linked acrylamide-acrylamidophenylboronic acid copolymer membrane. The imprinted membranes, associated with pH-sensitive field-effect transistors (ISFETs) or Au-quartz piezoelectric crystals, enable the potentiometric or microgravimetric analysis of the oxidized NAD(P)+ cofactors and the reduced NAD(P)H cofactors, respectively. The NAD+- and NADP+-imprinted membranes associated with the ISFET allow the analysis of NAD+ and NADP+ with sensitivities that correspond to 15.0 and 18.0 mVdecade(-1) and detection limits of 4 x 10(-7) and 2 x 10(-7) M, respectively. The NADH- and NADPH-imprinted membranes associated with the ISFET device enable the analysis of NADH and NADPH with sensitivities that correspond to 24.2 and 21.8 mV x decade(-1) and lower detection limits that are 1 x 10(-7) and 2 x 10(-7) M, respectively. The ISFET devices functionalized with the NADH and NADPH membranes are employed in the analysis of the biocatalyzed oxidation of lactic acid and ethanol in the presence of lactate dehydrogenase and alcohol dehydrogenase, respectively. PMID:12585477

  1. TEMPOL increases NAD(+) and improves redox imbalance in obese mice.

    PubMed

    Yamato, Mayumi; Kawano, Kimika; Yamanaka, Yuki; Saiga, Misako; Yamada, Ken-Ichi

    2016-08-01

    Continuous energy conversion is controlled by reduction-oxidation (redox) processes. NAD(+) and NADH represent an important redox couple in energy metabolism. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) is a redox-cycling nitroxide that promotes the scavenging of several reactive oxygen species (ROS) and is reduced to hydroxylamine by NADH. TEMPOL is also involved in NAD(+) production in the ascorbic acid-glutathione redox cycle. We utilized the chemical properties of TEMPOL to investigate the effects of antioxidants and NAD(+)/NADH modulators on the metabolic imbalance in obese mice. Increases in the NAD(+)/NADH ratio by TEMPOL ameliorated the metabolic imbalance when combined with a dietary intervention, changing from a high-fat diet to a normal diet. Plasma levels of the superoxide marker dihydroethidium were higher in mice receiving the dietary intervention compared with a control diet, but were normalized with TEMPOL consumption. These findings provide novel insights into redox regulation in obesity. PMID:26942863

  2. Inhibition of the intrinsic NAD+ glycohydrolase activity of CD38 by carbocyclic NAD analogues.

    PubMed Central

    Wall, K A; Klis, M; Kornet, J; Coyle, D; Amé, J C; Jacobson, M K; Slama, J T

    1998-01-01

    Carba-NAD and pseudocarba-NAD are carbocyclic analogues of NAD+ in which a 2,3-dihydroxycyclopentane methanol replaces the beta-d-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ [Slama and Simmons (1988) Biochemistry 27, 183-193]. These carbocyclic NAD+ analogues, related to each other as diastereomers, have been tested as inhibitors of the intrinsic NAD+ glycohydrolase activity of human CD38, dog spleen NAD+ glycohydrolase, mouse CD38 and Aplysia californica cADP-ribose synthetase. Pseudocarba-NAD, the carbocyclic dinucleotide in which l-2,3-dihydroxycyclopentane methanol replaces the d-ribose of the nicotinamide riboside moiety of NAD+, was found to be the more potent inhibitor. Pseudocarba-NAD was shown to inhibit the intrinsic NAD+ glycohydrolase activity of human CD38 competitively, with Ki=148 microM determined for the recombinant extracellular protein domain and Ki=180 microM determined for the native protein expressed as a cell-surface enzyme on cultured Jurkat cells. Pseudocarba-NAD was shown to be a non-competitive inhibitor of the purified dog spleen NAD+ glycohydrolase, with Kis=47 miroM and Kii=198 microM. Neither pseudocarba-NAD nor carba-NAD inhibited mouse CD38 or Aplysia californica cADP-ribose synthetase significantly at concentrations up to 1 mM. The results underscore significant species differences in the sensitivity of these enzymes to inhibition, and indicate that pseudocarba-NAD will be useful as an inhibitor of the enzymic activity of human but not mouse CD38 in studies using cultured cells. PMID:9794804

  3. The Importance of NAD in Multiple Sclerosis

    PubMed Central

    Penberthy, W. Todd; Tsunoda, Ikuo

    2009-01-01

    The etiology of multiple sclerosis (MS) is unknown but it manifests as a chronic inflammatory demyelinating disease in the central nervous system (CNS). During chronic CNS inflammation, nicotinamide adenine dinucleotide (NAD) concentrations are altered by (T helper) Th1-derived cytokines through the coordinated induction of both indoleamine 2,3-dioxygenase (IDO) and the ADP cyclase CD38 in pathogenic microglia and lymphocytes. While IDO activation may keep auto-reactive T cells in check, hyper-activation of IDO can leave neuronal CNS cells starving for extracellular sources of NAD. Existing data indicate that glia may serve critical functions as an essential supplier of NAD to neurons during times of stress. Administration of pharmacological doses of non-tryptophan NAD precursors ameliorates pathogenesis in animal models of MS. Animal models of MS involve artificially stimulated autoimmune attack of myelin by experimental autoimmune encephalomyelitis (EAE) or by viral-mediated demyelination using Thieler's murine encephalomyelitis virus (TMEV). The WldS mouse dramatically resists razor axotomy mediated axonal degeneration. This resistance is due to increased efficiency of NAD biosynthesis that delays stress-induced depletion of axonal NAD and ATP. Although the WldS genotype protects against EAE pathogenesis, TMEV-mediated pathogenesis is exacerbated. In this review, we contrast the role of NAD in EAE versus TMEV demyelinating pathogenesis to increase our understanding of the pharmacotherapeutic potential of NAD signal transduction pathways. We speculate on the importance of increased SIRT1 activity in both PARP-1 inhibition and the potentially integral role of neuronal CD200 interactions through glial CD200R with induction of IDO in MS pathogenesis. A comprehensive review of immunomodulatory control of NAD biosynthesis and degradation in MS pathogenesis is presented. Distinctive pharmacological approaches designed for NAD-complementation or targeting NAD

  4. Crystal growth of calcium sulphate dihydrate at low supersaturation

    NASA Astrophysics Data System (ADS)

    Christoffersen, M. R.; Christoffersen, J.; Weijnen, M. P. C.; Van Rosmalen, G. M.

    1982-08-01

    The growth rate of calcium sulphate dihydrate crystals, gypsum, in aqueous suspension has been shown to be screw dislocation controlled in the supersaturation range 1.03< {C}/{C s}<1.15 . Constant composition experiments show that the overall rate of growth decreases with increasing mass of the crystals. A combination of normal spiral growth, growth of cooperating spirals with non-parallel Burgers vectors, and growth of grain boundary spirals, together with partial outgrowth of concave parts of the crystals, can explain the rate of growth found for different preparations of gypsum crytals.

  5. Tophaceous calcium pyrophosphate dihydrate deposition disease of the temporomandibular joint.

    PubMed

    Reynolds, Jennifer L; Matthew, Ian R; Chalmers, Andrew

    2008-04-01

    Tophaceous pseudogout is a rare manifestation of calcium pyrophosphate dihydrate (CPPD) deposition disease that particularly affects the temporomandibular joint (TMJ). We describe a case of tophaceous pseudogout and review the literature. Thirty-four cases of chronic CPPD deposition disease affecting the TMJ are described. Symptoms usually included pain and swelling. Most patients required surgery because of extensive crystal deposits, usually localized to the joint and adjacent structures but occasionally invasive. For many patients, malignancy was the preoperative diagnosis. Although patients with acute pseudogout of the TMJ may have involvement of other joints, tophaceous pseudogout was predominantly isolated to the TMJ.

  6. Vitamins and aging: pathways to NAD+ synthesis.

    PubMed

    Denu, John M

    2007-05-01

    Recent genetic evidence reveals additional salvage pathways for NAD(+) synthesis. In this issue, Belenky et al. (2007) report that nicotinamide riboside, a new NAD(+) precursor, regulates Sir2 deacetylase activity and life span in yeast. The ability of nicotinamide riboside to enhance life span does not depend on calorie restriction. PMID:17482537

  7. Why NAD(+) Declines during Aging: It's Destroyed.

    PubMed

    Schultz, Michael B; Sinclair, David A

    2016-06-14

    NAD(+) is required not only for life but for a long life. In this issue, Camacho-Pereira et al. (2016) implicate CD38 in the decline of NAD(+) during aging, with implications for combating age-related diseases. PMID:27304496

  8. Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans.

    PubMed

    Inoue, Hiroyuki; Tamura, Takashi; Ehara, Nagisa; Nishito, Akira; Nakayama, Yumi; Maekawa, Makiko; Imada, Katsumi; Tanaka, Hidehiko; Inagaki, Kenji

    2002-08-27

    An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M(r) 45000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase.

  9. Layered compounds derived from vanadyl phosphate dihydrate

    SciTech Connect

    Melanova, K.; Votinsky, J.; Benes, L.; Zima, V.

    1995-09-01

    The layered crystalline solids stable in air with general formula [M(H{sub 2}O)]{sub x}(VO){sub 1{minus}x}PO{sub 4{sup {sm_bullet}}}2H{sub 2}O (M = Al, Cr, Fe, Ga; x = 0.15--0.20) were prepared by reaction of solid V{sub 2}O{sub 5} with a boiling aqueous solution of phosphoric acid and the corresponding metal salt. The elementary cells of these compounds are tetragonal (space symmetry group either P4/n or P4/nmm). The lattice parameters and densities were determined. The results of TG, DTA and magnetic susceptibility measurements are given.

  10. Nicotinamide riboside kinase structures reveal new pathways to NAD+.

    PubMed

    Tempel, Wolfram; Rabeh, Wael M; Bogan, Katrina L; Belenky, Peter; Wojcik, Marzena; Seidle, Heather F; Nedyalkova, Lyudmila; Yang, Tianle; Sauve, Anthony A; Park, Hee-Won; Brenner, Charles

    2007-10-01

    The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+. PMID:17914902

  11. Niacin status, NAD distribution and ADP-ribose metabolism.

    PubMed

    Kirkland, James B

    2009-01-01

    Dietary niacin deficiency, and pharmacological excesses of nicotinic acid or nicotinamide, have dramatic effects on cellular NAD pools, ADP-ribose metabolism, tissue function and health. ADP-ribose metabolism is providing new targets for pharmacological intervention, and it is important to consider how the supply of vitamin B3 may directly influence ADP-ribosylation reactions, or create interactions with other drugs designed to influence these pathways. In addition to its redox roles, NAD+ is used as a substrate for mono-, poly- and cyclic ADP-ribose formation. During niacin deficiency, not all of these processes can be maintained, and dramatic changes in tissue function and clinical condition take place. Conversely, these reactions may be differentially enhanced by pharmacological intakes of vitamin B3, and potentially by changing expression of specific NAD generating enzymes. A wide range of metabolic changes can take place following pharmacological supplementation of nicotinic acid or nicotinamide. As niacin status decreases towards a deficient state, the function of other types of pharmaceutical agents may be modified, including those that target ADP-ribosylation reactions, apoptosis and inflammation. This article will explore what is known and yet to be learned about the response of tissues, cells and subcellular compartments to excessive and limiting supplies of niacin, and will discuss the etiology of the resulting pathologies.

  12. Less is more: Nutrient limitation induces cross-talk of nutrient sensing pathways with NAD+ homeostasis and contributes to longevity

    PubMed Central

    TSANG, Felicia; LIN, Su-Ju

    2016-01-01

    Nutrient sensing pathways and their regulation grant cells control over their metabolism and growth in response to changing nutrients. Factors that regulate nutrient sensing can also modulate longevity. Reduced activity of nutrient sensing pathways such as glucose-sensing PKA, nitrogen-sensing TOR and S6 kinase homolog Sch9 have been linked to increased life span in the yeast, Saccharomyces cerevisiae, and higher eukaryotes. Recently, reduced activity of amino acid sensing SPS pathway was also shown to increase yeast life span. Life span extension by reduced SPS activity requires enhanced NAD+ (nicotinamide adenine dinucleotide, oxidized form) and nicotinamide riboside (NR, a NAD+ precursor) homeostasis. Maintaining adequate NAD+ pools has been shown to play key roles in life span extension, but factors regulating NAD+ metabolism and homeostasis are not completely understood. Recently, NAD+ metabolism was also linked to the phosphate (Pi)-sensing PHO pathway in yeast. Canonical PHO activation requires Pi-starvation. Interestingly, NAD+ depletion without Pi-starvation was sufficient to induce PHO activation, increasing NR production and mobilization. Moreover, SPS signaling appears to function in parallel with PHO signaling components to regulate NR/NAD+ homeostasis. These studies suggest that NAD+ metabolism is likely controlled by and/or coordinated with multiple nutrient sensing pathways. Indeed, cross-regulation of PHO, PKA, TOR and Sch9 pathways was reported to potentially affect NAD+ metabolism; though detailed mechanisms remain unclear. This review discusses yeast longevity-related nutrient sensing pathways and possible mechanisms of life span extension, regulation of NAD+ homeostasis, and cross-talk among nutrient sensing pathways and NAD+ homeostasis.

  13. Less is more: Nutrient limitation induces cross-talk of nutrient sensing pathways with NAD+ homeostasis and contributes to longevity

    PubMed Central

    TSANG, Felicia; LIN, Su-Ju

    2016-01-01

    Nutrient sensing pathways and their regulation grant cells control over their metabolism and growth in response to changing nutrients. Factors that regulate nutrient sensing can also modulate longevity. Reduced activity of nutrient sensing pathways such as glucose-sensing PKA, nitrogen-sensing TOR and S6 kinase homolog Sch9 have been linked to increased life span in the yeast, Saccharomyces cerevisiae, and higher eukaryotes. Recently, reduced activity of amino acid sensing SPS pathway was also shown to increase yeast life span. Life span extension by reduced SPS activity requires enhanced NAD+ (nicotinamide adenine dinucleotide, oxidized form) and nicotinamide riboside (NR, a NAD+ precursor) homeostasis. Maintaining adequate NAD+ pools has been shown to play key roles in life span extension, but factors regulating NAD+ metabolism and homeostasis are not completely understood. Recently, NAD+ metabolism was also linked to the phosphate (Pi)-sensing PHO pathway in yeast. Canonical PHO activation requires Pi-starvation. Interestingly, NAD+ depletion without Pi-starvation was sufficient to induce PHO activation, increasing NR production and mobilization. Moreover, SPS signaling appears to function in parallel with PHO signaling components to regulate NR/NAD+ homeostasis. These studies suggest that NAD+ metabolism is likely controlled by and/or coordinated with multiple nutrient sensing pathways. Indeed, cross-regulation of PHO, PKA, TOR and Sch9 pathways was reported to potentially affect NAD+ metabolism; though detailed mechanisms remain unclear. This review discusses yeast longevity-related nutrient sensing pathways and possible mechanisms of life span extension, regulation of NAD+ homeostasis, and cross-talk among nutrient sensing pathways and NAD+ homeostasis. PMID:27683589

  14. Sequence-motif Detection of NAD(P)-binding Proteins: Discovery of a Unique Antibacterial Drug Target

    NASA Astrophysics Data System (ADS)

    Hua, Yun Hao; Wu, Chih Yuan; Sargsyan, Karen; Lim, Carmay

    2014-09-01

    Many enzymes use nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (NAD(P)) as essential coenzymes. These enzymes often do not share significant sequence identity and cannot be easily detected by sequence homology. Previously, we determined all distinct locally conserved pyrophosphate-binding structures (3d motifs) from NAD(P)-bound protein structures, from which 1d sequence motifs were derived. Here, we aim to establish the precision of these 3d and 1d motifs to annotate NAD(P)-binding proteins. We show that the pyrophosphate-binding 3d motifs are characteristic of NAD(P)-binding proteins, as they are rarely found in nonNAD(P)-binding proteins. Furthermore, several 1d motifs could distinguish between proteins that bind only NAD and those that bind only NADP. They could also distinguish between NAD(P)-binding proteins from nonNAD(P)-binding ones. Interestingly, one of the pyrophosphate-binding 3d and corresponding 1d motifs was found only in enoyl-acyl carrier protein reductases, which are enzymes essential for bacterial fatty acid biosynthesis. This unique 3d motif serves as an attractive novel drug target, as it is conserved across many bacterial species and is not found in human proteins.

  15. ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

    PubMed Central

    Fouquerel, Elise; Sobol, Robert W.

    2014-01-01

    Nicotinamide adenine dinucleotide, NAD+, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD+ and NADH). NAD+ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalysed by the sirtuins (SIRTs) which use NAD+ as a substrate. In this review, we highlight the significance of NAD+ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked and dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischemia/reperfusion. PMID:25283336

  16. Phosphate-responsive signaling pathway is a novel component of NAD+ metabolism in Saccharomyces cerevisiae.

    PubMed

    Lu, Shu-Ping; Lin, Su-Ju

    2011-04-22

    Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in various cellular biochemical reactions. To date the signaling pathways that regulate NAD(+) metabolism remain unclear due to the dynamic nature and complexity of the NAD(+) metabolic pathways and the difficulty of determining the levels of the interconvertible pyridine nucleotides. Nicotinamide riboside (NmR) is a key pyridine metabolite that is excreted and re-assimilated by yeast and plays important roles in the maintenance of NAD(+) pool. In this study we establish a NmR-specific reporter system and use it to identify yeast mutants with altered NmR/NAD(+) metabolism. We show that the phosphate-responsive signaling (PHO) pathway contributes to control NAD(+) metabolism. Yeast strains with activated PHO pathway show increases in both the release rate and internal concentration of NmR. We further identify Pho8, a PHO-regulated vacuolar phosphatase, as a potential NmR production factor. We also demonstrate that Fun26, a homolog of human ENT (equilibrative nucleoside transporter), localizes to the vacuolar membrane and establishes the size of the vacuolar and cytosolic NmR pools. In addition, the PHO pathway responds to depletion of cellular nicotinic acid mononucleotide (NaMN) and mediates nicotinamide mononucleotide (NMN) catabolism, thereby contributing to both NmR salvage and phosphate acquisition. Therefore, NaMN is a putative molecular link connecting the PHO signaling and NAD(+) metabolic pathways. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes. PMID:21349851

  17. Differential activation of NAD kinase by plant calmodulin isoforms. The critical role of domain I.

    PubMed

    Lee, S H; Seo, H Y; Kim, J C; Heo, W D; Chung, W S; Lee, K J; Kim, M C; Cheong, Y H; Choi, J Y; Lim, C O; Cho, M J

    1997-04-01

    NAD kinase is a Ca2+/calmodulin (CaM)-dependent enzyme capable of converting cellular NAD to NADP. The enzyme purified from pea seedlings can be activated by highly conserved soybean CaM, SCaM-1, but not by the divergent soybean CaM isoform, SCaM-4 (Lee, S. H., Kim, J. C., Lee, M. S., Heo, W. D., Seo, H. Y., Yoon, H. W., Hong, J. C., Lee, S. Y., Bahk, J. D., Hwang, I., and Cho, M. J. (1995) J. Biol. Chem. 270, 21806-21812). To determine which domains were responsible for this differential activation of NAD kinase, a series of chimeric SCaMs were generated by exchanging functional domains between SCaM-4 and SCaM-1. SCaM-4111, a chimeric SCaM-1 that contains the first domain of SCaM-4, was severely impaired (only 40% of maximal) in its ability to activate NAD kinase. SCaM-1444, a chimeric SCaM-4 that contains the first domain of SCaM-1 exhibited nearly full ( approximately 70%) activation of NAD kinase. Only chimeras containing domain I of SCaM-1 produced greater than half-maximal activation of NAD kinase. To define the amino acid residue(s) in domain I that were responsible for this differential activation, seven single residue substitution mutants of SCaM-1 were generated and tested for NAD kinase activation. Among these mutants, only K30E and G40D showed greatly reduced NAD kinase activation. Also a double residue substitution mutant, K30E/G40D, containing these two mutations in combination was severely impaired in its NAD kinase-activating potential, reaching only 20% of maximal activation. Furthermore, a triple mutation, K30E/M36I/G40D, completely abolished NAD kinase activation. Thus, our data suggest that domain I of CaM plays a key role in the differential activation of NAD kinase exhibited by SCaM-1 and SCaM-4. Further, the residues Lys30 and Glu40 of SCaM-1 are critical for this function.

  18. A Case of Randall's Plugs Associated to Calcium Oxalate Dihydrate Calculi.

    PubMed

    Grases, Felix; Söhnel, Otakar; Costa-Bauza, Antonia; Servera, Antonio; Benejam, Juan

    2016-07-01

    A case of a patient who developed multiple calcium oxalate dihydrate calculi, some of them connected to intratubular calcifications (Randall's plugs), is presented. Randall's plugs were isolated and studied. The mechanism of Randall's plug development is also suggested. PMID:27335788

  19. Lisinopril dihydrate: single-crystal x-ray structure and physicochemical characterization of derived solid forms.

    PubMed

    Sorrenti, Milena; Catenacci, Laura; Cruickshank, Dyanne L; Caira, Mino R

    2013-10-01

    Screening for new solid forms of the antihypertensive lisinopril was performed by recrystallization of the commercial form, lisinopril dihydrate, from various solvents and by exposing the product of its dehydration to a series of vapors under controlled conditions. Modifications other than the dihydrate encountered in the study included new anhydrous and amorphous forms, with intrinsic dissolution rates significantly greater than that of the dihydrate. Further physicochemical characterization included constant and programmed temperature powder X-ray diffraction, differential scanning calorimetry, thermogravimetry, and Fourier transform infrared spectroscopy. In the course of this study, the single-crystal X-ray structure of lisinopril dihydrate, [a = 14.550(2), b = 5.8917(8), c = 14.238(2) Å, β = 112.832(3)° at T = 173(2) K, space group P21 , Z = 2], was determined for the first time, revealing its double zwitterionic character in the solid state. PMID:23873413

  20. Peganine hydrochloride dihydrate an orally active antileishmanial agent.

    PubMed

    Khaliq, Tanvir; Misra, Pragya; Gupta, Swati; Reddy, K Papi; Kant, Ruchir; Maulik, P R; Dube, Anuradha; Narender, T

    2009-05-01

    Protozoic infections caused by genus Leishmania pose an enormous public health threat in developing countries, compounded by the toxicity and resistance to current therapies. Under the aegis of our ongoing program on drug discovery and development on antileishmanial agents from plants, we carried out bioassay guided fractionation on Peganum harmala seeds which resulted in the isolation of 1 as an antileishmanial agent. 2D-NMR spectral data and single crystal X-ray crystallography data indicated 1 as peganine hydrochloride in dihydrated form. The compound 1 exhibited in-vitro activity against both extracellular promastigotes as well as intracellular amastigotes residing within murine macrophages in Leishmania donovani. Furthermore, 1 also exhibited in-vivo activity, 79.6 (+/-8.07)% against established VL in hamsters at a dose of 100mg/kgb.wt. PMID:19339182

  1. Binding of NAD+ to pertussis toxin.

    PubMed

    Lobban, M D; Irons, L I; van Heyningen, S

    1991-06-24

    The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, Km, of 30 +/- 5 microM for NAD+ (when the toxin is catalysing the ADP-ribosylation of water and of dithiothreitol). The intrinsic fluorescence of pertussis toxin was quenched by up to 60% on titration with NAD+, and after correction for dilution and inner filter effects, a Kd value of 27 microM at 30 degrees C was obtained, agreeing well with that found by equilibrium dialysis. The binding constants were measured at a number of temperatures using both techniques, and from this the enthalpy of binding of NAD+ to toxin was determined to be 30 kJ.mol-1, a typical value for a protein-ligand interaction. There is one binding site for NAD+ per toxin molecule. PMID:1648404

  2. Do NAD and NAT form in liquid stratospheric aerosols by pseudoheterogeneous nucleation?

    PubMed

    Knopf, Daniel A

    2006-05-01

    Laboratory data of the freezing of nitric acid hydrates (NAD, NAT) from HNO(3)/H(2)O and HNO(3)/H(2)SO(4)/H(2)O solution droplets have been evaluated with respect to a "pseudoheterogeneous" (surface-induced) nucleation mechanism of NAD and NAT, which has been argued to possibly lead to the formation of polar stratospheric clouds (PSCs). In addition, a parametrization of pseudoheterogeneous nucleation of NAD and NAT suggested recently (Tabazadeh et al. J. Phys. Chem. A 2002, 106, 10238-10246) has been analyzed, showing that this parametrization should not be used in stratospheric modeling studies. The analysis of several laboratory data sets yields an upper limit of the pseudoheterogeneous nucleation rate coefficient of NAD of 2.2 x 10(-5) cm(-2) s(-1). In contrast, the upper limit of the pseudoheterogeneous nucleation rate coefficient of NAT could not be constrained satisfactorily, since formation of NAT has not been observed at stratospheric conditions in laboratory experiments applying small droplets. Maximum NAD production rates of 9.6 x 10(-9) cm(-3) (air) h(-1) in the stratosphere have been estimated assuming a pseudoheterogeneous nucleation mechanism that is constrained by the experimental observations. If maximum NAD supersaturation persisted for 4 weeks in the polar stratosphere the corresponding NAD particle number densities are estimated to be about 6 x 10(-6) cm(-3). These particle number densities are 3 orders of magnitude lower than particle number densities recently observed in the stratosphere. In conclusion, on the basis of laboratory data it is found that a pseudoheterogeneous nucleation mechanism is not sufficient to explain recent observations of large nitric acid containing particles in the polar stratosphere.

  3. A laboratory study of the nucleation kinetics of nitric acid hydrates under stratospheric conditions

    NASA Astrophysics Data System (ADS)

    James, Alexander D.; Murray, Benjamin J.; Plane, John M. C.

    2016-04-01

    Measurements of the kinetics of crystallisation of ternary H2O-H2SO4-HNO3 mixtures to produce nitric acid hydrate phases, as occurs in the lower stratosphere, have been a long-standing challenge for investigators in the laboratory. Understanding polar stratospheric chlorine chemistry and thereby ozone depletion is increasingly limited by descriptions of nucleation processes. Meteoric smoke particles have been considered in the past as heterogeneous nuclei, however recent studies suggest that these particles will largely dissolve, leaving mainly silica and alumina as solid inclusions. In this study the nucleation kinetics of nitric acid hydrate phases have been measured in microliter droplets at polar stratospheric cloud (PSC) temperatures, using a droplet freezing assay. A clear heterogeneous effect was observed when silica particles were added. A parameterisation based on the number of droplets activated per nuclei surface area (ns) has been developed and compared to global model data. Nucleation experiments on identical droplets have been performed in an X-Ray Diffractometer (XRD) to determine the nature of the phase which formed. β-Nitric Acid Trihydrate (NAT) was observed alongside a mixture of Nitric Acid Dihydrate (NAD) phases. It is not possible to determine whether NAT nucleates directly or is formed by a phase transition from NAD (likely requiring the presence of a mediating liquid phase). Regardless, these results demonstrate the possibility of forming NAT on laboratory timescales. In the polar stratosphere, sulfuric acid (present at several weight percent of the liquid under equilibrium conditions) could provide such a liquid phase. This study therefor provides insight into previous discrepancies between phases formed in the laboratory and those observed in the atmosphere. It also provides a basis for future studies into atmospheric nucleation of solid PSCs.

  4. Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148.

    PubMed

    Carroll, S F; McCloskey, J A; Crain, P F; Oppenheimer, N J; Marschner, T M; Collier, R J

    1985-11-01

    Irradiation of mixtures of diphtheria toxin fragment A and [carbonyl-14C]NAD with UV light (253.7 nm) is known to induce efficient transfer of the radiolabel to position 148, corresponding to glutamic acid in the unmodified protein. Here we report the structure of the photoproduct at position 148, as determined by chemical and photochemical methods, fast-atom-bombardment mass spectrometry, and nuclear magnetic resonance. The photoproduct [an alpha-amino-gamma-(6-nicotin-amidyl)butyric acid residue] contains the entire nicotinamide moiety of NAD linked via its number 6 carbon to the decarboxylated gamma-methylene carbon of Glu-148. No portion of the ADP-ribosyl group of NAD is present. These findings are consistent with the idea that Glu-148 lies at or near the catalytic center of diphtheria toxin. PMID:3864158

  5. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences.

    PubMed

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-03-01

    NAD is an essential metabolite that exists in NAD(+) or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD(+)/NADH redox state and modulating cellular signaling processes through the activity of the NAD(+)-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD(+) and NADH contents and the NAD(+)/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD(+), total NAD contents, and NAD(+)/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.

  6. MtrR control of a transcriptional regulatory pathway in Neisseria meningitidis that influences expression of a gene (nadA) encoding a vaccine candidate.

    PubMed

    Cloward, Jason M; Shafer, William M

    2013-01-01

    The surface-exposed NadA adhesin produced by a subset of capsular serogroup B strains of Neisseria meningitidis is currently being considered as a vaccine candidate to prevent invasive disease caused by a hypervirulent lineage of meningococci. Levels of NadA are known to be controlled by both transcriptional regulatory factors and a component of human saliva, 4-hydroxyphenylacetic acid. Herein, we confirmed the capacity of a DNA-binding protein termed FarR to negatively control nadA expression. We also found that a known transcriptional regulator of farR in N. gonorrhoeae termed MtrR can have a negative regulatory impact on farR and nadA expression, especially when over-expressed. MtrR-mediated repression of nadA was found to be direct, and its binding to a target DNA sequence containing the nadA promoter influenced formation and/or stability of FarR::nadA complexes. The complexity of the multi-layered regulation of nadA uncovered during this investigation suggests that N. meningitidis modulates NadA adhesin protein levels for the purpose of interacting with host cells yet avoiding antibody directed against surface exposed epitopes.

  7. Disruption of NAD+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

    PubMed Central

    Phadke, Manali; Krynetskaia, Natalia; Mishra, Anurag; Barrero, Carlos; Merali, Salim; Gothe, Scott A; Krynetskiy, Evgeny

    2015-01-01

    AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+ (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners. PMID:26629320

  8. PGC1α-dependent NAD biosynthesis links oxidative metabolism to renal protection

    PubMed Central

    Tran, Mei T.; Zsengeller, Zsuzsanna K.; Berg, Anders H.; Khankin, Eliyahu V.; Bhasin, Manoj K.; Kim, Wondong; Clish, Clary B.; Stillman, Isaac E.; Karumanchi, S. Ananth; Rhee, Eugene P.; Parikh, Samir M.

    2016-01-01

    The energetic burden of continuously concentrating solutes against gradients along the tubule may render the kidney especially vulnerable to ischemia. Indeed, acute kidney injury (AKI) affects 3% of all hospitalized patients.1,2 Here we show that the mitochondrial biogenesis regulator, PGC1α,3,4 is a pivotal determinant of renal recovery from injury by regulating NAD biosynthesis. Following renal ischemia, PGC1α−/− mice developed local deficiency of the NAD precursor niacinamide (Nam), marked fat accumulation, and failure to re-establish normal function. Remarkably, exogenous Nam improved local NAD levels, fat accumulation, and renal function in post-ischemic PGC1α−/− mice. Inducible tubular transgenic mice (iNephPGC1α) recapitulated the effects of Nam supplementation, including more local NAD and less fat accumulation with better renal function after ischemia. PGC1α coordinately upregulated the enzymes that synthesize NAD de novo from amino acids whereas PGC1α deficiency or AKI attenuated the de novo pathway. Nam enhanced NAD via the enzyme NAMPT and augmented production of the fat breakdown product beta-hydroxybutyrate (β-OHB), leading to increased prostaglandin PGE2, a secreted autocoid that maintains renal function.5 Nam treatment reversed established ischemic AKI and also prevented AKI in an unrelated toxic model. Inhibition of β-OHB signaling or prostaglandins similarly abolished PGC1α-dependent renoprotection. Given the importance of mitochondrial health in aging and the function of metabolically active organs, the results implicate Nam and NAD as key effectors for achieving PGC1α-dependent stress resistance. PMID:26982719

  9. PGC1α drives NAD biosynthesis linking oxidative metabolism to renal protection.

    PubMed

    Tran, Mei T; Zsengeller, Zsuzsanna K; Berg, Anders H; Khankin, Eliyahu V; Bhasin, Manoj K; Kim, Wondong; Clish, Clary B; Stillman, Isaac E; Karumanchi, S Ananth; Rhee, Eugene P; Parikh, Samir M

    2016-03-24

    The energetic burden of continuously concentrating solutes against gradients along the tubule may render the kidney especially vulnerable to ischaemia. Acute kidney injury (AKI) affects 3% of all hospitalized patients. Here we show that the mitochondrial biogenesis regulator, PGC1α, is a pivotal determinant of renal recovery from injury by regulating nicotinamide adenine dinucleotide (NAD) biosynthesis. Following renal ischaemia, Pgc1α(-/-) (also known as Ppargc1a(-/-)) mice develop local deficiency of the NAD precursor niacinamide (NAM, also known as nicotinamide), marked fat accumulation, and failure to re-establish normal function. Notably, exogenous NAM improves local NAD levels, fat accumulation, and renal function in post-ischaemic Pgc1α(-/-) mice. Inducible tubular transgenic mice (iNephPGC1α) recapitulate the effects of NAM supplementation, including more local NAD and less fat accumulation with better renal function after ischaemia. PGC1α coordinately upregulates the enzymes that synthesize NAD de novo from amino acids whereas PGC1α deficiency or AKI attenuates the de novo pathway. NAM enhances NAD via the enzyme NAMPT and augments production of the fat breakdown product β-hydroxybutyrate, leading to increased production of prostaglandin PGE2 (ref. 5), a secreted autacoid that maintains renal function. NAM treatment reverses established ischaemic AKI and also prevented AKI in an unrelated toxic model. Inhibition of β-hydroxybutyrate signalling or prostaglandin production similarly abolishes PGC1α-dependent renoprotection. Given the importance of mitochondrial health in ageing and the function of metabolically active organs, the results implicate NAM and NAD as key effectors for achieving PGC1α-dependent stress resistance.

  10. Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae

    SciTech Connect

    Sporty, J; Kabir, M M; Turteltaub, K; Ognibene, T; Lin, S; Bench, G

    2008-01-10

    A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, approximately 10{sup 8} yeast cells were harvested by centrifugation and then lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH{sub 3}CN + 50-mM ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet precipitated proteins, organic solvent removal was performed on supernatants by chloroform extraction. The remaining aqueous phase was dried and resuspended in 50-mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. NAD and NADH contents are similar to previously reported levels in yeast obtained using enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (1) applicable to quantification of these metabolites in mammalian and bacterial cell cultures; and (2) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.

  11. PGC1α drives NAD biosynthesis linking oxidative metabolism to renal protection.

    PubMed

    Tran, Mei T; Zsengeller, Zsuzsanna K; Berg, Anders H; Khankin, Eliyahu V; Bhasin, Manoj K; Kim, Wondong; Clish, Clary B; Stillman, Isaac E; Karumanchi, S Ananth; Rhee, Eugene P; Parikh, Samir M

    2016-03-24

    The energetic burden of continuously concentrating solutes against gradients along the tubule may render the kidney especially vulnerable to ischaemia. Acute kidney injury (AKI) affects 3% of all hospitalized patients. Here we show that the mitochondrial biogenesis regulator, PGC1α, is a pivotal determinant of renal recovery from injury by regulating nicotinamide adenine dinucleotide (NAD) biosynthesis. Following renal ischaemia, Pgc1α(-/-) (also known as Ppargc1a(-/-)) mice develop local deficiency of the NAD precursor niacinamide (NAM, also known as nicotinamide), marked fat accumulation, and failure to re-establish normal function. Notably, exogenous NAM improves local NAD levels, fat accumulation, and renal function in post-ischaemic Pgc1α(-/-) mice. Inducible tubular transgenic mice (iNephPGC1α) recapitulate the effects of NAM supplementation, including more local NAD and less fat accumulation with better renal function after ischaemia. PGC1α coordinately upregulates the enzymes that synthesize NAD de novo from amino acids whereas PGC1α deficiency or AKI attenuates the de novo pathway. NAM enhances NAD via the enzyme NAMPT and augments production of the fat breakdown product β-hydroxybutyrate, leading to increased production of prostaglandin PGE2 (ref. 5), a secreted autacoid that maintains renal function. NAM treatment reverses established ischaemic AKI and also prevented AKI in an unrelated toxic model. Inhibition of β-hydroxybutyrate signalling or prostaglandin production similarly abolishes PGC1α-dependent renoprotection. Given the importance of mitochondrial health in ageing and the function of metabolically active organs, the results implicate NAM and NAD as key effectors for achieving PGC1α-dependent stress resistance. PMID:26982719

  12. Occurrence of Metastudtite (Uranium Peroxide Dihydrate) at a FUSRAP Site

    SciTech Connect

    Young, C.M.; Nelson, K.A.; Stevens, G.T.; Grassi, V.J.

    2006-07-01

    Uranium concentrations in groundwater in a localized area of a site exceed the USEPA Maximum Contaminant Level (MCL) by a factor of one thousand. Although the groundwater seepage velocity ranges up to 0.7 meters per day (m/day), data indicate that the uranium is not migrating in groundwater. We believe that the uranium is not mobile because of local geochemical conditions and the unstable nature of the uranium compound present at the site; uranium peroxide dihydrate (metastudtite). Metastudtite [UO{sub 4}.2(H{sub 2}O) or (U(O{sub 2})|O|(OH){sub 2}).3H{sub 2}O] has been identified at other sites as an alteration product in casks of spent nuclear fuel, but neither enriched nor depleted uranium were present at this site. Metastudtite was first identified as a natural mineral in 1983, although documented occurrences in the environment are uncommon. The U.S. Army Corps of Engineers (USACE) is conducting a remedial investigation at the DuPont Chambers Works in Deep water New Jersey under the Formerly Utilized Sites Remedial Action Program (FUSRAP) to evaluate radioactive contamination resulting from historical activities conducted in support of Manhattan Engineering District operations. From 1942 to 1947, Chambers Works converted uranium oxides to uranium tetrafluoride and uranium metal. More than half of the production at this facility resulted from the recovery process, where uranium-bearing dross and scrap were reacted with hydrogen peroxide to produce uranium peroxide dihydrate. The 280-hectare Chambers Works has produced some 600 products, including petrochemicals, aromatics, fluoro-chemicals, polymers, and elastomers. Contaminants resulting from these processes, including separate-phase petrochemicals, have also been detected within the boundaries of the FUSRAP investigation. USACE initiated remedial investigation field activities in 2002. The radionuclides of concern are natural uranium (U{sub nat}) and its short-lived progeny. Areas of impacted soil generally

  13. The plant mitochondrial mat-r gene/nad1 gene complex

    SciTech Connect

    Wolstenhome, D.R.

    1996-12-31

    We have completed sequencing segments of the maize mitochondrial (mt) DNA that contains all five of the exons (A-E) of the gene (nad1) for subunit I of the respiratory chain NADH dehydrogenase. Analysis of these sequences indicates that exons B and C are joined by a continuous group II intron, but the remaining exons are associated with partial group II introns and are encoded at widely separated locations in the maize mtDNA molecule. We have shown that mature transcripts of the maize nad1 gene contain 23 edited nucleotides, and that transcripts of maize and soybean mat-r genes contain 15 and 14 edits, respectively. The majority of edits in nad1 transcripts result in amino acid replacements that increase similarity between the maize NAD1 protein and NAD1 proteins of other plant species and of animal species. We found that the intron between exons b and c is not edited. From data obtained using PCR and sequencing we have shown that transcripts containing all possible exon combinations exist in maize mitochondria.

  14. The ANKH gene and familial calcium pyrophosphate dihydrate deposition disease.

    PubMed

    Netter, Patrick; Bardin, Thomas; Bianchi, Arnaud; Richette, Pascal; Loeuille, Damien

    2004-09-01

    Familial calcium pyrophosphate dihydrate deposition (CPPD) disease is a chronic condition in which CPPD microcrystals deposit in the joint fluid, cartilage, and periarticular tissues. Two forms of familial CPPD disease have been identified: CCAL1 and CCAL2. The CCAL1 locus is located on the long arm of chromosome 8 and is associated with CPPD and severe osteoarthritis. The CCAL2 locus has been mapped to the short arm of chromosome 5 and identified in families from the Alsace region of France and the United Kingdom. The ANKH protein is involved in pyrophosphate metabolism and, more specifically, in pyrophosphate transport from the intracellular to the extracellular compartment. Numerous ANKH gene mutations cause familial CCAL2; they enhance ANKH protein activity, thereby elevating extracellular pyrophosphate levels and promoting the formation of pyrophosphate crystals, which produce the manifestations of the disease. Recent studies show that growth factors and cytokines can modify the expression of the normal ANKH protein. These results suggest a role for ANKH in sporadic CPPD disease and in CPPD associated with degenerative disease.

  15. Plant Defensins NaD1 and NaD2 Induce Different Stress Response Pathways in Fungi.

    PubMed

    Dracatos, Peter M; Payne, Jennifer; Di Pietro, Antonio; Anderson, Marilyn A; Plummer, Kim M

    2016-09-03

    Nicotiana alata defensins 1 and 2 (NaD1 and NaD2) are plant defensins from the ornamental tobacco that have antifungal activity against a variety of fungal pathogens. Some plant defensins interact with fungal cell wall O-glycosylated proteins. Therefore, we investigated if this was the case for NaD1 and NaD2, by assessing the sensitivity of the three Aspergillus nidulans (An) O-mannosyltransferase (pmt) knockout (KO) mutants (An∆pmtA, An∆pmtB, and An∆pmtC). An∆pmtA was resistant to both defensins, while An∆pmtC was resistant to NaD2 only, suggesting NaD1 and NaD2 are unlikely to have a general interaction with O-linked side chains. Further evidence of this difference in the antifungal mechanism was provided by the dissimilarity of the NaD1 and NaD2 sensitivities of the Fusarium oxysporum f. sp. lycopersici (Fol) signalling knockout mutants from the cell wall integrity (CWI) and high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathways. HOG pathway mutants were sensitive to both NaD1 and NaD2, while CWI pathway mutants only displayed sensitivity to NaD2.

  16. Plant Defensins NaD1 and NaD2 Induce Different Stress Response Pathways in Fungi

    PubMed Central

    Dracatos, Peter M.; Payne, Jennifer; Di Pietro, Antonio; Anderson, Marilyn A.; Plummer, Kim M.

    2016-01-01

    Nicotiana alata defensins 1 and 2 (NaD1 and NaD2) are plant defensins from the ornamental tobacco that have antifungal activity against a variety of fungal pathogens. Some plant defensins interact with fungal cell wall O-glycosylated proteins. Therefore, we investigated if this was the case for NaD1 and NaD2, by assessing the sensitivity of the three Aspergillus nidulans (An) O-mannosyltransferase (pmt) knockout (KO) mutants (An∆pmtA, An∆pmtB, and An∆pmtC). An∆pmtA was resistant to both defensins, while An∆pmtC was resistant to NaD2 only, suggesting NaD1 and NaD2 are unlikely to have a general interaction with O-linked side chains. Further evidence of this difference in the antifungal mechanism was provided by the dissimilarity of the NaD1 and NaD2 sensitivities of the Fusarium oxysporum f. sp. lycopersici (Fol) signalling knockout mutants from the cell wall integrity (CWI) and high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathways. HOG pathway mutants were sensitive to both NaD1 and NaD2, while CWI pathway mutants only displayed sensitivity to NaD2. PMID:27598152

  17. Plant Defensins NaD1 and NaD2 Induce Different Stress Response Pathways in Fungi.

    PubMed

    Dracatos, Peter M; Payne, Jennifer; Di Pietro, Antonio; Anderson, Marilyn A; Plummer, Kim M

    2016-01-01

    Nicotiana alata defensins 1 and 2 (NaD1 and NaD2) are plant defensins from the ornamental tobacco that have antifungal activity against a variety of fungal pathogens. Some plant defensins interact with fungal cell wall O-glycosylated proteins. Therefore, we investigated if this was the case for NaD1 and NaD2, by assessing the sensitivity of the three Aspergillus nidulans (An) O-mannosyltransferase (pmt) knockout (KO) mutants (An∆pmtA, An∆pmtB, and An∆pmtC). An∆pmtA was resistant to both defensins, while An∆pmtC was resistant to NaD2 only, suggesting NaD1 and NaD2 are unlikely to have a general interaction with O-linked side chains. Further evidence of this difference in the antifungal mechanism was provided by the dissimilarity of the NaD1 and NaD2 sensitivities of the Fusarium oxysporum f. sp. lycopersici (Fol) signalling knockout mutants from the cell wall integrity (CWI) and high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathways. HOG pathway mutants were sensitive to both NaD1 and NaD2, while CWI pathway mutants only displayed sensitivity to NaD2. PMID:27598152

  18. Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa; Chang, Andrew; Gerratana, Barbara

    2012-08-31

    Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligand complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

  19. The human NAD metabolome: Functions, metabolism and compartmentalization

    PubMed Central

    Nikiforov, Andrey; Kulikova, Veronika; Ziegler, Mathias

    2015-01-01

    Abstract The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD+ and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools. PMID:25837229

  20. Evidence for a Direct Effect of the NAD+ Precursor Acipimox on Muscle Mitochondrial Function in Humans

    PubMed Central

    van de Weijer, Tineke; Phielix, Esther; Bilet, Lena; Williams, Evan G.; Ropelle, Eduardo R.; Bierwagen, Alessandra; Livingstone, Roshan; Nowotny, Peter; Sparks, Lauren M.; Paglialunga, Sabina; Szendroedi, Julia; Havekes, Bas; Moullan, Norman; Pirinen, Eija; Hwang, Jong-Hee; Schrauwen-Hinderling, Vera B.; Hesselink, Matthijs K.C.; Auwerx, Johan

    2015-01-01

    Recent preclinical studies showed the potential of nicotinamide adenine dinucleotide (NAD+) precursors to increase oxidative phosphorylation and improve metabolic health, but human data are lacking. We hypothesize that the nicotinic acid derivative acipimox, an NAD+ precursor, would directly affect mitochondrial function independent of reductions in nonesterified fatty acid (NEFA) concentrations. In a multicenter randomized crossover trial, 21 patients with type 2 diabetes (age 57.7 ± 1.1 years, BMI 33.4 ± 0.8 kg/m2) received either placebo or acipimox 250 mg three times daily dosage for 2 weeks. Acipimox treatment increased plasma NEFA levels (759 ± 44 vs. 1,135 ± 97 μmol/L for placebo vs. acipimox, P < 0.01) owing to a previously described rebound effect. As a result, skeletal muscle lipid content increased and insulin sensitivity decreased. Despite the elevated plasma NEFA levels, ex vivo mitochondrial respiration in skeletal muscle increased. Subsequently, we showed that acipimox treatment resulted in a robust elevation in expression of nuclear-encoded mitochondrial gene sets and a mitonuclear protein imbalance, which may indicate activation of the mitochondrial unfolded protein response. Further studies in C2C12 myotubes confirmed a direct effect of acipimox on NAD+ levels, mitonuclear protein imbalance, and mitochondrial oxidative capacity. To the best of our knowledge, this study is the first to demonstrate that NAD+ boosters can also directly affect skeletal muscle mitochondrial function in humans. PMID:25352640

  1. Physiology-based kinetic modeling of neuronal energy metabolism unravels the molecular basis of NAD(P)H fluorescence transients

    PubMed Central

    Berndt, Nikolaus; Kann, Oliver; Holzhütter, Hermann-Georg

    2015-01-01

    Imaging of the cellular fluorescence of the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is one of the few metabolic readouts that enable noninvasive and time-resolved monitoring of the functional status of mitochondria in neuronal tissues. Stimulation-induced transient changes in NAD(P)H fluorescence intensity frequently display a biphasic characteristic that is influenced by various molecular processes, e.g., intracellular calcium dynamics, tricarboxylic acid cycle activity, the malate–aspartate shuttle, the glycerol-3-phosphate shuttle, oxygen supply or adenosine triphosphate (ATP) demand. To evaluate the relative impact of these processes, we developed and validated a detailed physiologic mathematical model of the energy metabolism of neuronal cells and used the model to simulate metabolic changes of single cells and tissue slices under different settings of stimulus-induced activity and varying nutritional supply of glucose, pyruvate or lactate. Notably, all experimentally determined NAD(P)H responses could be reproduced with one and the same generic cellular model. Our computations reveal that (1) cells with quite different metabolic status may generate almost identical NAD(P)H responses and (2) cells of the same type may quite differently contribute to aggregate NAD(P)H responses recorded in brain slices, depending on the spatial location within the tissue. Our computational approach reconciles different and sometimes even controversial experimental findings and improves our mechanistic understanding of the metabolic changes underlying live-cell NAD(P)H fluorescence transients. PMID:25899300

  2. CD73 protein as a source of extracellular precursors for sustained NAD+ biosynthesis in FK866-treated tumor cells.

    PubMed

    Grozio, Alessia; Sociali, Giovanna; Sturla, Laura; Caffa, Irene; Soncini, Debora; Salis, Annalisa; Raffaelli, Nadia; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina

    2013-09-01

    NAD(+) is mainly synthesized in human cells via the "salvage" pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the "salvage" pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD(+) or NAD(+) precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD(+) precursors for NAD(+) biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD(+) biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors. PMID:23880765

  3. Preservation of calcium pyrophosphate dihydrate crystals: effect of Mayer's haematoxylin staining period

    PubMed Central

    Ohira, T; Ishikawa, K

    2001-01-01

    OBJECTIVE—To clarify the deleterious effects of Mayer's haematoxylin staining procedure which result in a decrease in, or complete loss of, the number of calcium pyrophosphate dihydrate (CPPD) crystals, and to determine the proper staining period for preserving the crystals in a histological paraffin section of articular tissues.
METHODS—Paraffin sections of CPPD crystal-bearing articular tissues of six patients were stained with Mayer's haematoxylin for 3, 8, or 15 minutes, and subsequently with eosin for one minute. The specimens were examined with an Olympus BHS polarised light microscope. The pH of Mayer's haematoxylin solution was measured with a TOA pH meter.
RESULTS—Positive birefringent CPPD crystals were seen clearly in all specimens stained with Mayer's haematoxylin for three minutes. The specimens stained for eight minutes showed a reduced number of crystals. No crystals were seen in the specimens stained for 15 minutes. Ordinary light microscopy showed no notable differences in the stainability of nucleus, cell membrane, and their surrounding tissues among specimens when stained with Mayer's haematoxylin for either 3, 8, or 15 minutes. The pH of Mayer's haematoxylin solution was 2.31.
CONCLUSIONS—To find CPPD crystals in the paraffin sections of articular tissues, the staining period with Mayer's haematoxylin should be limited to three minutes. The longer the staining period, the greater the reduction in the number of crystals owing to the strong acidity of the haematoxylin solution. A staining period of 15 minutes causes a complete loss of CPPD crystals.

 PMID:11114290

  4. A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71

    PubMed Central

    Wu, Ming-Cai; Tian, Chang-Qing; Cheng, Hong-Mei; Xu, Lei; Wang, Peng; Zhu, Guo-Ping

    2015-01-01

    In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD+-specific (NAD-IDH) or NADP+-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD+-specific and showed no detectable activity with NADP+. The Km values of the enzyme for NAD+ were 262.6±7.4 μM or 309.1±11.2 μM with Mg2+ or Mn2+ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP+ was approximately 24-fold higher than that for NAD+, suggesting that ClIDH is an NAD+-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn2+. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD+-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs. PMID:25942017

  5. NADS - Nuclear And Atomic Data System

    SciTech Connect

    McKinley, M S; Beck, B; McNabb, D

    2004-09-17

    We have developed NADS (Nuclear and Atomic Data System), a web-based graphical interface for viewing pointwise and grouped cross-sections and distributions. Our implementation is a client / server model. The client is a Java applet that displays the graphical interface, which has interactive 2-D, 3-D, and 4-D plots and tables. The server, which can serve and perform computations the data, has been implemented in Python using the FUDGE package developed by Bret Beck at LLNL. Computational capabilities include algebraic manipulation of nuclear evaluated data in databases such as LLNL's ENDL-99, ENDF/B-V and ENDF/B-VI as well as user data. Processed data used in LLNL's transport codes are accessible as well. NADS is available from http://nuclear.llnl.gov/

  6. NADS - Nuclear and Atomic Data System

    SciTech Connect

    McKinley, Michael S.; Beck, Bret; McNabb, Dennis

    2005-05-24

    We have developed NADS (Nuclear and Atomic Data System), a web-based graphical interface for viewing pointwise and grouped cross sections and distributions. Our implementation is a client / server model. The client is a Java applet that displays the graphical interface, which has interactive 2-D, 3-D, and 4-D plots and tables. The server, which can serve and perform computations of the data, has been implemented in Python using the FUDGE package developed by Bret Beck at LLNL. Computational capabilities include algebraic manipulation of nuclear evaluated data in databases such as LLNL's ENDL-99, ENDF/B-V, and ENDF/B-VI, as well as user data. Processed data used in LLNL's transport codes are accessible as well. NADS is available from http://nuclear.llnl.gov/.

  7. NadA diversity and carriage in Neisseria meningitidis.

    PubMed

    Comanducci, Maurizio; Bambini, Stefania; Caugant, Dominique A; Mora, Marirosa; Brunelli, Brunella; Capecchi, Barbara; Ciucchi, Laura; Rappuoli, Rino; Pizza, Mariagrazia

    2004-07-01

    NadA is a novel vaccine candidate recently identified in Neisseria meningitidis and involved in adhesion to host tissues. The nadA gene has been found in approximately 50% of the strains isolated from patients and in three of the four hypervirulent lineages of non-serogroup A strains. Here we investigated the presence of the nadA gene in 154 meningococcal strains isolated from healthy people (carrier strains). Only 25 (16.2%) of the 154 carrier isolates harbored the nadA gene. The commensal species Neisseria lactamica was also found not to have the nadA gene. Eighteen of the carrier strains belonged to the ET-5 and ET-37 hypervirulent clusters, indicating that only the 5.1% of the genuine carrier population actually harbored nadA (7 of 136 strains). Five of the seven strains harbored a novel allele of the nadA gene that was designated nadA4. The NadA4 protein was present on the bacterial surface as heat-stable high-molecular-weight oligomers. Antibodies against the recombinant NadA4 protein were bactericidal against homologous strains, whereas the activity against other NadA alleles was weak. In conclusion, the nadA gene segregates differently in the population of strains isolated from healthy individuals and in the population of strains isolated from patients. The presence of NadA can therefore be used as a tool to study the dynamics of meningococcal infections and understand why this bacterium, which is mostly a commensal, can become a severe pathogen.

  8. Gene transfers shaped the evolution of de novo NAD+ biosynthesis in eukaryotes.

    PubMed

    Ternes, Chad M; Schönknecht, Gerald

    2014-09-01

    NAD(+) is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD(+) biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD(+) biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD(+) biosynthesis in eukaryotes was shaped by numerous gene transfers.

  9. [Enzymes related with NAD synthesis promote conversion of 1,4-butanediol to 4-hydroxybutyrate].

    PubMed

    Zhang, Xin; Chen, Guoqiang

    2011-12-01

    Besides medical application, 4-hydroxybutyrate (4-HB) is a precursor of P3HB4HB, a bioplastic showing excellent physical properties and degradability. Escherichia coli S17-1 (pZL-dhaT-aldD) can transform 1, 4-butanediol (1,4-BD) into 4HB with participation of cofactor NAD. To enhance productivity, nicotinic acid phosphoribosyltransferase (PncB) and nicotinamide adenine dinucleotide synthetase (NadE) were overexpressed to increase intracellular nicotinamide adenine dinucleotide concentration and promote reaction process. The shake flask fermentation result showed that the conversion rate increased by 13.03% with help of PncB-NadE, leading to 4.87 g/L 4HB from 10 g/L 1,4-BD, and productivity was increased by 40.91% to 1.86 g/g. These results demonstrated that expression of PncB and NadE is beneficial for conversion of 1,4-BD to 4HB. PMID:22506415

  10. Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

    PubMed Central

    Ternes, Chad M.; Schönknecht, Gerald

    2014-01-01

    NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

  11. NAD(P)H regeneration is the key for heterolactic fermentation of hexoses in Oenococcus oeni.

    PubMed

    Maicas, Sergi; Ferrer, Sergi; Pardo, Isabel

    2002-01-01

    Oenococcus oeni (formerly Leuconostoc oenos) can perform malolactic fermentation, converting L-malate to L-lactate and carbon dioxide, in wines. The energy and redox potential required to support the growth of the micro-organism are supplied mainly by the consumption of carbohydrates via the heterolactic pathway. In the first steps of hexose metabolism two molecules of NAD(P)(+) are consumed, which must be regenerated in later reactions. The aim of this work was to test if aerobic growth of O. oeni promotes higher cell yields than anaerobic conditions, as has been shown for other lactic acid bacteria. O. oeni M42 was found to grow poorly under aerobic conditions with glucose as the only carbohydrate in the medium. It was demonstrated that O(2) inactivates the enzymes of the ethanol-forming pathway, one of the two pathways which reoxidizes NAD(P)(+) cofactors in the heterolactic catabolism of glucose. These results suggest that the regeneration of cofactors is the limiting factor for the aerobic consumption of glucose. When external electron acceptors, such as fructose or pyruvate, were added to glucose-containing culture medium the growth of O. oeni was stimulated slightly; fructose was converted to mannitol, oxidizing two molecules of NAD(P)H, and pyruvate was transformed to lactate, enabling the regeneration of NAD(+). The addition of cysteine seemed to suppress the inactivation of the ethanol-forming pathway enzymes by O(2), enabling glucose consumption in aerobic conditions to reach similar rates to those found in anaerobic conditions.

  12. Regulation of NAD biosynthetic enzymes modulates NAD-sensing processes to shape mammalian cell physiology under varying biological cues.

    PubMed

    Ruggieri, Silverio; Orsomando, Giuseppe; Sorci, Leonardo; Raffaelli, Nadia

    2015-09-01

    In addition to its role as a redox coenzyme, NAD is a substrate of various enzymes that split the molecule to either catalyze covalent modifications of target proteins or convert NAD into biologically active metabolites. The coenzyme bioavailability may be significantly affected by these reactions, with ensuing major impact on energy metabolism, cell survival, and aging. Moreover, through the activity of the NAD-dependent deacetylating sirtuins, NAD behaves as a beacon molecule that reports the cell metabolic state, and accordingly modulates transcriptional responses and metabolic adaptations. In this view, NAD biosynthesis emerges as a highly regulated process: it enables cells to preserve NAD homeostasis in response to significant NAD-consuming events and it can be modulated by various stimuli to induce, via NAD level changes, suitable NAD-mediated metabolic responses. Here we review the current knowledge on the regulation of mammalian NAD biosynthesis, with focus on the relevant rate-limiting enzymes. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. PMID:25770681

  13. Regulation of NAD biosynthetic enzymes modulates NAD-sensing processes to shape mammalian cell physiology under varying biological cues.

    PubMed

    Ruggieri, Silverio; Orsomando, Giuseppe; Sorci, Leonardo; Raffaelli, Nadia

    2015-09-01

    In addition to its role as a redox coenzyme, NAD is a substrate of various enzymes that split the molecule to either catalyze covalent modifications of target proteins or convert NAD into biologically active metabolites. The coenzyme bioavailability may be significantly affected by these reactions, with ensuing major impact on energy metabolism, cell survival, and aging. Moreover, through the activity of the NAD-dependent deacetylating sirtuins, NAD behaves as a beacon molecule that reports the cell metabolic state, and accordingly modulates transcriptional responses and metabolic adaptations. In this view, NAD biosynthesis emerges as a highly regulated process: it enables cells to preserve NAD homeostasis in response to significant NAD-consuming events and it can be modulated by various stimuli to induce, via NAD level changes, suitable NAD-mediated metabolic responses. Here we review the current knowledge on the regulation of mammalian NAD biosynthesis, with focus on the relevant rate-limiting enzymes. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  14. Phosphorescence maxima and triplet state lifetimes of NAD+ and epsilon-NAD+ in ternary complexes with horse liver alcohol dehydrogenase.

    PubMed

    Rousslang, K; Allen, L; Ross, J B

    1989-02-01

    This paper describes the phosphorescence emission and decay times of NAD+ and its fluorescent etheno derivative, epsilon-NAD+, in the pyrazole ternary complex with horse liver alcohol dehydrogenase (ADH). We show that the epsilon-NAD+ triplet state, as well as the tryptophan triplet state, can be utilized to monitor the coenzyme-enzyme interaction. The decays of NAD+ and AMP are single exponential, and the lifetimes are the same within experimental error. The phosphorescence lifetimes, evaluated as single exponentials, are slightly shorter in epsilon-NAD+ than they are in epsilon-AMP. Whereas the decay of epsilon-AMP was adequately fit by a single exponential with a time constant of very close to 0.5 s, it was necessary to fit the decay of epsilon-NAD+ to a double exponential. Ternary complexes with NAD+ excited at 297 nm exhibit decay kinetics nearly identical to those of ADH by itself. On the other hand, when excitation of the epsilon-NAD+ ternary complex is provided at 313 nm, where there is very little absorption by either tryptophan residue, the decay law of the ternary complex is similar to that of epsilon-NAD+ in solution. Our results demonstrate that NAD+ and epsilon-NAD+ quench tryptophan phosphorescence in ADH. Normalizing the phosphorescence intensity to the 0-0 vibronic band assigned to Trp-15 (blue-edge), we calculate a 21% decrease in the phosphorescence associated with Trp-314 at stoichiometric saturation of the coenzyme binding sites with NAD+ in the ternary complex. When the active sites are saturated with epsilon-NAD+, the relative phosphorescence due to Trp-314 decreases by 63%.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Critical role for NAD glycohydrolase in regulation of erythropoiesis by hematopoietic stem cells through control of intracellular NAD content.

    PubMed

    Nam, Tae-Sik; Park, Kwang-Hyun; Shawl, Asif Iqbal; Kim, Byung-Ju; Han, Myung-Kwan; Kim, Youngho; Moss, Joel; Kim, Uh-Hyun

    2014-06-01

    NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD. PMID:24759100

  16. Calcium pyrophosphate dihydrate and hydroxyapatite crystal deposition in the joint: new developments relevant to the clinician.

    PubMed

    Pay, Salih; Terkeltaub, Robert

    2003-06-01

    The major types of crystals containing calcium, which causes arthropathy and periarticular disease, are calcium pyrophosphate dihydrate and basic calcium phosphates, including hydroxyapatite. Exciting advances include the identification of mutations in the gene ANKH associated with disordered inorganic pyrophosphate (PPi) transport in some kindred with familial chondrocalcinosis linked to chromosome 5p. In addition, central basic mechanisms governing cartilage calcification and their relationship to aging and osteoarthritis have now been elucidated. These include the role of plasma cell glycoprotein-1, the PPi-generating ecto-enzyme, in chondrocalcinosis and the linkage of low- grade inflammation to expression and activation of two cartilage-expressed transglutaminase isoenzymes with direct calcification-stimulating activity. This review discusses clinically pertinent new information on pathogenesis. The authors also address, in detail, current diagnostic and therapeutic issues pertaining to calcium pyrophosphate dihydrate and hydroxyapatite crystal deposition in the joint, as well as possible therapeutic directions for the future.

  17. Agents for replacement of NAD+/NADH system in enzymatic reactions

    DOEpatents

    Fish, Richard H.; Kerr, John B.; Lo, Christine H.

    2004-04-06

    Novel agents acting as co-factors for replacement of NAD(P).sup.+ /NAD(P)H co-enzyme systems in enzymatic oxido-reductive reactions. Agents mimicking the action of NAD(P).sup.+ /NAD(P)H system in enzymatic oxidation/reduction of substrates into reduced or oxidized products. A method for selection and preparation of the mimicking agents for replacement of NAD(P).sup.+ /NAD(P)H system and a device comprising co-factors for replacement of NAD(P).sup.+ /NAD(P)H system.

  18. Strength and nature of hydrogen bonding interactions in mono- and di-hydrated formamide complexes.

    PubMed

    Angelina, Emilio L; Peruchena, Nélida M

    2011-05-12

    In this work, mono- and di-hydrated complexes of the formamide were studied. The calculations were performed at the MP2/6-311++G(d,p) level of approximation. The atoms in molecules theory (AIM), based on the topological properties of the electronic density distribution, was used to characterize the different types of bonds. The analysis of the hydrogen bonds (H-bonds) in the most stable mono- and di-hydrated formamide complexes shows a mutual reinforcement of the interactions, and some of these complexes can be considered as "bifunctional hydrogen bonding hydration complexes". In addition, we analyzed how the strength and the nature of the interactions, in mono-hydrated complexes, are modified by the presence of a second water molecule in di-hydrated formamide complexes. Structural changes, cooperativity, and electron density redistributions demonstrate that the H-bonds are stronger in the di-hydrated complexes than in the corresponding mono-hydrated complexes, wherein the σ- and π-electron delocalization were found. To explain the nature of such interactions, we carried out the atoms in molecules theory in conjunction with reduced variational space self-consistent field (RVS) decomposition analysis. On the basis of the local Virial theorem, the characteristics of the local electron energy density components at the bond critical points (BCPs) (the 1/4∇ (2)ρ(b) component of electron energy density and the kinetic energy density) were analyzed. These parameters were used in conjunction with the electron density and the Laplacian of the electron density to analyze the characteristics of the interactions. The analysis of the interaction energy components for the systems considered indicates that the strengthening of the hydrogen bonds is manifested by an increased contribution of the electrostatic energy component represented by the kinetic energy density at the BCP. PMID:21506592

  19. Solid-vapor interactions: influence of environmental conditions on the dehydration of carbamazepine dihydrate.

    PubMed

    Surana, Rahul; Pyne, Abira; Suryanarayanan, Raj

    2003-12-31

    The goal of this research was a phenomenological study of the effect of environmental factors on the dehydration behavior of carbamazepine dihydrate. Dehydration experiments were performed in an automated vapor sorption apparatus under a variety of conditions, and weight loss was monitored as a function of time. In addition to lattice water, carbamazepine dihydrate contained a significant amount of physically bound water. Based on the kinetics of water loss, it was possible to differentiate between the removal of physically bound water and the lattice water. The activation energy for the 2 processes was 44 and 88 kJ/mol, respectively. As expected, the dehydration rate of carbamazepine dihydrate decreased with an increase in water vapor pressure. While dehydration at 0% relative humidity (RH) resulted in an amorphous anhydrate, the crystallinity of the anhydrate increased as a function of the RH of dehydration. A method was developed for in situ crystallinity determination of the anhydrate formed. Dehydration in the presence of the ethanol vapor was a 2-step process, and the fraction dehydrated at each step was a function of the ethanol vapor pressure. We hypothesize the formation of an intermediate lower hydrate phase with unknown water stoichiometry. An increase in the ethanol vapor pressure first led to a decrease in the dehydration rate followed by an increase. In summary, the dehydration behavior of carbamazepine dihydrate was evaluated at different vapor pressures of water and ethanol. Using the water sorption apparatus, it was possible to (1) differentiate between the removal of physically bound and lattice water, and (2) develop a method for quantifying, in situ, the crystallinity of the product (anhydrate) phase. PMID:15198563

  20. Strength and nature of hydrogen bonding interactions in mono- and di-hydrated formamide complexes.

    PubMed

    Angelina, Emilio L; Peruchena, Nélida M

    2011-05-12

    In this work, mono- and di-hydrated complexes of the formamide were studied. The calculations were performed at the MP2/6-311++G(d,p) level of approximation. The atoms in molecules theory (AIM), based on the topological properties of the electronic density distribution, was used to characterize the different types of bonds. The analysis of the hydrogen bonds (H-bonds) in the most stable mono- and di-hydrated formamide complexes shows a mutual reinforcement of the interactions, and some of these complexes can be considered as "bifunctional hydrogen bonding hydration complexes". In addition, we analyzed how the strength and the nature of the interactions, in mono-hydrated complexes, are modified by the presence of a second water molecule in di-hydrated formamide complexes. Structural changes, cooperativity, and electron density redistributions demonstrate that the H-bonds are stronger in the di-hydrated complexes than in the corresponding mono-hydrated complexes, wherein the σ- and π-electron delocalization were found. To explain the nature of such interactions, we carried out the atoms in molecules theory in conjunction with reduced variational space self-consistent field (RVS) decomposition analysis. On the basis of the local Virial theorem, the characteristics of the local electron energy density components at the bond critical points (BCPs) (the 1/4∇ (2)ρ(b) component of electron energy density and the kinetic energy density) were analyzed. These parameters were used in conjunction with the electron density and the Laplacian of the electron density to analyze the characteristics of the interactions. The analysis of the interaction energy components for the systems considered indicates that the strengthening of the hydrogen bonds is manifested by an increased contribution of the electrostatic energy component represented by the kinetic energy density at the BCP.

  1. A case of chronic calcium pyrophosphate dihydrate crystal disease (tophaceous pseudogout) in the temporomandibular joint.

    PubMed

    Meul, B; Ernestus, K; Neugebauer, J; Kuebler, A C

    2005-03-01

    Pseudogout is a rare joint disease which is characterized by the presence of calcium pyrophosphate dihydrate crystals in the intraarticular and periarticular tissue. The crystals tend to attach to fibrocartilage tissue. Pseudogout principally affects the knee and wrist joints. Involvement of the temporomandibular joint (TMJ) is very rare. There have been <20 cases reported world-wide. Both acute and chronic manifestations have been described. We present here an unusual case that necessitated a high condylectomy.

  2. Boosting NAD(+) for the prevention and treatment of liver cancer.

    PubMed

    Djouder, Nabil

    2015-01-01

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide yet has limited therapeutic options. We recently demonstrated that inhibition of de novo nicotinamide adenine dinucleotide (NAD(+)) synthesis is responsible for DNA damage, thereby initiating hepatocarcinogenesis. We propose that boosting NAD(+) levels might be used as a prophylactic or therapeutic approach in HCC. PMID:27308492

  3. Reassessing the role of NAD as a prosurvival factor

    PubMed Central

    Preyat, Nicolas; Leo, Oberdan

    2016-01-01

    ABSTRACT The mechanisms regulating the choice of cell demise remain largely unknown. NAD+, a key metabolite with well-known roles in cell metabolism, has been shown to counteract apoptosis while promoting necroptosis, a form of proinflammatory cell death. This observation identifies NAD+ availability as an important parameter with contrasting roles in the regulation of distinct regulated cell death programs. PMID:27308572

  4. Lung contains an inhibitor for nicotinatemononucleotide pyrophosphorylase (carboxylating) of NAD biosynthesis.

    PubMed

    Seither, R L; Brown, O R; Babu, B V

    1991-01-01

    Rat, cow and foal lung extracts contained an inhibitor for the liver NAD biosynthetic-pathway enzyme, nicotinatemononucleotide pyrophosphorylase (carboxylating) [EC 2.4.2.19]. The inhibitor was not dialyzable, was labile at 100 degrees C, was retained by a 30,000 dalton pore size Amicon membrane and, when partially purified by precipitation at 40-100% ammonium sulfate, inhibited the enzyme stoichiometrically. Lung reportedly does not contain nicotinate-mononucleotide pyrophosphorylase or make NAD de novo. However, the inhibitor would mask detection of the enzyme in lung extracts. We detected a low nicotinatemononucleotide pyrophosphorylase-like activity (0.003 +/- 0.001 nanomoles CO2 produced from quinolinic acid per mg of extract protein) in rat lung but none in foal or cow lung.

  5. Isolation of a GPD gene from Debaryomyces hansenii encoding a glycerol 3-phosphate dehydrogenase (NAD+).

    PubMed

    Thomé, Patricia E

    2004-01-30

    A gene homologous to GPD1, coding for glycerol-3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), has been isolated from the halophilic yeast Debaryomyces hansenii by complementation of a Saccharomyces cerevisiae gpd1 Delta mutant. DNA sequencing of the complementing genomic clone indicated the existence of an open reading frame encoding a protein with 369 amino acids. Comparative analysis of the deduced amino acid sequence showed high similarity to homologous genes described for other eukaryotic GPD enzymes. The sequence has been submitted to the GenBank database under Accession No. AY333427.

  6. NAD + -dependent Formate Dehydrogenase from Plants

    PubMed Central

    Alekseeva, A.A.; Savin, S.S.; Tishkov, V.I.

    2011-01-01

    NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) widely occurs in nature. FDH consists of two identical subunits and contains neither prosthetic groups nor metal ions. This type of FDH was found in different microorganisms (including pathogenic ones), such as bacteria, yeasts, fungi, and plants. As opposed to microbiological FDHs functioning in cytoplasm, plant FDHs localize in mitochondria. Formate dehydrogenase activity was first discovered as early as in 1921 in plant; however, until the past decade FDHs from plants had been considerably less studied than the enzymes from microorganisms. This review summarizes the recent results on studying the physiological role, properties, structure, and protein engineering of plant formate dehydrogenases. PMID:22649703

  7. Antidepressants may lead to a decrease in niacin and NAD in patients with poor dietary intake.

    PubMed

    Viljoen, Margaretha; Swanepoel, Annie; Bipath, Priyesh

    2015-03-01

    The term niacin is the generic name for the two compounds nicotinic acid and nicotinamide, the major dietary precursors for two important coenzymes, nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP. Niacin is important for the maintenance of cellular integrity and energy production and is involved in more than 500 intracellular reactions. Deficiencies of niacin may contribute to neuropsychiatric and neurodegenerative disorders. Patients who develop nutritional deficiencies as a result of poor dietary intake, especially inadequate intake of proteins and vitamins, could potentially suffer from niacin deficiency and NAD depletion. However, de novo synthesis of niacin and NAD in the kynurenine pathway of tryptophan metabolism may compensate for impaired dietary intake. The rate of synthesis of NAD and niacin from tryptophan oxidation depends on the induction of the enzyme indoleamine 2,3-dioxygenase (IDO) by pro-inflammatory cytokines such as interferon-gamma. Niacin synthesis is not limited by a decrease in tryptophan and excessive IDO activity may therefore lead to a decline in tryptophan levels. Antidepressants have an anti-inflammatory effect, including reduction of interferon-gamma and therefore inhibition of IDO, the rate-limiting enzyme of the kynurenine pathway. In theory, this could account for increased serotonin as more tryptophan becomes available for serotonin synthesis. However, the downside may be that less NAD and niacin are synthesised downstream, which could exacerbate common psychiatric problems. It is our hypothesis that patients with poor dietary intake, who are treated with antidepressants, are at risk of developing niacin/NAD deficiency with possible development of associated neuropsychiatric symptoms. We therefore propose that niacin supplementation be considered in patients with inadequate diets who are treated with antidepressants. We believe that if this does not happen, a subclinical niacin deficiency may result

  8. Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions.

    PubMed

    de Souza, Robson Francisco; Aravind, L

    2012-06-01

    Nicotinamide adenine dinucleotide (NAD) is a ubiquitous cofactor participating in numerous redox reactions. It is also a substrate for regulatory modifications of proteins and nucleic acids via the addition of ADP-ribose moieties or removal of acyl groups by transfer to ADP-ribose. In this study, we use in-depth sequence, structure and genomic context analysis to uncover new enzymes and substrate-binding proteins in NAD-utilizing metabolic and macromolecular modification systems. We predict that Escherichia coli YbiA and related families of domains from diverse bacteria, eukaryotes, large DNA viruses and single strand RNA viruses are previously unrecognized components of NAD-utilizing pathways that probably operate on ADP-ribose derivatives. Using contextual analysis we show that some of these proteins potentially act in RNA repair, where NAD is used to remove 2'-3' cyclic phosphodiester linkages. Likewise, we predict that another family of YbiA-related enzymes is likely to comprise a novel NAD-dependent ADP-ribosylation system for proteins, in conjunction with a previously unrecognized ADP-ribosyltransferase. A similar ADP-ribosyltransferase is also coupled with MACRO or ADP-ribosylglycohydrolase domain proteins in other related systems, suggesting that all these novel systems are likely to comprise pairs of ADP-ribosylation and ribosylglycohydrolase enzymes analogous to the DraG-DraT system, and a novel group of bacterial polymorphic toxins. We present evidence that some of these coupled ADP-ribosyltransferases/ribosylglycohydrolases are likely to regulate certain restriction modification enzymes in bacteria. The ADP-ribosyltransferases found in these, the bacterial polymorphic toxin and host-directed toxin systems of bacteria such as Waddlia also throw light on the evolution of this fold and the origin of eukaryotic polyADP-ribosyltransferases and NEURL4-like ARTs, which might be involved in centrosomal assembly. We also infer a novel biosynthetic pathway that

  9. Enzymology of mammalian NAD metabolism in health and disease.

    PubMed

    Magni, Giulio; Orsomando, Giuseppe; Raffelli, Nadia; Ruggieri, Silverio

    2008-01-01

    Mounting evidence attests to the paramount importance of the non-redox NAD functions. Indeed, NAD homeostasis is related to the free radicals-mediated production of reactive oxygen species responsible for irreversible cellular damage in infectious disease, diabetes, inflammatory syndromes, neurodegeneration and cancer. Because the cellular redox status depends on both the absolute concentration of pyridine dinucleotides and their respective ratios of oxidized and reduced forms (i.e., NAD/NADH and NADP/NADPH), it is conceivable that an altered regulation of the synthesis and degradation of NAD impairs the cell redox state and likely contributes to the mechanisms underlying the pathogenesis of the above mentioned diseases. Taking into account the recent appearance in the literature of comprehensive reviews covering different aspects of the significance of NAD metabolism, with particular attention to the enzymes involved in NAD cleavage, this monograph includes the most recent results on NAD biosynthesis in mammals and humans. Due to recent findings on nicotinamide riboside as a nutrient, its inclusion under "niacins" is proposed. Here, the enzymes involved in the de novo and reutilization pathways are overviewed. PMID:18508649

  10. Reduced Ssy1-Ptr3-Ssy5 (SPS) signaling extends replicative life span by enhancing NAD+ homeostasis in Saccharomyces cerevisiae.

    PubMed

    Tsang, Felicia; James, Christol; Kato, Michiko; Myers, Victoria; Ilyas, Irtqa; Tsang, Matthew; Lin, Su-Ju

    2015-05-15

    Attenuated nutrient signaling extends the life span in yeast and higher eukaryotes; however, the mechanisms are not completely understood. Here we identify the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway as a novel longevity factor. A null mutation of SSY5 (ssy5Δ) increases replicative life span (RLS) by ∼50%. Our results demonstrate that several NAD(+) homeostasis factors play key roles in this life span extension. First, expression of the putative malate-pyruvate NADH shuttle increases in ssy5Δ cells, and deleting components of this shuttle, MAE1 and OAC1, largely abolishes RLS extension. Next, we show that Stp1, a transcription factor of the SPS pathway, directly binds to the promoter of MAE1 and OAC1 to regulate their expression. Additionally, deletion of SSY5 increases nicotinamide riboside (NR) levels and phosphate-responsive (PHO) signaling activity, suggesting that ssy5Δ increases NR salvaging. This increase contributes to NAD(+) homeostasis, partially ameliorating the NAD(+) deficiency and rescuing the short life span of the npt1Δ mutant. Moreover, we observed that vacuolar phosphatase, Pho8, is partially required for ssy5Δ-mediated NR increase and RLS extension. Together, our studies present evidence that supports SPS signaling is a novel NAD(+) homeostasis factor and ssy5Δ-mediated life span extension is likely due to concomitantly increased mitochondrial and vacuolar function. Our findings may contribute to understanding the molecular basis of NAD(+) metabolism, cellular life span, and diseases associated with NAD(+) deficiency and aging. PMID:25825491

  11. Rosuvastatin prevents angiotensin II-induced vascular changes by inhibition of NAD(P)H oxidase and COX-1

    PubMed Central

    Colucci, Rocchina; Fornai, Matteo; Duranti, Emiliano; Antonioli, Luca; Rugani, Ilaria; Aydinoglu, Fatma; Ippolito, Chiara; Segnani, Cristina; Bernardini, Nunzia; Taddei, Stefano; Blandizzi, Corrado; Virdis, Agostino

    2013-01-01

    Background and Purpose NAD(P)H oxidase and COX-1 participate in vascular damage induced by angiotensin II. We investigated the effect of rosuvastatin on endothelial dysfunction, vascular remodelling, changes in extracellular matrix components and mechanical properties of small mesenteric arteries from angiotensin II-infused rats. Experimental Approach Male rats received angiotensin II (120 ng·kg−1·min−1, subcutaneously) for 14 days with or without rosuvastatin (10 mg·kg−1·day−1, oral gavage) or vehicle. Vascular functions and morphological parameters were assessed by pressurized myography. Key Results In angiotensin II-infused rats, ACh-induced relaxation was attenuated compared with controls, less sensitive to L-NAME, enhanced by SC-560 (COX-1 inhibitor) or SQ-29548 (prostanoid TP receptor antagonist), and normalized by the antioxidant ascorbic acid or NAD(P)H oxidase inhibitors. After rosuvastatin, relaxations to ACh were normalized, fully sensitive to L-NAME, and no longer affected by SC-560, SQ-29548 or NAD(P)H oxidase inhibitors. Angiotensin II enhanced intravascular superoxide generation, eutrophic remodelling, collagen and fibronectin depositions, and decreased elastin content, resulting in increased vessel stiffness. All these changes were prevented by rosuvastatin. Angiotensin II increased phosphorylation of NAD(P)H oxidase subunit p47phox and its binding to subunit p67phox, effects inhibited by rosuvastatin. Rosuvastatin down-regulated vascular Nox4/NAD(P)H isoform and COX-1 expression, attenuated the vascular release of 6-keto-PGF1α, and enhanced copper/zinc-superoxide dismutase expression. Conclusion and Implications Rosuvastatin prevents angiotensin II-induced alterations in resistance arteries in terms of function, structure, mechanics and composition. These effects depend on restoration of NO availability, prevention of NAD(P)H oxidase-derived oxidant excess, reversal of COX-1 induction and its prostanoid production, and stimulation of

  12. Reduced Ssy1-Ptr3-Ssy5 (SPS) Signaling Extends Replicative Life Span by Enhancing NAD+ Homeostasis in Saccharomyces cerevisiae*

    PubMed Central

    Tsang, Felicia; James, Christol; Kato, Michiko; Myers, Victoria; Ilyas, Irtqa; Tsang, Matthew; Lin, Su-Ju

    2015-01-01

    Attenuated nutrient signaling extends the life span in yeast and higher eukaryotes; however, the mechanisms are not completely understood. Here we identify the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway as a novel longevity factor. A null mutation of SSY5 (ssy5Δ) increases replicative life span (RLS) by ∼50%. Our results demonstrate that several NAD+ homeostasis factors play key roles in this life span extension. First, expression of the putative malate-pyruvate NADH shuttle increases in ssy5Δ cells, and deleting components of this shuttle, MAE1 and OAC1, largely abolishes RLS extension. Next, we show that Stp1, a transcription factor of the SPS pathway, directly binds to the promoter of MAE1 and OAC1 to regulate their expression. Additionally, deletion of SSY5 increases nicotinamide riboside (NR) levels and phosphate-responsive (PHO) signaling activity, suggesting that ssy5Δ increases NR salvaging. This increase contributes to NAD+ homeostasis, partially ameliorating the NAD+ deficiency and rescuing the short life span of the npt1Δ mutant. Moreover, we observed that vacuolar phosphatase, Pho8, is partially required for ssy5Δ-mediated NR increase and RLS extension. Together, our studies present evidence that supports SPS signaling is a novel NAD+ homeostasis factor and ssy5Δ-mediated life span extension is likely due to concomitantly increased mitochondrial and vacuolar function. Our findings may contribute to understanding the molecular basis of NAD+ metabolism, cellular life span, and diseases associated with NAD+ deficiency and aging. PMID:25825491

  13. Isolation of a Histoplasma capsulatum cDNA that complements a mitochondrial NAD(+)-isocitrate dehydrogenase subunit I-deficient mutant of Saccharomyces cerevisiae.

    PubMed

    Johnson, C H; McEwen, J E

    1999-06-30

    A cDNA library was prepared from Histoplasma capsulatum strain G-217B yeast cells and an apparently full-length cDNA for a subunit of the citric acid cycle enzyme NAD(+)-isocitrate dehydrogenase was identified by sequence analysis. Its predicted amino acid sequence is more similar to the IDH1 regulatory subunit of S. cerevisiae NAD(+)-isocitrate dehydrogenase than to the IDH2 catalytic subunit. After expression in S. cerevisiae from an S. cerevisiae promoter, it was shown to functionally complement an S. cerevisiae idh1 mutant, but not an idh2 mutant, for growth on acetate as a carbon source and for production of NAD(+)-isocitrate dehydrogenase enzyme activity. These results confirm that the H. capsulatum cDNA encodes a homologue of subunit I of the S. cerevisiae mitochondrial isocitrate dehydrogenase isozyme that functions in the citric acid cycle.

  14. A Nampt inhibitor FK866 mimics vitamin B3 deficiency by causing senescence of human fibroblastic Hs68 cells via attenuation of NAD(+)-SIRT1 signaling.

    PubMed

    Song, Tuzz-Ying; Yeh, Shu-Lan; Hu, Miao-Lin; Chen, Mei-Yau; Yang, Nae-Cherng

    2015-12-01

    Vitamin B3 (niacin) deficiency can cause pellagra with symptoms of dermatitis, diarrhea and dementia. However, it is unclear whether the vitamin B3 deficiency causes human aging. FK866 (a Nampt inhibitor) can reduce intracellular NAD(+) level and induce senescence of human Hs68 cells. However, the mechanisms underlying FK866-induced senescence of Hs68 cells are unclear. In this study, we used FK866 to mimic the effects of vitamin B3 deficiency to reduce the NAD(+) level and investigated the mechanisms of FK866-induced senescence of Hs68 cells. We hypothesized that FK866 induced the senescence of Hs68 cells via an attenuation of NAD(+)-silent information regulator T1 (SIRT1) signaling. We found that FK866 induced cell senescence and diminished cellular NAD(+) levels and SIRT1 activity (detected by acetylation of p53), and these effects were dramatically antagonized by co-treatment with nicotinic acid, nicotinamide, or NAD(+). In contrast, the protein expression of SIRT1, AMP-activated protein kinase, mammalian target of rapamycin, and nicotinamide phosphoribosyltransferase (Nampt) was not affected by FK866. In addition, the role of GSH in the FK866-induced cells senescence may be limited, as N-acetylcysteine did not antagonize FK866-induced cell senescence. These results suggest that FK866 induces cell senescence via attenuation of NAD(+)-SIRT1 signaling. The effects of vitamin B3 deficiency on human aging warrant further investigation.

  15. Simulations of the Vertical Redistribution of HNO3 by NAT or NAD PSCs: The Sensitivity to the Number of Cloud Particles Formed and the Cloud Lifetime

    NASA Technical Reports Server (NTRS)

    Jensen, Eric J.; Tabazadeh, Azadeh; Drdla, Katja; Toon, Owen B.; Gore, Warren J. (Technical Monitor)

    2000-01-01

    Recent satellite and in situ measurements have indicated that limited denitrification can occur in the Arctic stratosphere. In situ measurements from the SOLVE campaign indicate polar stratospheric clouds (PSCs) composed of small numbers (about 3 x 10^ -4 cm^-3) of 10-20 micron particles (probably NAT or NAD). These observations raise the issue of whether low number density NAT PSCs can substantially denitrify the air with reasonable cloud lifetimes. In this study, we use a one dimensional cloud model to investigate the verticle redistribution of HNO3 by NAT/NAD PSCs. The cloud formation is driven by a temperature oscillation which drops the temperature below the NAT/NAD formation threshold (about 195 K) for a few days. We assume that a small fraction of the available aerosols act as NAT nuclei when the saturation ratio of HNO3 over NAT(NAD) exceeds 10(l.5). The result is a cloud between about 16 and 20 km in the model, with NAT/NAD particle effective radii as large as about 10 microns (in agreement with the SOLVE data). We find that for typical cloud lifetimes of 2-3 days or less, the net depletion of HNO3 is no more than 1-2 ppbv, regardless of the NAT or NAD particle number density. Repeated passes of the air column through the cold pool build up the denitrification to 3-4 ppbv, and the cloud altitude steadily decreases due to the downward transport of nitric acid. Increasing the cloud lifetime results in considerably more effective denitrification, even with very low cloud particle number densities. As expected, the degree of denitrification by NAT clouds is much larger than that by NAD Clouds. Significant denitrification by NAD Clouds is only possible if the cloud lifetime is several days or more. The clouds also cause a local maximum HNO3 mixing ratio at cloud base where the cloud particles sublimate.

  16. Faster Rubisco Is the Key to Superior Nitrogen-Use Efficiency in NADP-Malic Enzyme Relative to NAD-Malic Enzyme C4 Grasses1

    PubMed Central

    Ghannoum, Oula; Evans, John R.; Chow, Wah Soon; Andrews, T. John; Conroy, Jann P.; von Caemmerer, Susanne

    2005-01-01

    In 27 C4 grasses grown under adequate or deficient nitrogen (N) supplies, N-use efficiency at the photosynthetic (assimilation rate per unit leaf N) and whole-plant (dry mass per total leaf N) level was greater in NADP-malic enzyme (ME) than NAD-ME species. This was due to lower N content in NADP-ME than NAD-ME leaves because neither assimilation rates nor plant dry mass differed significantly between the two C4 subtypes. Relative to NAD-ME, NADP-ME leaves had greater in vivo (assimilation rate per Rubisco catalytic sites) and in vitro Rubisco turnover rates (kcat; 3.8 versus 5.7 s−1 at 25°C). The two parameters were linearly related. In 2 NAD-ME (Panicum miliaceum and Panicum coloratum) and 2 NADP-ME (Sorghum bicolor and Cenchrus ciliaris) grasses, 30% of leaf N was allocated to thylakoids and 5% to 9% to amino acids and nitrate. Soluble protein represented a smaller fraction of leaf N in NADP-ME (41%) than in NAD-ME (53%) leaves, of which Rubisco accounted for one-seventh. Soluble protein averaged 7 and 10 g (mmol chlorophyll)−1 in NADP-ME and NAD-ME leaves, respectively. The majority (65%) of leaf N and chlorophyll was found in the mesophyll of NADP-ME and bundle sheath of NAD-ME leaves. The mesophyll-bundle sheath distribution of functional thylakoid complexes (photosystems I and II and cytochrome f) varied among species, with a tendency to be mostly located in the mesophyll. In conclusion, superior N-use efficiency of NADP-ME relative to NAD-ME grasses was achieved with less leaf N, soluble protein, and Rubisco having a faster kcat. PMID:15665246

  17. Membrane-Associated NAD-Dependent Isocitrate Dehydrogenase in Potato Mitochondria 1

    PubMed Central

    Laties, George G.

    1983-01-01

    The oxidation isotherms for citrate and isocitrate by potato (Solanum tuberosum var. Russet Burbank) mitochondria in the presence of NAD differ markedly. Citrate oxidation shows positively cooperative kinetics with a sigmoid isotherm, whereas isocitrate oxidation shows Michaelis-Menten kinetics at concentrations up to 3 millimolar, and cooperative kinetics thereafter up to 30 millimolar. In the absence of exogenous NAD, the isocitrate isotherm is sigmoid throughout. The dual isotherm for isocitrate oxidation in the presence of exogenous NAD reflects the operation of two forms of isocitrate dehydrogenase, one in the matrix and one associated with the inner mitochondrial membrane. Whereas in intact mitochondria the activity of the membrane-bound enzyme is insensitive to rotenone, and to butylmalonate, an inhibitor of organic acid transport, isocitrate oxidation by the soluble matrix enzyme is inhibited by both. The membrane-bound isocitrate dehydrogenase does not operate through the NADH dehydrogenase on the outer face of the inner mitochondrial membrane, and is thus considered to face inward. The regulatory potential of isocitrate dehydrogenase in potato mitochondria may be realized by the apportionment of the enzyme between its soluble and bound forms. PMID:16663145

  18. Heterogeneous kinetics of H2O, HNO3 and HCl on HNO3 hydrates (α-NAT, β-NAT, NAD) in the range 175-200 K

    NASA Astrophysics Data System (ADS)

    Iannarelli, Riccardo; Rossi, Michel J.

    2016-09-01

    Experiments on the title compounds have been performed using a multidiagnostic stirred-flow reactor (SFR) in which the gas phase as well as the condensed phase has been simultaneously investigated under stratospheric temperatures in the range 175-200 K. Wall interactions of the title compounds have been taken into account using Langmuir adsorption isotherms in order to close the mass balance between deposited and desorbed (recovered) compounds. Thin solid films at 1 µm typical thickness have been used as a proxy for atmospheric ice particles and have been deposited on a Si window of the cryostat, with the optical element being the only cold point in the deposition chamber. Fourier transform infrared (FTIR) absorption spectroscopy in transmission as well as partial and total pressure measurement using residual gas mass spectrometry (MS) and sensitive pressure gauges have been employed in order to monitor growth and evaporation processes as a function of temperature using both pulsed and continuous gas admission and monitoring under SFR conditions. Thin solid H2O ice films were used as the starting point throughout, with the initial spontaneous formation of α-NAT (nitric acid trihydrate) followed by the gradual transformation of α- to β-NAT at T > 185 K. Nitric acid dihydrate (NAD) was spontaneously formed at somewhat larger partial pressures of HNO3 deposited on pure H2O ice. In contrast to published reports, the formation of α-NAT proceeded without prior formation of an amorphous HNO3 / H2O layer and always resulted in β-NAT. For α- and β-NAT, the temperature-dependent accommodation coefficient α(H2O) and α(HNO3), the evaporation flux Jev(H2O) and Jev(HNO3) and the resulting saturation vapor pressure Peq(H2O) and Peq(HNO3) were measured and compared to binary phase diagrams of HNO3 / H2O in order to afford a thermochemical check of the kinetic parameters. The resulting kinetic and thermodynamic parameters of activation energies for evaporation (Eev) and

  19. Calcium pyrophosphate dihydrate deposition in the intervertebral discs in a case of Wilson's disease.

    PubMed

    McClure, J; Smith, P S

    1983-07-01

    The vertebral column from a known case of Wilson's disease (hepatolenticular degeneration) was examined by radiological, histological, histochemical and x-ray microanalytical techniques which demonstrated the presence of focal depositions of calcium pyrophosphate dihydrate (CPPD) in the intervertebral discs. These deposits were present in both the annulus fibrosus and the nucleus pulposus but in certain discs the deposits were concentrated near the interface between disc and vertebral body bone endplates. At these sites there was new bone formation with narrowing of the discs, irregularity and sclerosis of the bone endplates and exostosis.

  20. Dosimetric and kinetic investigations of γ-irradiated sodium tartrate dihydrate.

    PubMed

    Tuner, H; Kayikçi, M A

    2012-03-01

    Effects of gamma radiation on solid sodium tartrate dihydrate (NaTA) were studied using electron spin resonance (ESR) spectroscopy. One main singlet located at g = 2.0034 and many weak lines located at low and high magnetic field sides were found in the irradiated samples. Dosimetric and kinetic features of the radical species responsible for the experimental ESR spectra were explored through the variations in the signal intensities with respect to applied microwave power, temperature and storage time. Activation energies of the involved radical species were also determined using data derived from annealing studies.

  1. Synovial chondromatosis of the temporomandibular joint with calcium pyrophosphate dihydrate crystal deposition disease (pseudogout)

    PubMed Central

    Matsumura, Y; Nomura, J; Nakanishi, K; Yanase, S; Kato, H; Tagawa, T

    2012-01-01

    This report describes a very rare case of synovial chondromatosis with deposition of calcium pyrophosphate dihydrate (CPPD) crystals (pseudogout) in the temporomandibular joint (TMJ) of a 46-year-old male patient. Synovial chondromatosis is a non-neoplastic disease characterized by metaplasia of the connective tissue leading to chondrogenesis in the synovial membrane. Pseudogout is an inflammatory disease of the joints caused by the deposition of CPPD, producing similar symptoms to those observed in gout but not hyperuricaemia. Both diseases commonly affect the knee, hip and elbow joints, but rarely affect the TMJ. PMID:23166363

  2. Tumoral calcium pyrophosphate dihydrate crystal deposition disease of the temporomandibular joint: identification on crystallography.

    PubMed

    Mikami, Toshinari; Takeda, Yasunori; Ohira, Akinori; Hoshi, Hideki; Sugiyama, Yoshiki; Yoshida, Yasuo; Ambo, Junichi

    2008-11-01

    This paper reports a case of calcium pyrophosphate dihydrate (CPPD) crystal deposition in the temporomandibular joint (TMJ) of a 59-year-old man with the chief complaint of severe pain in the left TMJ. On CT a radiopaque area was seen around the condylar process of the left TMJ with irregular destructive bony changes. A provisional diagnosis of crystalline-induced arthritis was made on histopathology of a biopsy specimen. Electron probe microanalysis (EPMA), scanning electron microscopy (SEM) and X-ray diffraction showed both CPPD and hydroxyapatite (HA) in the crystalline materials. Identification of these two types of crystal in crystal deposition disease of TMJ, using crystallography, is discussed.

  3. NAD+/NADH and skeletal muscle mitochondrial adaptations to exercise

    PubMed Central

    White, Amanda T.

    2012-01-01

    The pyridine nucleotides, NAD+ and NADH, are coenzymes that provide oxidoreductive power for the generation of ATP by mitochondria. In skeletal muscle, exercise perturbs the levels of NAD+, NADH, and consequently, the NAD+/NADH ratio, and initial research in this area focused on the contribution of redox control to ATP production. More recently, numerous signaling pathways that are sensitive to perturbations in NAD+(H) have come to the fore, as has an appreciation for the potential importance of compartmentation of NAD+(H) metabolism and its subsequent effects on various signaling pathways. These pathways, which include the sirtuin (SIRT) proteins SIRT1 and SIRT3, the poly(ADP-ribose) polymerase (PARP) proteins PARP1 and PARP2, and COOH-terminal binding protein (CtBP), are of particular interest because they potentially link changes in cellular redox state to both immediate, metabolic-related changes and transcriptional adaptations to exercise. In this review, we discuss what is known, and not known, about the contribution of NAD+(H) metabolism and these aforementioned proteins to mitochondrial adaptations to acute and chronic endurance exercise. PMID:22436696

  4. NMNAT expression and its relation to NAD metabolism.

    PubMed

    Jayaram, H N; Kusumanchi, P; Yalowitz, J A

    2011-01-01

    Nicotinamide mononucleotide adenylyltransferease (NMNAT), a rate-limiting enzyme present in all organisms, reversibly catalyzes the important step in the biosynthesis of NAD from ATP and NMN. NAD and NADP are used reversibly in anabolic and catabolic reactions. NAD is necessary for cell survival in oxidative stress and DNA damage. Based on their localization, three different NMNAT's have been recognized, NMNAT-1 (homohexamer) in the nucleus (chromosome 1 p32-35), NMNAT-2 (homodimer) in the cytoplasm (chromosome 1q25) and NMNAT-3 (homotetramer) in the mitochondria. NMNAT also catalyzes the metabolic conversion of potent antitumor prodrugs like tiazofurin and benzamide riboside to their active forms which are analogs of NAD. NAD synthase-NMNAT acts as a chaperone to protect against neurodegeneration, injury-induced axonal degeneration and also correlates with DNA synthesis during cell cycle. Since its activity is rather low in tumor cells it can be exploited as a source for therapeutic targeting. Steps involved in NAD synthesis are being utilized as targets for chemoprevention, radiosensitization and therapy of wide range of diseases, such as cancer, multiple sclerosis, neurodegeneration and Huntington's disease. PMID:21517776

  5. New Therapeutic Concept of NAD Redox Balance for Cisplatin Nephrotoxicity

    PubMed Central

    Oh, Gi-Su; Kim, Hyung-Jin; Shen, AiHua; Lee, Su-Bin; Yang, Sei-Hoon; Shim, Hyeok; Cho, Eun-Young; Kwon, Kang-Beom; Kwak, Tae Hwan; So, Hong-Seob

    2016-01-01

    Cisplatin is a widely used chemotherapeutic agent for the treatment of various tumors. In addition to its antitumor activity, cisplatin affects normal cells and may induce adverse effects such as ototoxicity, nephrotoxicity, and peripheral neuropathy. Various mechanisms such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammatory responses are closely associated with cisplatin-induced nephrotoxicity; however, the precise mechanism remains unclear. The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of cellular energy metabolism and homeostasis. Recent studies have demonstrated associations between disturbance in intracellular NAD+ levels and clinical progression of various diseases through the production of reactive oxygen species and inflammation. Furthermore, we demonstrated that reduction of the intracellular NAD+/NADH ratio is critically involved in cisplatin-induced kidney damage through inflammation and oxidative stress and that increase of the cellular NAD+/NADH ratio suppresses cisplatin-induced kidney damage by modulation of potential damage mediators such as oxidative stress and inflammatory responses. In this review, we describe the role of NAD+ metabolism in cisplatin-induced nephrotoxicity and discuss a potential strategy for the prevention or treatment of cisplatin-induced adverse effects with a particular focus on NAD+-dependent cellular pathways. PMID:26881219

  6. Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N(6)-monooxygenase.

    PubMed

    Abdelwahab, Heba; Robinson, Reeder; Rodriguez, Pedro; Adly, Camelia; El-Sohaimy, Sohby; Sobrado, Pablo

    2016-09-15

    l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

  7. Ethanol Enhances Hepatitis C Virus Replication through Lipid Metabolism and Elevated NADH/NAD+*

    PubMed Central

    Seronello, Scott; Ito, Chieri; Wakita, Takaji; Choi, Jinah

    2010-01-01

    Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD+ ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD+. Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting β-oxidation with β-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. Conclusion: Therefore, lipid metabolism and alteration of cellular NADH/NAD+ ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress. PMID:19910460

  8. Metabolic control of cell division in α-proteobacteria by a NAD-dependent glutamate dehydrogenase.

    PubMed

    Beaufay, François; De Bolle, Xavier; Hallez, Régis

    2016-01-01

    Prior to initiate energy-consuming processes, such as DNA replication or cell division, cells need to evaluate their metabolic status. We have recently identified and characterized a new connection between metabolism and cell division in the α-proteobacterium Caulobacter crescentus. We showed that an NAD-dependent glutamate dehydrogenase (GdhZ) coordinates growth with cell division according to its enzymatic activity. Here we report the conserved role of GdhZ in controlling cell division in another α-proteobacterium, the facultative intracellular pathogen Brucella abortus. We also discuss the importance of amino acids as a main carbon source for α-proteobacteria.

  9. Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle.

    PubMed

    Frederick, David W; Loro, Emanuele; Liu, Ling; Davila, Antonio; Chellappa, Karthikeyani; Silverman, Ian M; Quinn, William J; Gosai, Sager J; Tichy, Elisia D; Davis, James G; Mourkioti, Foteini; Gregory, Brian D; Dellinger, Ryan W; Redpath, Philip; Migaud, Marie E; Nakamaru-Ogiso, Eiko; Rabinowitz, Joshua D; Khurana, Tejvir S; Baur, Joseph A

    2016-08-01

    NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function. PMID:27508874

  10. Unusual effect of water vapor pressure on dehydration of dibasic calcium phosphate dihydrate.

    PubMed

    Kaushal, Aditya M; Vangala, Venu R; Suryanarayanan, Raj

    2011-04-01

    Dibasic calcium phosphate occurs as an anhydrate (DCPA; CaHPO₄) and as a dihydrate (DCPD; CaHPO₄•2H₂O). Our objective was to investigate the unusual behavior of these phases. Dibasic calcium phosphate dihydrate was dehydrated in a (i) differential scanning calorimeter (DSC) in different pan configurations; (ii) variable-temperature X-ray diffractometer (XRD) at atmospheric and under reduced pressure, and in sealed capillaries; and (iii) water vapor sorption analyzer at varying temperature and humidity conditions. Dehydration was complete by 210°C in an open DSC pan and under atmospheric pressure in the XRD. Unlike "conventional" hydrates, the dehydration of DCPD was facilitated in the presence of water vapor. Variable-temperature XRD in a sealed capillary and DSC in a hermetic pan with pinhole caused complete dehydration by 100°C and 140°C, respectively. Under reduced pressure, conversion to the anhydrate was incomplete even at 300°C. The increase in dehydration rate with increase in water vapor pressure has been explained by the Smith-Topley effect. Under "dry" conditions, a coating of poorly crystalline product is believed to form on the surface of particles and act as a barrier to further dehydration. However, in the presence of water vapor, recrystallization occurs, creating cracks and channels and facilitating continued dehydration.

  11. A very rare benign tumour in the parotid region: calcium pyrophosphate dihydrate crystal deposition disease.

    PubMed

    Olin, H B; Pedersen, K; Francis, D; Hansen, H; Poulsen, F W

    2001-06-01

    Calcium pyrophosphate dihydrate crystal deposition disease, exhibits several clinical manifestations, from absence of symptoms to severely destructive arthropathy or conditions simulating neoplasm, which is frequently related to the temporomandibular joint. Fifteen of the 31 reported cases of tophaceous pseudogout were found in the head and neck region. A patient presented with a parotid swelling, which initially was suspected to be malignant because of the following findings: radiodensity, progression into the joint, osseous destruction of the major ala of the sphenoid and a fine needle aspirate with crystals, osteoblasts, megakaryocytes and irregular cells of varying size. At surgery there was found a tumour consisting of a white, firm gritty material. It progressed to the skull base where material had to be left, because of the presence of the nerves and vessels. A frozen specimen was reported to be benign. Histological examination showed inflammatory cells, macrophages, a chondroid material with embedded metaplastic chondroid cells and giant cells of foreign body type. Crystal examination of X-ray diffraction revealed calcium pyrophosphate dihydrate.

  12. High-resolution structures of Thermus thermophilus enoyl-acyl carrier protein reductase in the apo form, in complex with NAD+ and in complex with NAD+ and triclosan.

    PubMed

    Otero, José M; Noël, Ann Josée; Guardado-Calvo, Pablo; Llamas-Saiz, Antonio L; Wende, Wolfgang; Schierling, Benno; Pingoud, Alfred; van Raaij, Mark J

    2012-10-01

    Enoyl-acyl carrier protein reductase (ENR; the product of the fabI gene) is an important enzyme that is involved in the type II fatty-acid-synthesis pathway of bacteria, plants, apicomplexan protozoa and mitochondria. Harmful pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum use the type II fatty-acid-synthesis system, but not mammals or fungi, which contain a type I fatty-acid-synthesis pathway consisting of one or two multifunctional enzymes. For this reason, specific inhibitors of ENR are attractive antibiotic candidates. Triclosan, a broad-range antibacterial agent, binds to ENR, inhibiting fatty-acid synthesis. As humans do not have an ENR enzyme, they are not affected. Here, high-resolution structures of Thermus thermophilus (Tth) ENR in the apo form, bound to NAD(+) and bound to NAD(+) plus triclosan are reported. Differences from and similarities to other known ENR structures are reported; in general, the structures are very similar. The cofactor-binding site is also very similar to those of other ENRs and, as reported for other species, triclosan leads to greater ordering of the loop that covers the cofactor-binding site, which, together with the presence of triclosan itself, presumably provides tight binding of the dinucleotide, preventing cycling of the cofactor. Differences between the structures of Tth ENR and other ENRs are the presence of an additional β-sheet at the N-terminus and a larger number of salt bridges and side-chain hydrogen bonds. These features may be related to the high thermal stability of Tth ENR.

  13. High-resolution structures of Thermus thermophilus enoyl-acyl carrier protein reductase in the apo form, in complex with NAD+ and in complex with NAD+ and triclosan

    PubMed Central

    Otero, José M.; Noël, Ann-Josée; Guardado-Calvo, Pablo; Llamas-Saiz, Antonio L.; Wende, Wolfgang; Schierling, Benno; Pingoud, Alfred; van Raaij, Mark J.

    2012-01-01

    Enoyl-acyl carrier protein reductase (ENR; the product of the fabI gene) is an important enzyme that is involved in the type II fatty-acid-synthesis pathway of bacteria, plants, apicomplexan protozoa and mitochondria. Harmful pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum use the type II fatty-acid-synthesis system, but not mammals or fungi, which contain a type I fatty-acid-synthesis pathway consisting of one or two multifunctional enzymes. For this reason, specific inhibitors of ENR are attractive antibiotic candidates. Triclosan, a broad-range antibacterial agent, binds to ENR, inhibiting fatty-acid synthesis. As humans do not have an ENR enzyme, they are not affected. Here, high-resolution structures of Thermus thermophilus (Tth) ENR in the apo form, bound to NAD+ and bound to NAD+ plus triclosan are reported. Differences from and similarities to other known ENR structures are reported; in general, the structures are very similar. The cofactor-binding site is also very similar to those of other ENRs and, as reported for other species, triclosan leads to greater ordering of the loop that covers the cofactor-binding site, which, together with the presence of triclosan itself, presumably provides tight binding of the dinucleotide, preventing cycling of the cofactor. Differences between the structures of Tth ENR and other ENRs are the presence of an additional β-sheet at the N-terminus and a larger number of salt bridges and side-chain hydrogen bonds. These features may be related to the high thermal stability of Tth ENR. PMID:23027736

  14. Structure and function of NAD kinase and NADP phosphatase: key enzymes that regulate the intracellular balance of NAD(H) and NADP(H).

    PubMed

    Kawai, Shigeyuki; Murata, Kousaku

    2008-04-01

    The functions of NAD(H) (NAD(+) and NADH) and NADP(H) (NADP(+) and NADPH) are undoubtedly significant and distinct. Hence, regulation of the intracellular balance of NAD(H) and NADP(H) is important. The key enzymes involved in the regulation are NAD kinase and NADP phosphatase. In 2000, we first succeeded in identifying the gene for NAD kinase, thereby facilitating worldwide studies of this enzyme from various organisms, including eubacteria, archaea, yeast, plants, and humans. Molecular biological study has revealed the physiological function of this enzyme, that is to say, the significance of NADP(H), in some model organisms. Structural research has elucidated the tertiary structure of the enzyme, the details of substrate-binding sites, and the catalytic mechanism. Research on NAD kinase also led to the discovery of archaeal NADP phosphatase. In this review, we summarize the physiological functions, applications, and structure of NAD kinase, and the way we discovered archaeal NADP phosphatase.

  15. Identification and characterization of a human mitochondrial NAD kinase.

    PubMed

    Ohashi, Kazuto; Kawai, Shigeyuki; Murata, Kousaku

    2012-01-01

    NAD kinase is the sole NADP(+) biosynthetic enzyme. Despite the great significance of NADP(+), to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP(+) remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP(+). The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists carry C5orf33 homologues as their sole NADP(+) biosynthetic enzyme, whereas plants and fungi possess no C5orf33 homologue. These observations provide insights into eukaryotic NADP(+) biosynthesis, which has pivotal roles in cells and organelles.

  16. Suppression of NDA-type alternative mitochondrial NAD(P)H dehydrogenases in arabidopsis thaliana modifies growth and metabolism, but not high light stimulation of mitochondrial electron transport.

    PubMed

    Wallström, Sabá V; Florez-Sarasa, Igor; Araújo, Wagner L; Escobar, Matthew A; Geisler, Daniela A; Aidemark, Mari; Lager, Ida; Fernie, Alisdair R; Ribas-Carbó, Miquel; Rasmusson, Allan G

    2014-05-01

    The plant respiratory chain contains several pathways which bypass the energy-conserving electron transport complexes I, III and IV. These energy bypasses, including type II NAD(P)H dehydrogenases and the alternative oxidase (AOX), may have a role in redox stabilization and regulation, but current evidence is inconclusive. Using RNA interference, we generated Arabidopsis thaliana plants simultaneously suppressing the type II NAD(P)H dehydrogenase genes NDA1 and NDA2. Leaf mitochondria contained substantially reduced levels of both proteins. In sterile culture in the light, the transgenic lines displayed a slow growth phenotype, which was more severe when the complex I inhibitor rotenone was present. Slower growth was also observed in soil. In rosette leaves, a higher NAD(P)H/NAD(P)⁺ ratio and elevated levels of lactate relative to sugars and citric acid cycle metabolites were observed. However, photosynthetic performance was unaffected and microarray analyses indicated few transcriptional changes. A high light treatment increased AOX1a mRNA levels, in vivo AOX and cytochrome oxidase activities, and levels of citric acid cycle intermediates and hexoses in all genotypes. However, NDA-suppressing plants deviated from the wild type merely by having higher levels of several amino acids. These results suggest that NDA suppression restricts citric acid cycle reactions, inducing a shift towards increased levels of fermentation products, but do not support a direct association between photosynthesis and NDA proteins.

  17. Disulfide Bond Formation in Yeast NAD+-Specific Isocitrate Dehydrogenase†

    PubMed Central

    Garcia, Joshua A.; Minard, Karyl I.; Lin, An-Ping; McAlister-Henn, Lee

    2009-01-01

    The tricarboxylic acid cycle NAD+-specific isocitrate dehydrogenase (IDH) of Saccharomyces cerevisiae is an octameric enzyme composed of four heterodimers of regulatory IDH1 and catalytic IDH2 subunits. Recent structural analyses revealed the close proximity of Cys-150 residues from IDH2 in adjacent heterodimers, and features of the structure for the ligand-free enzyme suggested that formation of a disulfide bond between these residues might stabilize an inactive form of the enzyme. We constructed two mutant forms of IDH, one containing a C150S substitution in IDH2 and the other containing C56S/C242S substitutions in IDH2 leaving Cys-150 as the sole cysteine residue. Treatment of the affinity-purified enzymes with diamide resulted in the formation of disulfide bonds and in decreased activities for the wild-type and C56S/C242S enzymes. Both effects were reversible by the addition of dithiothreitol. Diamide had no effect on the C150S mutant enzyme, suggesting that Cys-150 is essential for the formation of a disulfide bond that inhibits IDH activity. Diamide-induced formation of the Cys-150 disulfide bond was also observed in vivo for yeast transformants expressing the wild-type or C56S/C242S enzymes but not for a transformant expressing the C150S enzyme. Finally, natural formation of the Cys-150 disulfide bond with a concomitant decrease in cellular IDH activity was observed during the stationary phase for the parental strain and for transformants expressing wild-type or C56S/C242S enzymes but not for a transformant expressing the C150S enzyme. A reduction in viability for the latter strain suggests that a decrease in IDH activity is important for metabolic changes in stationary phase cells. PMID:19645416

  18. Time-resolved fluorescence spectroscopy investigation of the effect of 4-hydroxynonenal on endogenous NAD(P)H in living cardiac myocytes

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Aneba, Swida; Mateasik, Anton; Chorvat, Dusan; Comte, Blandine

    2013-06-01

    Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P)H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states.

  19. The Tuberculosis Necrotizing Toxin kills macrophages by hydrolyzing NAD

    PubMed Central

    Sun, Jim; Siroy, Axel; Lokareddy, Ravi K.; Speer, Alexander; Doornbos, Kathryn S.; Cingolani, Gino; Niederweis, Michael

    2015-01-01

    Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb utilizes the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT) that induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential co-enzyme nicotinamide adenine dinucleotide (NAD+). Expression or injection of a non-catalytic TNT mutant showed no cytotoxicity in macrophages or zebrafish zygotes, respectively, demonstrating that the NAD+-glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT-IFT complex revealed a novel NAD+-glycohydrolase fold of TNT, which constitutes the founding member of a toxin family wide-spread in pathogenic microorganisms. PMID:26237511

  20. NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy.

    PubMed

    Goody, Michelle F; Kelly, Meghan W; Reynolds, Christine J; Khalil, Andre; Crawford, Bryan D; Henry, Clarissa A

    2012-01-01

    Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex- or integrin alpha7-deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha

  1. The high-resolution crystal structure of periplasmic Haemophilus influenzae NAD nucleotidase reveals a novel enzymatic function of human CD73 related to NAD metabolism.

    PubMed

    Garavaglia, Silvia; Bruzzone, Santina; Cassani, Camilla; Canella, Laura; Allegrone, Gianna; Sturla, Laura; Mannino, Elena; Millo, Enrico; De Flora, Antonio; Rizzi, Menico

    2012-01-01

    Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its nicotinamide dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN (NAD nucleotidase), which plays a central role by degrading NAD into adenosine and NR (nicotinamide riboside), the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN which determined that the enzyme is a Zn2+-dependent 5'-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3 Å resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate specificity, recognizing either mono- or di-nucleotide nicotinamides and different adenosine phosphates with a maximal activity on 5'-adenosine monophosphate. Sequence and structural analysis of H. influenzae NadN led us to discover that human CD73 is capable of processing both NAD and NMN, therefore disclosing a possible novel function of human CD73 in systemic NAD metabolism. Our data may prove to be useful for inhibitor design and disclosed unanticipated fascinating evolutionary relationships. PMID:21933152

  2. NAD+ metabolism and the control of energy homeostasis - a balancing act between mitochondria and the nucleus

    PubMed Central

    Cantó, Carles; Menzies, Keir; Auwerx, Johan

    2015-01-01

    NAD+ has emerged as a vital cofactor that can rewire metabolism, activate sirtuins and maintain mitochondrial fitness through mechanisms such as the mitochondrial unfolded protein response. This improved understanding of NAD+ metabolism revived interest in NAD+ boosting strategies to manage a wide spectrum of diseases, ranging from diabetes to cancer. In this review, we summarize how NAD+ metabolism links energy status with adaptive cellular and organismal responses and how this knowledge can be therapeutically exploited. PMID:26118927

  3. NAD(+) Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus.

    PubMed

    Cantó, Carles; Menzies, Keir J; Auwerx, Johan

    2015-07-01

    NAD(+) has emerged as a vital cofactor that can rewire metabolism, activate sirtuins, and maintain mitochondrial fitness through mechanisms such as the mitochondrial unfolded protein response. This improved understanding of NAD(+) metabolism revived interest in NAD(+)-boosting strategies to manage a wide spectrum of diseases, ranging from diabetes to cancer. In this review, we summarize how NAD(+) metabolism links energy status with adaptive cellular and organismal responses and how this knowledge can be therapeutically exploited.

  4. Growth, photoluminescence, thermal and mechanical behaviour of Ethyltriphenylphosphonium bromide dihydrate crystal

    NASA Astrophysics Data System (ADS)

    Parthasarathy, M.; Gopalakrishnan, R.

    2013-10-01

    Single crystal of Ethyltriphenylphosphonium bromide dihydrate (ETPB) was grown by slow evaporation solution growth technique. The grown crystal was confirmed by single crystal X-ray diffraction. The functional groups and vibrational frequencies were identified using FT-IR and FT-Raman spectral analyses. Optical properties were studied by UV-Visible and photoluminescence spectroscopic techniques to explore its efficacy towards device fabrication. Thermal characteristics of ETPB were studied using the TGA/DTA and DSC response curves. The mechanical behaviour of the grown crystal was studied using Vicker's microhardness tester and the work hardening coefficient was evaluated. The second harmonic generation of the title compound was tested by Kurtz-Perry powder technique.

  5. Crystallization of calcium sulfate dihydrate in the presence of some metal ions

    NASA Astrophysics Data System (ADS)

    Hamdona, Samia K.; Al Hadad, Umaima A.

    2007-02-01

    Crystallization of calcium sulfate dihydrate (CaSO 4·2H 2O gypsum) in sodium chloride solutions in the presence of some metal ions, and over a range of relative super-saturation has been studied. The addition of metal ions, even at relatively low concentration (10 -6 mol l -1), markedly retard the rate of crystallization of gypsum. Retardation effect was enhanced with increase in the additives contents. Moreover, the effect was enhanced as the relative super-saturation decreases. Influence of mixed additives on the rate of crystallization (Cd 2++Arg, Cd 2++H 3PO 4 and Cd 2++PAA) has also been studied. Direct adsorption experiments of these metal ions on the surface of gypsum crystals have been made for comparison.

  6. Dual roles of borax in kinetics of calcium sulfate dihydrate formation.

    PubMed

    Jiang, Wenge; Pan, Haihua; Tao, Jinhui; Xu, Xurong; Tang, Ruikang

    2007-04-24

    An additive is not exclusively retardant or promoter for a crystallization system. The kinetic studies of calcium sulfate dihydrate (CSD) crystal growth demonstrated that borax played dual roles in the reaction, which accelerated CSD formations at the low concentration levels but inhibited the crystal growth at the high ones. In situ atomic force microscopy studies revealed that borax modulated the CSD crystallization via two different pathways: promoted the secondary nucleation to increase the step density on the growing crystal faces but simultaneously retarded the spread of these growth steps by the Langmuir adsorption. These two contradictory factors were incorporated in the crystallization, and their balance was regulated by the borax concentration. Both the macroscopic and microscopic experimental data nicely displayed the crystallization model of birth and spread that was able to account for the behaviors of borax in CSD formations.

  7. Gallium Arsenate Dihydrate under Pressure: Elastic Properties, Compression Mechanism, and Hydrogen Bonding.

    PubMed

    Spencer, Elinor C; Soghomonian, Victoria; Ross, Nancy L

    2015-08-01

    Gallium arsenate dihydrate is a member of a class of isostructural compounds, with the general formula M(3+)AsO4·2H2O (M(3+) = Fe, Al, In, or Ga), which are being considered as potential solid-state storage media for the sequestration of toxic arsenic cations. We report the first high-pressure structural analysis of a metal arsenate dihydrate, namely, GaAsO4·2H2O. This compound crystallizes in the orthorhombic space group Pbca with Z = 8. Accurate unit cell parameters as a function of pressure were obtained by high-pressure single-crystal X-ray diffraction, and a bulk modulus of 51.1(3) GPa for GaAsO4·2H2O was determined from a third-order Birch-Murnaghan equation of state fit to the P-V data. Assessment of the pressure dependencies of the unit cell lengths showed that the compressibility of the structure along the axial directions increases in the order of [010] < [100] < [001]. This order was found to correlate well with the proposed compression mechanism for GaAsO4·2H2O, which involves deformation of the internal channel void spaces of the polyhedral helices that lie parallel to the [010] direction, and increased distortion of the GaO6 octahedra. The findings of the high-pressure diffraction experiment were further supported by the results from variable-pressure Raman analysis of GaAsO4·2H2O. Moreover, we propose a revised and more complex model for the hydrogen-bonding scheme in GaAsO4·2H2O, and on the basis of this revision, we reassigned the peaks in the OH stretching regions of previously published Raman spectra of this compound.

  8. Expression of the meningococcal adhesin NadA is controlled by a transcriptional regulator of the MarR family.

    PubMed

    Schielke, Stephanie; Huebner, Claudia; Spatz, Carolin; Nägele, Virginie; Ackermann, Nikolaus; Frosch, Matthias; Kurzai, Oliver; Schubert-Unkmeir, Alexandra

    2009-05-01

    Two closely related pathogenic species have evolved in the genus Neisseria: N. meningitidis and N. gonorrhoeae, which occupy different host niches and cause different clinical entities. In contrast to the pathogen N. gonorrhoeae, N. meningitidis is a commensal and only rarely becomes invasive. Little is known about the genetic background of the entirely different lifestyles in these closely related species. Meningococcal NMB1843 encodes a transcriptional regulator of the MarR family. The gonococcal homologue FarR regulates expression of farAB, mediating fatty acid resistance. We show that NmFarR also directly interacts with NmfarAB. Yet, by contrast to N. gonorrhoeae, no significant sensitivity to fatty acids was observed in a DeltafarR mutant due to intrinsic resistance of meningococci. Further analyses identified an NmFarR-repressed protein absent from N. gonorrhoeae. This protein is the meningococcus-specific adhesin and vaccine component NadA that has most likely been acquired by horizontal gene transfer. NmFarR binds to a 16 base pair palindromic repeat within the nadA promoter. De-repression of nadA resulted in significantly higher association of a DeltafarR strain with epithelial cells. Hence NmFarR has gained control over a meningococcus-specific gene involved in host colonization and thus contributed to divergent niche adaptation in pathogenic Neisseriae.

  9. Analysis of Polymorphic Residues Reveals Distinct Enzymatic and Cytotoxic Activities of the Streptococcus pyogenes NAD+ Glycohydrolase*

    PubMed Central

    Chandrasekaran, Sukantha; Ghosh, Joydeep; Port, Gary C.; Koh, Eun-ik; Caparon, Michael G.

    2013-01-01

    The Streptococcus pyogenes NAD+ glycohydrolase (SPN) is secreted from the bacterial cell and translocated into the host cell cytosol where it contributes to cell death. Recent studies suggest that SPN is evolving and has diverged into NAD+ glycohydrolase-inactive variants that correlate with tissue tropism. However, the role of SPN in both cytotoxicity and niche selection are unknown. To gain insight into the forces driving the adaptation of SPN, a detailed comparison of representative glycohydrolase activity-proficient and -deficient variants was conducted. Of a total 454 amino acids, the activity-deficient variants differed at only nine highly conserved positions. Exchanging residues between variants revealed that no one single residue could account for the inability of the deficient variants to cleave the glycosidic bond of β-NAD+ into nicotinamide and ADP-ribose; rather, reciprocal changes at 3 specific residues were required to both abolish activity of the proficient version and restore full activity to the deficient variant. Changing any combination of 1 or 2 residues resulted in intermediate activity. However, a change to any 1 residue resulted in a significant decrease in enzyme efficiency. A similar pattern involving multiple residues was observed for comparison with a second highly conserved activity-deficient variant class. Remarkably, despite differences in glycohydrolase activity, all versions of SPN were equally cytotoxic to cultured epithelial cells. These data indicate that the glycohydrolase activity of SPN may not be the only contribution the toxin has to the pathogenesis of S. pyogenes and that both versions of SPN play an important role during infection. PMID:23689507

  10. Interaction Signatures Stabilizing the NAD(P)-Binding Rossmann Fold: A Structure Network Approach

    PubMed Central

    Bhattacharyya, Moitrayee; Upadhyay, Roopali; Vishveshwara, Saraswathi

    2012-01-01

    The fidelity of the folding pathways being encoded in the amino acid sequence is met with challenge in instances where proteins with no sequence homology, performing different functions and no apparent evolutionary linkage, adopt a similar fold. The problem stated otherwise is that a limited fold space is available to a repertoire of diverse sequences. The key question is what factors lead to the formation of a fold from diverse sequences. Here, with the NAD(P)-binding Rossmann fold domains as a case study and using the concepts of network theory, we have unveiled the consensus structural features that drive the formation of this fold. We have proposed a graph theoretic formalism to capture the structural details in terms of the conserved atomic interactions in global milieu, and hence extract the essential topological features from diverse sequences. A unified mathematical representation of the different structures together with a judicious concoction of several network parameters enabled us to probe into the structural features driving the adoption of the NAD(P)-binding Rossmann fold. The atomic interactions at key positions seem to be better conserved in proteins, as compared to the residues participating in these interactions. We propose a “spatial motif” and several “fold specific hot spots” that form the signature structural blueprints of the NAD(P)-binding Rossmann fold domain. Excellent agreement of our data with previous experimental and theoretical studies validates the robustness and validity of the approach. Additionally, comparison of our results with statistical coupling analysis (SCA) provides further support. The methodology proposed here is general and can be applied to similar problems of interest. PMID:23284738

  11. TEMPOL increases NAD+ and improves redox imbalance in obese mice

    PubMed Central

    Yamato, Mayumi; Kawano, Kimika; Yamanaka, Yuki; Saiga, Misako; Yamada, Ken-ichi

    2016-01-01

    Continuous energy conversion is controlled by reduction–oxidation (redox) processes. NAD+ and NADH represent an important redox couple in energy metabolism. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) is a redox-cycling nitroxide that promotes the scavenging of several reactive oxygen species (ROS) and is reduced to hydroxylamine by NADH. TEMPOL is also involved in NAD+ production in the ascorbic acid–glutathione redox cycle. We utilized the chemical properties of TEMPOL to investigate the effects of antioxidants and NAD+/NADH modulators on the metabolic imbalance in obese mice. Increases in the NAD+/NADH ratio by TEMPOL ameliorated the metabolic imbalance when combined with a dietary intervention, changing from a high-fat diet to a normal diet. Plasma levels of the superoxide marker dihydroethidium were higher in mice receiving the dietary intervention compared with a control diet, but were normalized with TEMPOL consumption. These findings provide novel insights into redox regulation in obesity. PMID:26942863

  12. NadA, a novel vaccine candidate of Neisseria meningitidis.

    PubMed

    Comanducci, Maurizio; Bambini, Stefania; Brunelli, Brunella; Adu-Bobie, Jeannette; Aricò, Beatrice; Capecchi, Barbara; Giuliani, Marzia Monica; Masignani, Vega; Santini, Laura; Savino, Silvana; Granoff, Dan M; Caugant, Dominique A; Pizza, Mariagrazia; Rappuoli, Rino; Mora, Marirosa

    2002-06-01

    Neisseria meningitidis is a human pathogen, which, in spite of antibiotic therapy, is still a major cause of mortality due to sepsis and meningitis. Here we describe NadA, a novel surface antigen of N. meningitidis that is present in 52 out of 53 strains of hypervirulent lineages electrophoretic types (ET) ET37, ET5, and cluster A4. The gene is absent in the hypervirulent lineage III, in N. gonorrhoeae and in the commensal species N. lactamica and N. cinerea. The guanine/cytosine content, lower than the chromosome, suggests acquisition by horizontal gene transfer and subsequent limited evolution to generate three well-conserved alleles. NadA has a predicted molecular structure strikingly similar to a novel class of adhesins (YadA and UspA2), forms high molecular weight oligomers, and binds to epithelial cells in vitro supporting the hypothesis that NadA is important for host cell interaction. NadA induces strong bactericidal antibodies and is protective in the infant rat model suggesting that this protein may represent a novel antigen for a vaccine able to control meningococcal disease caused by three hypervirulent lineages. PMID:12045242

  13. NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion

    PubMed Central

    Sasaki, Yo; Nakagawa, Takashi; Mao, Xianrong; DiAntonio, Aaron; Milbrandt, Jeffrey

    2016-01-01

    Overexpression of the NAD+ biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD+ or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD+ metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD+ synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD+ consumption that is central to axon degeneration. DOI: http://dx.doi.org/10.7554/eLife.19749.001 PMID:27735788

  14. Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA.

    PubMed

    Liguori, Alessia; Malito, Enrico; Lo Surdo, Paola; Fagnocchi, Luca; Cantini, Francesca; Haag, Andreas F; Brier, Sébastien; Pizza, Mariagrazia; Delany, Isabel; Bottomley, Matthew J

    2016-04-01

    Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in

  15. Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA

    PubMed Central

    Liguori, Alessia; Malito, Enrico; Lo Surdo, Paola; Fagnocchi, Luca; Cantini, Francesca; Haag, Andreas F.; Brier, Sébastien; Pizza, Mariagrazia; Delany, Isabel; Bottomley, Matthew J.

    2016-01-01

    Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in

  16. Ligand Binding and Structural Changes Associated with Allostery in Yeast NAD+-specific Isocitrate Dehydrogenase

    PubMed Central

    Lee, McAlister-Henn

    2011-01-01

    Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octameric enzyme composed of four each of regulatory IDH1 and catalytic IDH2 subunits that share 42% sequence identity. IDH2 contains catalytic isocitrate/Mg2+ and NAD+ binding sites whereas IDH1 contains homologous binding sites, respectively, for cooperative binding of isocitrate and for allosteric binding of AMP. Ligand binding is highly ordered in vitro, and IDH exhibits the unusual property of half-site binding for all ligands. The structures of IDH solved in the absence or presence of ligands have shown: (a) a heterodimer to be the basic structural/functional unit of the enzyme, (b) the organization of heterodimers to form tetramer and octamer structures, (c) structural differences that may underlie cooperative and allosteric regulatory mechanisms, and (d) the possibility for formation of a disulfide bond that could reduce catalytic activity. In vivo analyses of mutant enzymes have elucidated the physiological importance of catalytic activity and allosteric regulation of this tricarboxylic acid cycle enzyme. Other studies have established the importance of a disulfide bond in regulation of IDH activity in vivo, as well as contributions of this bond to the property of half-site ligand binding exhibited by the wild-type enzyme. PMID:22008468

  17. Characterization of NAD salvage pathways and their role in virulence in Streptococcus pneumoniae.

    PubMed

    Johnson, Michael D L; Echlin, Haley; Dao, Tina H; Rosch, Jason W

    2015-11-01

    NAD is a necessary cofactor present in all living cells. Some bacteria cannot de novo synthesize NAD and must use the salvage pathway to import niacin or nicotinamide riboside via substrate importers NiaX and PnuC, respectively. Although homologues of these two importers and their substrates have been identified in other organisms, limited data exist in Streptococcus pneumoniae, specifically, on its effect on overall virulence. Here, we sought to characterize the substrate specificity of NiaX and PnuC in Str. pneumoniae TIGR4 and the contribution of these proteins to virulence of the pathogen. Although binding affinity of each importer for nicotinamide mononucleotide may overlap, we found NiaX to specifically import nicotinamide and nicotinic acid, and PnuC to be primarily responsible for nicotinamide riboside import. Furthermore, a pnuC mutant is completely attenuated during both intranasal and intratracheal infections in mice. Taken together, these findings underscore the importance of substrate salvage in pneumococcal pathogenesis and indicate that PnuC could potentially be a viable small-molecule therapeutic target to alleviate disease progression in the host. PMID:26311256

  18. Characterization of NAD salvage pathways and their role in virulence in Streptococcus pneumoniae.

    PubMed

    Johnson, Michael D L; Echlin, Haley; Dao, Tina H; Rosch, Jason W

    2015-11-01

    NAD is a necessary cofactor present in all living cells. Some bacteria cannot de novo synthesize NAD and must use the salvage pathway to import niacin or nicotinamide riboside via substrate importers NiaX and PnuC, respectively. Although homologues of these two importers and their substrates have been identified in other organisms, limited data exist in Streptococcus pneumoniae, specifically, on its effect on overall virulence. Here, we sought to characterize the substrate specificity of NiaX and PnuC in Str. pneumoniae TIGR4 and the contribution of these proteins to virulence of the pathogen. Although binding affinity of each importer for nicotinamide mononucleotide may overlap, we found NiaX to specifically import nicotinamide and nicotinic acid, and PnuC to be primarily responsible for nicotinamide riboside import. Furthermore, a pnuC mutant is completely attenuated during both intranasal and intratracheal infections in mice. Taken together, these findings underscore the importance of substrate salvage in pneumococcal pathogenesis and indicate that PnuC could potentially be a viable small-molecule therapeutic target to alleviate disease progression in the host.

  19. Crystallization and preliminary X-ray crystallographic analysis of yeast NAD{sup +}-specific isocitrate dehydrogenase

    SciTech Connect

    Hu, Gang; Taylor, Alexander B.; McAlister-Henn, Lee; Hart, P. John

    2005-05-01

    Yeast NAD{sup +}-isocitrate dehydrogenase has been purified and crystallized using sodium citrate, a competitive inhibitor of the enzyme, as a precipitant. Preliminary X-ray analyses indicate the molecular boundaries of the molecule and large continuous solvent channels in the crystal. NAD{sup +}-specific isocitrate dehydrogenase (IDH; EC 1.1.1.41) is a complex allosterically regulated enzyme in the tricarboxylic acid cycle. Yeast IDH is believed to be an octamer containing four catalytic IDH2 and four regulatory IDH1 subunits. Crystals of yeast IDH have been obtained and optimized using sodium citrate, a competitive inhibitor of the enzyme, as the precipitating agent. The crystals belong to space group R3, with unit-cell parameters a = 302.0, c = 112.1 Å. Diffraction data were collected to 2.9 Å from a native crystal and to 4.0 Å using multiwavelength anomalous diffraction (MAD) methods from an osmium derivative. Initial electron-density maps reveal large solvent channels and the molecular boundaries of the allosteric IDH multimer.

  20. Dynamic conformations of the CD38-mediated NAD cyclization captured in a single crystal

    PubMed Central

    Zhang, HongMin; Graeff, Richard; Chen, Zhe; Zhang, LiangRen; Zhang, LiHe; Lee, HonCheung; Hao, Quan

    2010-01-01

    The extracellular domain of human CD38 is a multifunctional enzyme involved in the metabolism of two Ca2+ messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When NAD is used as a substrate, CD38 predominantly hydrolyzes it to ADP-ribose with a trace mount of cADPR produced through cyclization of the substrate. However, a mutation of a key residue at the active site, E146A, inhibits the hydrolysis activity of CD38 but greatly increases its cyclization activity. To understand the role of the residue, E146, in the catalytic process, we determined the crystal structure of the E146A mutant protein with a substrate analogue, ara-2’F-NAD. The structure captured the enzymatic reaction intermediates in six different conformations in a crystallographic asymmetric unit. The structural results indicate a folding-back process for the adenine ring of the substrate and provides the first multiple snap shots of the process. Our approach of utilizing multiple molecules in the crystallographic asymmetric unit should be generally applicable for capturing the dynamic nature of enzymatic catalysis. PMID:21134381

  1. An unassembled subunit of NAD(+)-dependent isocitrate dehydrogenase is insoluble and covalently modified.

    PubMed

    Gadde, D M; Yang, E; McCammon, M T

    1998-06-01

    The NAD(+)-dependent isocitrate dehydrogenase of Saccharomyces cerevisiae is an octamer composed of four Idh1p subunits and four Idh2p subunits. Isocitrate dehydrogenase functions in the tricarboxylic acid cycle and has also been reported to bind to the 5' nontranslated region of mitochondrially encoded mRNAs. Mutants defective in either or both of these subunits are unable to grow on the nonfermentable carbon source, acetate, but will utilize glycerol or ethanol. Mutant strains lacking Idh2p maintain normal if not elevated levels of mitochondrial Idh1p. In addition to the mature unassembled Idh1p subunit, a complex of bands in the 85- to 170-kDa range (Idh1p-Cpx) is observed using NAD-IDH antiserum. Both Idh1p and Idh1p-Cpx are insoluble within the mitochondrion and are associated with the mitochondrial inner membrane. A histidine-tagged form of Idh1p was expressed in yeast strains. Chemical amounts of the Idh1p-Cpx could be purified from strains lacking Idh2p but not from strains containing normal levels of Idh2p. The data indicate that Idh1p-Cpx is an aggregated and cross-linked form of Idh1p that may be oxidized within the mitochondrion as a consequence of its aborted assembly.

  2. NAD+ accumulation during pollen maturation in Arabidopsis regulating onset of germination.

    PubMed

    Hashida, Shin-nosuke; Takahashi, Hideyuki; Takahara, Kentaro; Kawai-Yamada, Maki; Kitazaki, Kazuyoshi; Shoji, Kazuhiro; Goto, Fumiyuki; Yoshihara, Toshihiro; Uchimiya, Hirofumi

    2013-01-01

    Although the nicotinamide nucleotides NAD(H) and NADP(H) are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well understood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD+ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immediately after contact with water. Importantly, excess of NAD+ impaired pollen tube growth. Moreover, NAD+ accumulation was retained after pollen was imbibed in the presence of NAD+-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD+ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther locule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD+ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD+ accumulation acts to negatively regulate germination and a decrease in NAD+ plays an important role in metabolic state transition.

  3. Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism.

    PubMed

    Frederick, David W; Davis, James G; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A; Nakamaru-Ogiso, Eiko; Baur, Joseph A

    2015-01-16

    The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. PMID:25411251

  4. Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism.

    PubMed

    Frederick, David W; Davis, James G; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A; Nakamaru-Ogiso, Eiko; Baur, Joseph A

    2015-01-16

    The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.

  5. Genetic characterization and regulation of the nadB locus of Salmonella typhimurium.

    PubMed Central

    Cookson, B T; Olivera, B M; Roth, J R

    1987-01-01

    The nadB locus encodes the first enzyme of NAD synthesis. It has been reported that this gene and nadA are regulated by a positive regulatory protein encoded in the nadB region. In pursuing this regulatory mechanism, we constructed a fine-structure genetic map of the nadB gene. The region appears to include a single complementation group; no evidence for a positive regulatory element was found. Several mutations causing resistance to the analog 6-aminonicotinamide mapped within the structural gene and probably cause resistance to feedback inhibition. Regulatory mutations for nadB were isolated. These mutants mapped far from nadB near the pnuA gene, which encodes a function required for nicotinamide mononucleotide transport. The regulatory mutations appear to affect a distinct function encoded in the same operon as pnuA. PMID:3305482

  6. Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae.

    PubMed

    Kato, Michiko; Lin, Su-Ju

    2014-11-01

    Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates

  7. Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae.

    PubMed

    Kato, Michiko; Lin, Su-Ju

    2014-11-01

    Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates

  8. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD+ and triclosan

    PubMed Central

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2010-01-01

    Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD+ has been solved to a resolution of 2.1 Å. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD+ reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD+ cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors. PMID:21045289

  9. NAD+ salvage pathway proteins suppress proteotoxicity in yeast models of neurodegeneration by promoting the clearance of misfolded/oligomerized proteins.

    PubMed

    Ocampo, Alejandro; Liu, Jingjing; Barrientos, Antoni

    2013-05-01

    Increased levels of nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT) act as a powerful suppressor of Wallerian degeneration and ataxin- and tau-induced neurodegeneration in flies and mice. However, the nature of the suppression mechanism/s remains controversial. Here, we show that in yeast models of proteinopathies, overexpression of the NMNAT yeast homologs, NMA1 and NMA2, suppresses polyglutamine (PolyQ) and α-synuclein-induced cytotoxicities. Unexpectedly, overexpression of other genes in the salvage pathway for NAD(+) biosynthesis, including QNS1, NPT1 and PNC1 also protected against proteotoxicity. Our data revealed that in all cases, this mechanism involves extensive clearance of the non-native protein. Importantly, we demonstrate that suppression by NMA1 does not require the presence of a functional salvage pathway for NAD(+) biosynthesis, SIR2 or an active mitochondrial oxidative phosphorylation (OXPHOS) system. Our results imply the existence of histone deacetylase- and OXPHOS-independent crosstalk between the proteins in the salvage pathway for NAD(+) biosynthesis and the proteasome that can be manipulated to achieve cellular protection against proteotoxic stress.

  10. Sources and implications of NADH/NAD+ redox imbalance in diabetes and its complications

    PubMed Central

    Wu, Jinzi; Jin, Zhen; Zheng, Hong; Yan, Liang-Jun

    2016-01-01

    NAD+ is a fundamental molecule in metabolism and redox signaling. In diabetes and its complications, the balance between NADH and NAD+ can be severely perturbed. On one hand, NADH is overproduced due to influx of hyperglycemia to the glycolytic and Krebs cycle pathways and activation of the polyol pathway. On the other hand, NAD+ can be diminished or depleted by overactivation of poly ADP ribose polymerase that uses NAD+ as its substrate. Moreover, sirtuins, another class of enzymes that also use NAD+ as their substrate for catalyzing protein deacetylation reactions, can also affect cellular content of NAD+. Impairment of NAD+ regeneration enzymes such as lactate dehydrogenase in erythrocytes and complex I in mitochondria can also contribute to NADH accumulation and NAD+ deficiency. The consequence of NADH/NAD+ redox imbalance is initially reductive stress that eventually leads to oxidative stress and oxidative damage to macromolecules, including DNA, lipids, and proteins. Accordingly, redox imbalance-triggered oxidative damage has been thought to be a major factor contributing to the development of diabetes and its complications. Future studies on restoring NADH/NAD+ redox balance could provide further insights into design of novel antidiabetic strategies. PMID:27274295

  11. The potential regulatory roles of NAD(+) and its metabolism in autophagy.

    PubMed

    Zhang, Dong-Xia; Zhang, Jia-Ping; Hu, Jiong-Yu; Huang, Yue-Sheng

    2016-04-01

    (Macro)autophagy mediates the bulk degradation of defective organelles, long-lived proteins and protein aggregates in lysosomes and plays a critical role in cellular and tissue homeostasis. Defective autophagy processes have been found to contribute to a variety of metabolic diseases. However, the regulatory mechanisms of autophagy are not fully understood. Increasing data indicate that nicotinamide adenine nucleotide (NAD(+)) homeostasis correlates intimately with autophagy. NAD(+) is a ubiquitous coenzyme that functions primarily as an electron carrier of oxidoreductase in multiple redox reactions. Both NAD(+) homeostasis and its metabolism are thought to play critical roles in regulating autophagy. In this review, we discuss how the regulation of NAD(+) and its metabolism can influence autophagy. We focus on the regulation of NAD(+)/NADH homeostasis and the effects of NAD(+) consumption by poly(ADP-ribose) (PAR) polymerase-1 (PARP-1), NAD(+)-dependent deacetylation by sirtuins and NAD(+) metabolites on autophagy processes and the underlying mechanisms. Future studies should provide more direct evidence for the regulation of autophagy processes by NAD(+). A better understanding of the critical roles of NAD(+) and its metabolites on autophagy will shed light on the complexity of autophagy regulation, which is essential for the discovery of new therapeutic tools for autophagy-related diseases. PMID:26975537

  12. Sources and implications of NADH/NAD(+) redox imbalance in diabetes and its complications.

    PubMed

    Wu, Jinzi; Jin, Zhen; Zheng, Hong; Yan, Liang-Jun

    2016-01-01

    NAD(+) is a fundamental molecule in metabolism and redox signaling. In diabetes and its complications, the balance between NADH and NAD(+) can be severely perturbed. On one hand, NADH is overproduced due to influx of hyperglycemia to the glycolytic and Krebs cycle pathways and activation of the polyol pathway. On the other hand, NAD(+) can be diminished or depleted by overactivation of poly ADP ribose polymerase that uses NAD(+) as its substrate. Moreover, sirtuins, another class of enzymes that also use NAD(+) as their substrate for catalyzing protein deacetylation reactions, can also affect cellular content of NAD(+). Impairment of NAD(+) regeneration enzymes such as lactate dehydrogenase in erythrocytes and complex I in mitochondria can also contribute to NADH accumulation and NAD(+) deficiency. The consequence of NADH/NAD(+) redox imbalance is initially reductive stress that eventually leads to oxidative stress and oxidative damage to macromolecules, including DNA, lipids, and proteins. Accordingly, redox imbalance-triggered oxidative damage has been thought to be a major factor contributing to the development of diabetes and its complications. Future studies on restoring NADH/NAD(+) redox balance could provide further insights into design of novel antidiabetic strategies. PMID:27274295

  13. Human CD38 is an authentic NAD(P)+ glycohydrolase.

    PubMed Central

    Berthelier, V; Tixier, J M; Muller-Steffner, H; Schuber, F; Deterre, P

    1998-01-01

    The leucoyte surface antigen CD38 has been shown to be an ecto-enzyme with multiple catalytic activities. It is principally a NAD+ glycohydrolase that transforms NAD+ into ADP-ribose and nicotinamide. CD38 is also able to produce small amounts of cyclic ADP-ribose (ADP-ribosyl cyclase activity) and to hydrolyse this cyclic metabolite into ADP-ribose (cyclic ADP-ribose hydrolase activity). To classify CD38 among the enzymes that transfer the ADP-ribosyl moiety of NAD+ to a variety of acceptors, we have investigated its substrate specificity and some characteristics of its kinetic and molecular mechanisms. We find that CD38-catalysed cleavage of the nicotinamide-ribose bond results in the formation of an E.ADP-ribosyl intermediary complex, which is common to all reaction pathways; this intermediate reacts (1) with acceptors such as water (hydrolysis), methanol (methanolysis) or pyridine (transglycosidation), and (2) intramolecularly, yielding cyclic ADP-ribose with a low efficiency. This reaction scheme is also followed when using nicotinamide guanine dinucleotide as an alternative substrate; in this case, however, the cyclization process is highly favoured. The results obtained here are not compatible with the prevailing model for the mode of action of CD38, according to which this enzyme produces first cyclic ADP-ribose which is then immediately hydrolysed into ADP-ribose (i.e. sequential ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities). We show instead that the cyclic metabolite was a reaction product of CD38 rather than an obligatory reaction intermediate during the glycohydrolase activity. Altogether our results lead to the conclusion that CD38 is an authentic 'classical' NAD(P)+ glycohydrolase (EC 3.2.2.6). PMID:9494110

  14. Experimental and theoretical electron density distribution of alpha,alpha-trehalose dihydrate.

    PubMed

    Stevens, Edwin D; Dowd, Michael K; Johnson, Glenn P; French, Alfred D

    2010-07-01

    alpha,alpha-Trehalose is of interest because of its cryoprotective and antidessicant properties, and because it possesses various technical anomalies such as (13)C NMR spectra that give misleading indications of intramolecular structural symmetry. It is a non-reducing disaccharide, with the glycosidic oxygen atom shared by the anomeric carbon atoms of the two glucose rings, and is therefore subject to a proposed 'overlapping'exo-anomeric effect. We report here a study of the electron density of trehalose with X-ray diffraction and quantum mechanics calculations, similar to a recent study of sucrose, also a non-reducing molecule. In particular we studied the electron density around the glycosidic linkage and the hydrogen bonding with both deformation density and Atoms in Molecules (AIM) analyses. A total of 129,952 single crystal X-ray intensity measurements were collected on alpha,alpha-trehalose dihydrate to a resolution of sintheta/lambda=1.18A(-1) at 100K and refined with an aspherical multipole model to a final agreement factor of R(1)=0.0160. Wavefunctions were calculated at three levels of theory. Redistribution of electron density due to anomeric effects was reduced in trehalose, compared to sucrose. Five new C-Hcdots, three dots, centeredO hydrogen bonds were confirmed with bond critical points and bond paths from AIM analyses, as were the previously proposed O-Hcdots, three dots, centeredO hydrogen bonds. PMID:20381017

  15. Molecular structure, vibrational spectra and DFT computational studies of melaminium N-acetylglycinate dihydrate

    NASA Astrophysics Data System (ADS)

    Tanak, H.; Pawlus, K.; Marchewka, M. K.

    2016-10-01

    Melaminium N-acetylglycinate dihydrate, an organic material has been synthesized and characterized by X-ray diffraction, FT-IR, and FT-Raman spectroscopies for the protiated and deuteriated crystals. The title complex crystallizes in the triclinic system, and the space group is P-1 with a = 5.642(1) Å, b = 7.773(2) Å, c = 15.775(3) Å, α = 77.28(1)°, β = 84.00(1)°, γ = 73.43(1)° and Z = 2. The molecular geometry, vibrational frequencies and intensity of the vibrational bands have been interpreted with the aid of structure optimization based on density functional method (B3LYP) with the 6-311++G(d,p) basis set. The obtained vibrational wavenumbers and optimized geometric parameters were seen to be in good agreement with the experimental data. The intermolecular hydrogen bonding interactions of the title compound have been investigated using the natural bonding orbital analysis. It reveals that the O-H···O, N-H···N and N-H···O intermolecular interactions significantly influence crystal packing of this molecule. The non-linear optical properties are also addressed theoretically. The predicted NLO properties of the title compound are much greater than ones of urea. In addition, DFT calculations of the title compound, molecular electrostatic potential, thermodynamic properties, frontier orbitals and chemical reactivity descriptors were also performed at 6-311++G(d,p) level of theory.

  16. Problems in polarized light microscopy observation of birefringence of calcium pyrophosphate dihydrate crystals.

    PubMed

    Omura, Yoko; Okamoto, Renzo; Konno, Minoru; Shiro, Motoo

    2010-12-01

    To reconsider the problems arising from the use of the phase plate as a test plate inserted into a polarized light microscope system for the analysis of triclinic calcium pyrophosphate dihydrate (t-CPPD) crystals, or Ca(2)P(2)O(7) x 2H(2)O in the synovial fluid of arthritis patients, we made the polarized light microscopy observations using a phase plate with a retardation of 530 nm for the synthesized t-CPPD crystals well-characterized by X-ray powder pattern indexing and single crystal X-ray diffraction measurements. The microscopy observations were made of crystals of different sizes, thicknesses and shapes. The retardation was assessed using the interference color chart at four extinction and diagonal positions both with and without the test plate. The addition and subtraction states produced by superimposing the retardations of two anisotropic materials, that is, the t-CPPD crystal and the 530 nm phase plate, were deduced from the interference color change by inserting the test plate at four diagonal positions. When the color change of a crystal at a diagonal position resulting from 90-degree rotation exhibits no clear birefringence, the interference color chart was shown to be useless. We suggested the use of a compensator whose retardation can be changed to obtain an accurate value for the retardation of the crystal.

  17. N'-[(E)-(3-Fluoro-pyridin-2-yl)methyl-idene]pyridine-3-carbohydrazide dihydrate.

    PubMed

    Nair, Yamuna; Sithambaresan, M; Nair, S Muraleedharan; Kurup, M R Prathapachandra

    2014-04-01

    The organic molecule in the title dihydrate, C12H9FN4O·2H2O, exists in the E conformation with respect to the azo-methane C=N double bond. The mol-ecule is approximately planar, with a maximum deviation of 0.117 (1) Å for the carbonyl O atom from the mean plane of the mol-ecule. Both pyridine rings are essentially coplanar with the central C(=O)N2C unit [dihedral angles = 1.99 (7) and 5.71 (8)°], exhibiting a significant difference in dihedral angles from its benzohydrazide analogue. The crystal packing features N-H⋯O, O-H⋯N and O-H⋯O hydrogen-bond inter-actions, which lead to the formation of a chain along the c-axis direction through one of the water mol-ecules present, and these chains are stacked one over the other by means of π-π inter-actions [with centroid-centroid distances of 3.7099 (10) and 3.6322 (10) Å] between the aromatic rings in neighbouring anti-parallel mol-ecules, building a three-dimensional supra-molecular network. PMID:24826177

  18. Mitogenic effects of hydroxyapatite and calcium pyrophosphate dihydrate crystals on cultured mammalian cells.

    PubMed

    Cheung, H S; Story, M T; McCarty, D J

    1984-06-01

    Synthetic hydroxyapatite (HA) crystals in 1% serum stimulated 3H thymidine uptake into quiescent canine synovial fibroblasts and human foreskin fibroblast cultures, as did 10% serum. The onset of stimulation and peak uptake of thymidine after crystal addition were delayed by 2-3 hours as compared with the effects produced by 10% serum. Stimulation of 3H thymidine uptake was proportional to the serum concentration used. HA crystals (50 micrograms/ml) stimulated nuclide uptake at each serum concentration used. 3H thymidine uptake was also proportional to the dose of HA or calcium pyrophosphate dihydrate crystals, although larger doses of the latter crystal were required to produce equivalent effects. Not all particulates were effective mitogenic agents. Latex beads and diamond crystals had no effect. Monosodium urate crystals modestly stimulated and calcium urate crystals markedly stimulated nuclide uptake. The more complex crystals found in a naturally occurring condition (calcinosis) were as mitogenic as the pure synthetic HA. The synovial cell hyperplasia sometimes associated with crystals might be explained in part by their mitogenic activity. PMID:6329235

  19. A Rare Case of Tumoral Calcium Pyrophosphate Dihydrate Crystal Deposition Disease of the Wrist Joint

    PubMed Central

    Nakamura, Osamu; Kaji, Yoshio; Yamagami, Yoshiki; Yamaguchi, Kounosuke; Nishimura, Hideki; Fukuoka, Natsuko; Yamamoto, Tetsuji

    2015-01-01

    Introduction. Tumoral calcium pyrophosphate dihydrate (CPPD) crystal deposition disease (CPPDCD), also known as tophaceous calcium pyrophosphate deposition disease (CPDD), is a tumorlike lesion, and it should be distinguished from usual CPDD that causes severe joint inflammation and arthralgia. A case of tumoral CPPDCD of the wrist joint that required differentiation from synovial osteochondromatosis is described. Case Presentation. The patient was a 78-year-old woman with a 5-year history of nodular lesions at the right wrist that had gradually increased in size. An excisional biopsy and a histological examination of the excised nodular lesions by hematoxylin and eosin (H&E) staining were performed, demonstrating numerous polarizable, rhabdoid, and rectangular crystals, surrounded by fibroblasts, macrophages, and foreign body-type giant cells, consistent with tumoral CPPDCD. Conclusion. Tumoral CPPDCD, especially at the wrist joint, is rare, and, to the best of our knowledge, only 2 articles have been published. This case seems to need further follow-up for recurrence, because tumoral CPPDCD may recur after complete or incomplete surgical excision. PMID:26783477

  20. Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing

    PubMed Central

    Leu, Kuan-Chieh; Hsieh, Ming-Hsiun; Wang, Huei-Jing; Hsieh, Hsu-Liang

    2016-01-01

    ABSTRACT The mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg300-to-Trp300 and Pro313-to-Ser313 amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles. PMID:27149614

  1. Regulation of active site coupling in glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    LaRonde-LeBlanc, Nicole; Resto, Melissa; Gerratana, Barbara

    2009-05-21

    NAD{sup +} is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD{sup +} biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD{sup +} synthetase, which catalyzes the ATP-dependent formation of NAD{sup +} at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD{sup +} synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD{sup +} synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-{angstrom} intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

  2. Dual emission fluorescent silver nanoclusters for sensitive detection of the biological coenzyme NAD+/NADH.

    PubMed

    Yuan, Yufeng; Huang, Kehan; Chang, Mengfang; Qin, Cuifang; Zhang, Sanjun; Pan, Haifeng; Chen, Yan; Xu, Jianhua

    2016-02-01

    Fluorescent silver nanoclusters (Ag NCs) displaying dual-excitation and dual-emission properties have been developed for the specific detection of NAD(+) (nicotinamide adenine dinucleotide, oxidized form). With the increase of NAD(+) concentrations, the longer wavelength emission (with the peak at 550 nm) was gradually quenched due to the strong interactions between the NAD(+) and Ag NCs, whereas the shorter wavelength emission (peaking at 395 nm) was linearly enhanced. More important, the dual-emission intensity ratio (I395/I550), fitting by a single-exponential decay function, can efficiently detect various NAD(+) levels from 100 to 4000 μM, as well as label NAD(+)/NADH (reduced form of NAD) ratios in the range of 1-50.

  3. Neisseria meningitidis adhesin NadA targets beta1 integrins: functional similarity to Yersinia invasin.

    PubMed

    Nägele, Virginie; Heesemann, Jürgen; Schielke, Stephanie; Jiménez-Soto, Luisa F; Kurzai, Oliver; Ackermann, Nikolaus

    2011-06-10

    Meningococci are facultative-pathogenic bacteria endowed with a set of adhesins allowing colonization of the human upper respiratory tract, leading to fulminant meningitis and septicemia. The Neisseria adhesin NadA was identified in about 50% of N. meningitidis isolates and is closely related to the Yersinia adhesin YadA, the prototype of the oligomeric coiled-coil adhesin (Oca) family. NadA is known to be involved in cell adhesion, invasion, and induction of proinflammatory cytokines. Because of the enormous diversity of neisserial cell adhesins the analysis of the specific contribution of NadA in meningococcal host interactions is limited. Therefore, we used a non-invasive Y. enterocolitica mutant as carrier to study the role of NadA in host cell interaction. NadA was shown to be efficiently produced and localized in its oligomeric form on the bacterial surface of Y. enterocolitica. Additionally, NadA mediated a β1 integrin-dependent adherence with subsequent internalization of yersiniae by a β1 integrin-positive cell line. Using recombinant NadA(24-210) protein and human and murine β1 integrin-expressing cell lines we could demonstrate the role of the β1 integrin subunit as putative receptor for NadA. Subsequent inhibition assays revealed specific interaction of NadA(24-210) with the human β1 integrin subunit. Cumulatively, these results indicate that Y. enterocolitica is a suitable toolbox system for analysis of the adhesive properties of NadA, revealing strong evidence that β1 integrins are important receptors for NadA. Thus, this study demonstrated for the first time a direct interaction between the Oca-family member NadA and human β1 integrins.

  4. Dynamics of NAD in cortical nephron segments: Effect of nicotinamide and of dietary phosphate intake

    SciTech Connect

    Yusufi, A.N.K.; Kiebzak, G.M.; Kusano, E.; Werness, J.L.; Homma, S.; Dousa, T.P. )

    1987-08-01

    NAD content and the rate of NAD hydrolysis were determined in proximal convoluted tubules (PCT), proximal straight tubules (PST), and adjacent cortical nephron segments microdissected from kidneys of tyroparathyroidectomized (TPTX) rats. In the basal state, rats fed a normal phosphate diet had an NAD content higher in PCT, PST, and in cortical ascending limb (CAL) than in glomeruli. After intraperitoneal injection of nicotinamide, the NAD content increased significantly in all nephron segments except CAL. In experiments conducted on TPTX rats stabilized on a low-phosphorus diet, NAD content increased in response to a nicotinamide injection in PCT, but did not change significantly in PST. The catabolism of NAD was determined by generation of ({sup 3}H)adenosine, a major metabolite of (adenine-2,8-{sup 3}H)NAD. The rate of ({sup 3}H)adenosine generation from ({sup 3}H)NAD was significantly higher in PST than in PCT. The authors conclude that, in response to nicotinamide administration in vivo, the NAD content increases more in PCT than in PST and that this difference may be, at least partly, due to a lower rate of NAD breakdown in PCT. In a state of dietary phosphate deprivation, NAD also increases significantly in response to intraperitoneal nicotinamide in PCT, but it does not increase significantly in PST. The nicotinamide-elicited increase of NAD content in proximal tubules, mainly in PCT, may be related to inhibition of Na{sup +}-gradient-dependent inorganic phosphate (P{sub i}) reabsorption across the brush-border membrane proximal tubules and to the phosphaturic effect of nicotinamide in rats red normal-P{sub i} diet.

  5. Study the effect of Vitamin K on intracellular NAD level in yeast by fluorescence spectrum

    NASA Astrophysics Data System (ADS)

    Yahong, Chen; Ruxiu, Cai; Ke, Zhang

    2007-05-01

    The intracellular NAD level plays a pivotal role in numerous biological processes such as rhythm, senescence, cancer and death. The study of the intracellular NAD level has been one of the "hotspots" in biomedical research. We investigated the effect of Vitamin K on intracellular NAD level in yeast by fluorescence spectrum in this paper. Plasma membrane redox system of yeast was found to be greatly promoted by the addition of Vitamin K 3 or Vitamin K 1. Ferricyanide reduction catalyzed by Vitamin K was accompanied by the decrease in intracellular NADH concentration and the increase in intracellular NAD level of yeast cells.

  6. Activating transhydrogenase and NAD kinase in combination for improving isobutanol production.

    PubMed

    Shi, Aiqin; Zhu, Xinna; Lu, Jiao; Zhang, Xueli; Ma, Yanhe

    2013-03-01

    Isobutanol is an excellent alternative biofuel. Fermentative production of isobutanol had been realized in several microorganisms by combining branched-chain amino acids synthetic pathway and Ehrlich pathway. In contrast to using plasmid overexpression and inducible promoters, genetically stable Escherichia coli strains for isobutanol production were constructed in this work by integrating essential genes into chromosome. A chromosome-based markerless gene modulation method was then developed for fine-tuning gene expression with multiple regulatory parts to improve isobutanol production. There was also a cofactor imbalance problem for anaerobic isobutanol synthesis. NADPH is the reducing equivalent required for isobutanol production, while the common reducing equivalent under anaerobic condition is NADH. Two strategies were used to modulate expression of transhydrogenase (pntAB) and NAD kinase (yfjB) genes to increase NADPH supply for improving isobutanol production. Plasmid overexpression of pntAB and yfjB genes either individually or in combination had little effect on isobutanol production. In contrast, modulating pntAB and yfjB gene expression in chromosome with multiple regulatory parts identified optimal modulators under aerobic and anaerobic conditions, respectively, and improved isobutanol production. Modulating pntAB gene alone led to 20% and 8% increase of anaerobic isobutanol titer and yield. Although modulating yfjB gene alone had nearly no effect, modulating pntAB and yfjB genes in combination led to 50% and 30% increase of isobutanol titer and yield in comparison with modulating pntAB gene alone. It was also found that increasing pntAB gene expression alone had a threshold for improving anaerobic isobutanol production, while activating NAD kinase could break through this threshold, leading to a yield of 0.92mol/mol. Our results suggested that transhydrogenase and NAD kinase had a synergistic effect on increasing NADPH supply and improving anaerobic

  7. The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA- OMVs, without further stimulating their proinflammatory activity on circulating monocytes.

    PubMed

    Tavano, Regina; Franzoso, Susanna; Cecchini, Paola; Cartocci, Elena; Oriente, Francesca; Aricò, Beatrice; Papini, Emanuele

    2009-07-01

    Hypervirulent MenB causing fatal human infections frequently display the oligomeric-coiled coil adhesin NadA, a 45-kDa intrinsic outer membrane protein implicated in binding to and invasion of respiratory epithelial cells. A recombinant soluble mutant lacking the 10-kDa COOH terminal membrane domain (NadA(Delta351-405)) also activates human monocytes/macrophages/DCs. As NadA is physiologically released during sepsis as part of OMVs, in this study, we tested the hypothesis that NadA(+) OMVs have an enhanced or modified proinflammatory/proimmune action compared with NadA(-) OMVs. To do this we investigated the activity of purified free NadA(Delta351-405) and of OMVs from MenB and Escherichia coli strains, expressing or not full-length NadA. NadA(Delta351-405) stimulated monocytes and macrophages to secrete cytokines (IL-1beta, TNF-alpha, IL-6, IL-12p40, IL-12p70, IL-10) and chemokines (IL-8, MIP-1alpha, MCP-1, RANTES), and full-length NadA improved MenB OMV activity, preferentially on macrophages, and only increased cytokine release. NadA(Delta351-405) induced the lymphocyte costimulant CD80 in monocytes and macrophages, and NadA(+) OMVs induced a wider set of molecules supporting antigen presentation (CD80, CD86, HLA-DR, and ICAM-1) more efficiently than NadA(-) OMVs only in macrophages. Moreover, membrane NadA effects, unlike NadA(Delta351-405) ones, were much less IFN-gamma-sensitive. The activity of NadA-positive E. coli OMVs was similar to that of control OMVs. NadA in MenB OMVs acted at adhesin concentrations approximately 10(6) times lower than those required to stimulate cells with free NadA(Delta351-405).

  8. Troxerutin improves hepatic lipid homeostasis by restoring NAD(+)-depletion-mediated dysfunction of lipin 1 signaling in high-fat diet-treated mice.

    PubMed

    Zhang, Zi-Feng; Fan, Shao-Hua; Zheng, Yuan-Lin; Lu, Jun; Wu, Dong-Mei; Shan, Qun; Hu, Bin

    2014-09-01

    Recent evidences suggest that NAD(+) depletion leads to abnormal hepatic lipid metabolism in high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD); however, the contributing mechanism is not well understood. Our previous study showed that troxerutin, a trihydroxyethylated derivative of natural bioflavonoid rutin, effectively inhibited obesity, and normalized hyperglycemia and hyperlipidemia in high-cholesterol diet-induced diabetic mice. Here we investigated whether troxerutin improved hepatic lipid metabolism via preventing NAD(+) depletion in HFD-induced NAFLD mouse model and the mechanisms underlying these effects. Our results showed that troxerutin markedly prevented obesity, liver steatosis and injury in HFD-fed mice. Troxerutin largely suppressed oxidative stress-mediated NAD(+)-depletion by increasing nicotinamide phosphoribosyltransferase (NAMPT) protein expression and decreasing poly (ADP-ribose) polymerase-1 (PARP1) protein expression and activity in HFD-treated mouse livers. Consequently, troxerutin remarkably restored Silent mating type information regulation 2 homolog1 (SirT1) protein expression and activity in HFD-treated mouse livers. Therefore, troxerutin promoted SirT1-mediated AMP-activated protein kinase (AMPK) activation to inhibit mammalian target of rapamycin complex 1 (mTORC1) signaling, which enhanced nuclear lipin 1 localization, lowered cytoplasmic lipin 1 localization and the ratio of hepatic Lpin 1β/α. Ultimately, troxerutin improved lipid homeostasis by enhancing fatty acid oxidation and triglyceride secretion, and suppressing lipogenesis in HFD-fed mouse livers. In conclusion, troxerutin displayed beneficial effects on hepatic lipid homeostasis in HFD-induced NAFLD by blocking oxidative stress to restore NAD(+)-depletion-mediated dysfunction of lipin 1 signaling. This study provides novel mechanistic insights into NAFLD pathogenesis and indicates that troxerutin is a candidate for pharmacological intervention of NAFLD

  9. Spatial Distribution of Trehalose Dihydrate Crystallization in Tablets by X-ray Diffractometry.

    PubMed

    Thakral, Naveen K; Yamada, Hiroyuki; Stephenson, Gregory A; Suryanarayanan, Raj

    2015-10-01

    Crystallization of trehalose dihydrate (C12H22O11·2H2O) was induced by storing tablets of amorphous anhydrous trehalose (C12H22O11) at 65% RH (RT). Our goal was to evaluate the advantages and limitations of two approaches of profiling spatial distribution of drug crystallization in tablets. The extent of crystallization, as a function of depth, was determined in tablets stored for different time-periods. The first approach was glancing angle X-ray diffractometry, where the penetration depth of X-rays was modulated by the incident angle. Based on the mass attenuation coefficient of the matrix, the depth of X-ray penetration was calculated as a function of incident angle, which in turn enabled us to "calculate" the extent of crystallization to different depths. In the second approach, the tablets were split into halves and the split surfaces were analyzed directly. Starting from the tablet surface and moving toward the midplane, XRD patterns were collected in 36 "regions", in increments of 0.05 mm. The results obtained by the two approaches were, in general, in good agreement. Additionally, the results obtained were validated by determining the "average" crystallization in the entire tablet by using synchrotron radiation in the transmission mode. The glancing angle method could detect crystallization up to ∼650 μm and had a "surface bias". Being a nondestructive technique, this method will permit repeated analyses of the same tablet at different time points, for example, during a stability study. However, split tablet analyses, while a "destructive" technique, provided comprehensive and unbiased depth profiling information.

  10. Catalases are NAD(P)H-dependent tellurite reductases.

    PubMed

    Calderón, Iván L; Arenas, Felipe A; Pérez, José Manuel; Fuentes, Derie E; Araya, Manuel A; Saavedra, Claudia P; Tantaleán, Juan C; Pichuantes, Sergio E; Youderian, Philip A; Vásquez, Claudio C

    2006-01-01

    Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO(3)(2-)) to the less toxic, insoluble metal, tellurium (Te(o)), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical. PMID:17183702

  11. Steroid Receptor Coactivator 1 is an Integrator of Glucose and NAD+/NADH Homeostasis

    PubMed Central

    Motamed, Massoud; Rajapakshe, Kimal I.; Hartig, Sean M.; Coarfa, Cristian; Moses, Robb E.; Lonard, David M.

    2014-01-01

    Steroid receptor coactivator 1 (SRC-1) drives diverse gene expression programs necessary for the dynamic regulation of cancer metastasis, inflammation and gluconeogenesis, pointing to its overlapping roles as an oncoprotein and integrator of cell metabolic programs. Nutrient utilization has been intensely studied with regard to cellular adaptation in both cancer and noncancerous cells. Nonproliferating cells consume glucose through the citric acid cycle to generate NADH to fuel ATP generation via mitochondrial oxidative phosphorylation. In contrast, cancer cells undergo metabolic reprogramming to support rapid proliferation. To generate lipids, nucleotides, and proteins necessary for cell division, most tumors switch from oxidative phosphorylation to glycolysis, a phenomenon known as the Warburg Effect. Because SRC-1 is a key coactivator responsible for driving a hepatic gluconeogenic program under fasting conditions, we asked whether SRC-1 responds to alterations in nutrient availability to allow for adaptive metabolism. Here we show SRC-1 is stabilized by the 26S proteasome in the absence of glucose. RNA profiling was used to examine the effects of SRC-1 perturbation on gene expression in the absence or presence of glucose, revealing that SRC-1 affects the expression of complex I of the mitochondrial electron transport chain, a set of enzymes responsible for the conversion of NADH to NAD+. NAD+ and NADH were subsequently identified as metabolites that underlie SRC-1's response to glucose deprivation. Knockdown of SRC-1 in glycolytic cancer cells abrogated their ability to grow in the absence of glucose consistent with SRC-1's role in promoting cellular adaptation to reduced glucose availability. PMID:24438340

  12. Engineering NAD+ availability for Escherichia coli whole-cell biocatalysis: a case study for dihydroxyacetone production

    PubMed Central

    2013-01-01

    Background Whole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them. Results In order to investigate the effects of cofactor availability on whole-cell redox biocatalysis, we devised recombinant Escherichia coli strains for the production of dihydroxyacetone (DHA) catalyzed by the NAD+-dependent glycerol dehydrogenase (GldA). In this model system, a water-forming NAD+ oxidase (NOX) and a NAD+ transporter (NTT4) were also co-expressed for cofactor regeneration and extracellular NAD+ uptake, respectively. We found that cellular cofactor level, NAD+/NADH ratio and NOX activity were not only strain-dependent, but also growth condition-dependent, leading to significant differences in specific DHA titer among different whole-cell biocatalysts. The host E. coli DH5α had the highest DHA specific titer of 0.81 g/gDCW with the highest NAD+/NADH ratio of 6.7 and NOX activity of 3900 U. The biocatalyst had a higher activity when induced with IPTG at 37°C for 8 h compared with those at 30°C for 8 h and 18 h. When cells were transformed with the ntt4 gene, feeding NAD+ during the cell culture stage increased cellular NAD(H) level by 1.44 fold and DHA specific titer by 1.58 fold to 2.13 g/gDCW. Supplementing NAD+ during the biotransformation stage was also beneficial to cellular NAD(H) level and DHA production, and the highest DHA productivity reached 0.76 g/gDCW/h. Cellular NAD(H) level, NAD+/NADH ratio, and NOX and GldA activity dropped over time during the biotransformation process. Conclusions High NAD+/NADH ratio driving by NOX was very important for DHA production. Once cofactor was

  13. Biochemical Issues in Estimation of Cytosolic Free NAD/NADH Ratio

    PubMed Central

    Xie, Jiansheng; Hu, Xun

    2012-01-01

    Cytosolic free NAD/NADH ratio is fundamentally important in maintaining cellular redox homeostasis but current techniques cannot distinguish between protein-bound and free NAD/NADH. Williamson et al reported a method to estimate this ratio by cytosolic lactate/pyruvate (L/P) based on the principle of chemical equilibrium. Numerous studies used L/P ratio to estimate the cytosolic free NAD/NADH ratio by assuming that the conversion in cells was at near-equilibrium but not verifying how near it was. In addition, it seems accepted that cytosolic free NAD/NADH ratio was a dependent variable responding to the change of L/P ratio. In this study, we show (1) that the change of lactate/glucose (percentage of glucose that converts to lactate by cells) and L/P ratio could measure the status of conversion between pyruvate + NADH and lactate + NAD that tends to or gets away from equilibrium; (2) that cytosolic free NAD/NADH could be accurately estimated by L/P only when the conversion is at or very close to equilibrium otherwise a calculation error by one order of magnitude could be introduced; (3) that cytosolic free NAD/NADH is stable and L/P is highly labile, that the highly labile L/P is crucial to maintain the homeostasis of NAD/NADH; (4) that cytosolic free NAD/NADH is dependent on oxygen levels. Our study resolved the key issues regarding accurate estimation of cytosolic free NAD/NADH ratio and the relationship between NAD/NADH and L/P. PMID:22570687

  14. Biochemical and Structural Studies of Uncharacterized Protein PA0743 from Pseudomonas aeruginosa Revealed NAD+-dependent l-Serine Dehydrogenase*

    PubMed Central

    Tchigvintsev, Anatoli; Singer, Alexander; Brown, Greg; Flick, Robert; Evdokimova, Elena; Tan, Kemin; Gonzalez, Claudio F.; Savchenko, Alexei; Yakunin, Alexander F.

    2012-01-01

    The β-hydroxyacid dehydrogenases form a large family of ubiquitous enzymes that catalyze oxidation of various β-hydroxy acid substrates to corresponding semialdehydes. Several known enzymes include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, 2-(hydroxymethyl)glutarate dehydrogenase, and phenylserine dehydrogenase, but the vast majority of β-hydroxyacid dehydrogenases remain uncharacterized. Here, we demonstrate that the predicted β-hydroxyisobutyrate dehydrogenase PA0743 from Pseudomonas aeruginosa catalyzes an NAD+-dependent oxidation of l-serine and methyl-l-serine but exhibits low activity against β-hydroxyisobutyrate. Two crystal structures of PA0743 were solved at 2.2–2.3-Å resolution and revealed an N-terminal Rossmann fold domain connected by a long α-helix to the C-terminal all-α domain. The PA0743 apostructure showed the presence of additional density modeled as HEPES bound in the interdomain cleft close to the predicted catalytic Lys-171, revealing the molecular details of the PA0743 substrate-binding site. The structure of the PA0743-NAD+ complex demonstrated that the opposite side of the enzyme active site accommodates the cofactor, which is also bound near Lys-171. Site-directed mutagenesis of PA0743 emphasized the critical role of four amino acid residues in catalysis including the primary catalytic residue Lys-171. Our results provide further insight into the molecular mechanisms of substrate selectivity and activity of β-hydroxyacid dehydrogenases. PMID:22128181

  15. Activities of citrate synthase and NAD+-linked and NADP+-linked isocitrate dehydrogenase in muscle from vertebrates and invertebrates.

    PubMed Central

    Alp, P R; Newsholme, E A; Zammit, V A

    1976-01-01

    1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles of both vertebrates and invertebrates contain high activities of citrate synthase and NADP+-liniked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation of isocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthease catalyses a near-equilibrium reaction. In other muscles

  16. Proteomic analysis of a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate: a case report.

    PubMed

    Kaneko, Kiyoko; Matsuta, Yosuke; Moriyama, Manabu; Yasuda, Makoto; Chishima, Noriharu; Yamaoka, Noriko; Fukuuchi, Tomoko; Miyazawa, Katsuhito; Suzuki, Koji

    2014-03-01

    The objective of the present study was to investigate the matrix protein of a rare urinary stone that contained calcium carbonate. A urinary stone was extracted from a 34-year-old male patient with metabolic alkalosis. After X-ray diffractometry and infrared analysis of the stone, proteomic analysis was carried out. The resulting mass spectra were evaluated with protein search software, and matrix proteins were identified. X-ray diffraction and infrared analysis confirmed that the stone contained calcium carbonate and calcium oxalate dihydrate. Of the identified 53 proteins, 24 have not been previously reported from calcium oxalate- or calcium phosphate-containing stones. The protease inhibitors and several proteins related to cell adhesion or the cytoskeleton were identified for the first time. We analyzed in detail a rare urinary stone composed of calcium carbonate and calcium oxalate dihydrate. Considering the formation of a calcium carbonate stone, the new identified proteins should play an important role on the urolithiasis process in alkaline condition.

  17. Effect of experimental and sample factors on dehydration kinetics of mildronate dihydrate: mechanism of dehydration and determination of kinetic parameters.

    PubMed

    Bērziņš, Agris; Actiņš, Andris

    2014-06-01

    The dehydration kinetics of mildronate dihydrate [3-(1,1,1-trimethylhydrazin-1-ium-2-yl)propionate dihydrate] was analyzed in isothermal and nonisothermal modes. The particle size, sample preparation and storage, sample weight, nitrogen flow rate, relative humidity, and sample history were varied in order to evaluate the effect of these factors and to more accurately interpret the data obtained from such analysis. It was determined that comparable kinetic parameters can be obtained in both isothermal and nonisothermal mode. However, dehydration activation energy values obtained in nonisothermal mode showed variation with conversion degree because of different rate-limiting step energy at higher temperature. Moreover, carrying out experiments in this mode required consideration of additional experimental complications. Our study of the different sample and experimental factor effect revealed information about changes of the dehydration rate-limiting step energy, variable contribution from different rate limiting steps, as well as clarified the dehydration mechanism. Procedures for convenient and fast determination of dehydration kinetic parameters were offered.

  18. Crystallization of calcium sulfate dihydrate and calcium sulfite hemihydrate from synthetic flue gas desulfurization solutions: Final report

    SciTech Connect

    Trofe, T.W.; Fishman, V.A.; Meserole, F.B.

    1986-10-01

    The precipitation of calcium sulfate dihydrate (CaSO/sub 4/.2H/sub 2/O) and calcium sulfite hemihydrate (CaSO/sub 3/.1/2H/sub 2/O) from high, up to 240,000 mg/L, total dissolved solids (TDS) solutions was studied at 50/sup 0/C. The solutions were selected to cover a range of solution compositions of magnesium, calcium, sodium, chloride, and sulfate. Precipitation rates along with crystal habit and size changes were measured to determine the effects of these dissolved species as compared to dilute solution conditions. Calcium sulfate dihydrate (gypsum) precipitation rate was accelerated in the high TDS solutions, especially those containing chloride ion. Alternatively, calcium sulfite hemihydrate precipitation rate was found to be faster in high sulfate ion containing solutions. Sodium ion appears to produce gypsum crystals more columnar in habit while solutions containing high amounts of calcium produced very lamellar gypsum crystals. Solutions containing magnesium produced acicular gypsum crystals. Calcium sulfite hemihydrate solids precipitated from solutions containing high sulfate concentrations were rod shaped and globular as compared to the lamellar calcium sulfite hemihydrate crystals precipitated from high chloride and dilute solution liquors. Calcium sulfate-calcium sulfite solid solutions were characterized using infrared spectroscopy. Ion scavenging of Na, Mg, and Cl by gypsum and calcium sulfite solids precipitated from these high TDS solutions was also investigated. 10 refs., 21 figs., 13 tabs.

  19. Influence of shot peening on corrosion properties of biocompatible magnesium alloy AZ31 coated by dicalcium phosphate dihydrate (DCPD).

    PubMed

    Mhaede, Mansour; Pastorek, Filip; Hadzima, Branislav

    2014-06-01

    Magnesium alloys are promising materials for biomedical applications because of many outstanding properties like biodegradation, bioactivity and their specific density and Young's modulus are closer to bone than the commonly used metallic implant materials. Unfortunately their fatigue properties and low corrosion resistance negatively influenced their application possibilities in the field of biomedicine. These problems could be diminished through appropriate surface treatments. This study evaluates the influence of a surface pre-treatment by shot peening and shot peening+coating on the corrosion properties of magnesium alloy AZ31. The dicalcium phosphate dihydrate coating (DCPD) was electrochemically deposited in a solution containing 0.1M Ca(NO3)2, 0.06M NH4H2PO4 and 10mL/L of H2O2. The effect of shot peening on the surface properties of magnesium alloy was evaluated by microhardness and surface roughness measurements. The influence of the shot peening and dicalcium phosphate dihydrate layer on the electrochemical characteristics of AZ31 magnesium alloy was evaluated by potentiodynamic measurements and electrochemical impedance spectroscopy in 0.9% NaCl solution at a temperature of 22±1°C. The obtained results were analyzed by the Tafel-extrapolation method and equivalent circuit method. The results showed that the application of shot peening process followed by DCPD coating improves the properties of the AZ31 surface from corrosion and mechanical point of view. PMID:24863232

  20. Activity of select dehydrogenases with Sepharose-immobilized N6-carboxymethyl-NAD

    PubMed Central

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N6-carboxymethyl-NAD (N6-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N6-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N6-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N6-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N6-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N6-amine group on NAD. PMID:25611453

  1. Detection of NAD(P)H-dependent enzyme activity with dynamic luminescence quenching of terbium complexes.

    PubMed

    Ito, Hiroki; Terai, Takuya; Hanaoka, Kenjiro; Ueno, Tasuku; Komatsu, Toru; Nagano, Tetsuo; Urano, Yasuteru

    2015-05-14

    We discovered that positively charged terbium complexes bearing 1,4,7,10-tetraazacyclododecane functionalized with amide ligands are highly sensitive to dynamic luminescence quenching by NAD(P)H. We exploited this phenomenon to establish a general time-resolved luminescence-based assay platform for sensitive detection of NAD(P)H-dependent enzyme activities.

  2. Exogenous NAD(+) administration significantly protects against myocardial ischemia/reperfusion injury in rat model.

    PubMed

    Zhang, Youjun; Wang, Ban; Fu, Xingli; Guan, Shaofeng; Han, Wenzheng; Zhang, Jie; Gan, Qian; Fang, Weiyi; Ying, Weihai; Qu, Xinkai

    2016-01-01

    Acute myocardial infarction is one of the leading causes for death around the world. Although essential for successful interventional therapy, it is inevitably complicated by reperfusion injury. Thus effective approaches to reduce ischemia/reperfusion (I/R) injury are still critically needed. To test our hypothesis that intravenous administration of NAD(+) can attenuate I/R injury by reducing apoptotic damage and enhancing antioxidant capacity, we used a rat mode of myocardial I/R. Our study found that administration of 10-20 mg/kg NAD(+) can dose dependently reduce myocardial infarct induced by I/R, with an approximately 85% reduction of the infarct at the dosage of 20 mg/kg NAD(+). We further found that the injection of NAD(+) can significantly decrease I/R-induced apoptotic damage in the heart: NAD(+) administration can both decrease the TUNEL signals, Bax, cleaved caspase-3 levels and increase the Bcl-XL levels in the rats that are subjected to myocardial I/R injury. NAD(+) administration can also significantly attenuate I/R-induced decreases in SOD activity and SOD-2 protein levels in the hearts. NAD(+) can profoundly decrease myocardial I/R injury at least partially by attenuating apoptotic damage and enhancing the antioxidant capacity, thus suggesting that NAD(+) may become a promising therapeutic agent for myocardial I/R injury. PMID:27648125

  3. 33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage...

  4. 33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage...

  5. 33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage...

  6. 33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage...

  7. 33 CFR 110.235 - Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). 110.235 Section 110.235 Navigation and Navigable Waters COAST... Pacific Ocean (Mamala Bay), Honolulu Harbor, Hawaii (Datum: NAD 83). (a) The anchorage...

  8. NAD(P) biosynthesis enzymes as potential targets for selective drug design.

    PubMed

    Magni, G; Di Stefano, M; Orsomando, G; Raffaelli, N; Ruggieri, S

    2009-01-01

    NAD(P) biosynthetic pathways can be considered a generous source of enzymatic targets for drug development. Key reactions for NAD(P) biosynthesis in all organisms, common to both de novo and salvage routes, are catalyzed by NMN/NaMN adenylyltransferase (NMNAT), NAD synthetase (NADS), and NAD kinase (NADK). These reactions represent a three-step pathway, present in the vast majority of living organisms, which is responsible for the generation of both NAD and NADP cellular pools. The validation of these enzymes as drug targets is based on their essentiality and conservation among a large variety of pathogenic microorganisms, as well as on their differential structural features or their differential metabolic contribution to NAD(P) homeostasis between microbial and human cell types. This review describes the structural and functional properties of eubacterial and human enzymes endowed with NMNAT, NADS, and NADK activities, as well as with nicotinamide phosphoribosyltransferase (NamPRT) and nicotinamide riboside kinase (NRK) activities, highlighting the species-related differences, with emphasis on their relevance for drug design. In addition, since the overall NMNAT activity in humans is accounted by multiple isozymes differentially involved in the metabolic activation of antineoplastic compounds, their individual diagnostic value for early therapy optimization is outlined. The involvement of human NMNAT in neurodegenerative disorders and its role in neuroprotection is also discussed. PMID:19355893

  9. Exogenous NAD+ administration significantly protects against myocardial ischemia/reperfusion injury in rat model

    PubMed Central

    Zhang, Youjun; Wang, Ban; Fu, Xingli; Guan, Shaofeng; Han, Wenzheng; Zhang, Jie; Gan, Qian; Fang, Weiyi; Ying, Weihai; Qu, Xinkai

    2016-01-01

    Acute myocardial infarction is one of the leading causes for death around the world. Although essential for successful interventional therapy, it is inevitably complicated by reperfusion injury. Thus effective approaches to reduce ischemia/reperfusion (I/R) injury are still critically needed. To test our hypothesis that intravenous administration of NAD+ can attenuate I/R injury by reducing apoptotic damage and enhancing antioxidant capacity, we used a rat mode of myocardial I/R. Our study found that administration of 10-20 mg/kg NAD+ can dose dependently reduce myocardial infarct induced by I/R, with an approximately 85% reduction of the infarct at the dosage of 20 mg/kg NAD+. We further found that the injection of NAD+ can significantly decrease I/R-induced apoptotic damage in the heart: NAD+ administration can both decrease the TUNEL signals, Bax, cleaved caspase-3 levels and increase the Bcl-XL levels in the rats that are subjected to myocardial I/R injury. NAD+ administration can also significantly attenuate I/R-induced decreases in SOD activity and SOD-2 protein levels in the hearts. NAD+ can profoundly decrease myocardial I/R injury at least partially by attenuating apoptotic damage and enhancing the antioxidant capacity, thus suggesting that NAD+ may become a promising therapeutic agent for myocardial I/R injury. PMID:27648125

  10. Exogenous NAD+ administration significantly protects against myocardial ischemia/reperfusion injury in rat model

    PubMed Central

    Zhang, Youjun; Wang, Ban; Fu, Xingli; Guan, Shaofeng; Han, Wenzheng; Zhang, Jie; Gan, Qian; Fang, Weiyi; Ying, Weihai; Qu, Xinkai

    2016-01-01

    Acute myocardial infarction is one of the leading causes for death around the world. Although essential for successful interventional therapy, it is inevitably complicated by reperfusion injury. Thus effective approaches to reduce ischemia/reperfusion (I/R) injury are still critically needed. To test our hypothesis that intravenous administration of NAD+ can attenuate I/R injury by reducing apoptotic damage and enhancing antioxidant capacity, we used a rat mode of myocardial I/R. Our study found that administration of 10-20 mg/kg NAD+ can dose dependently reduce myocardial infarct induced by I/R, with an approximately 85% reduction of the infarct at the dosage of 20 mg/kg NAD+. We further found that the injection of NAD+ can significantly decrease I/R-induced apoptotic damage in the heart: NAD+ administration can both decrease the TUNEL signals, Bax, cleaved caspase-3 levels and increase the Bcl-XL levels in the rats that are subjected to myocardial I/R injury. NAD+ administration can also significantly attenuate I/R-induced decreases in SOD activity and SOD-2 protein levels in the hearts. NAD+ can profoundly decrease myocardial I/R injury at least partially by attenuating apoptotic damage and enhancing the antioxidant capacity, thus suggesting that NAD+ may become a promising therapeutic agent for myocardial I/R injury.

  11. Activity of select dehydrogenases with sepharose-immobilized N(6)-carboxymethyl-NAD.

    PubMed

    Beauchamp, Justin; Vieille, Claire

    2015-01-01

    N(6)-carboxymethyl-NAD (N(6)-CM-NAD) can be used to immobilize NAD onto a substrate containing terminal primary amines. We previously immobilized N(6)-CM-NAD onto sepharose beads and showed that Thermotoga maritima glycerol dehydrogenase could use the immobilized cofactor with cofactor recycling. We now show that Saccharomyces cerevisiae alcohol dehydrogenase, rabbit muscle L-lactate dehydrogenase (type XI), bovine liver L-glutamic dehydrogenase (type III), Leuconostoc mesenteroides glucose-6-phosphate dehydro-genase, and Thermotoga maritima mannitol dehydrogenase are active with soluble N(6)-CM-NAD. The products of all enzymes but 6-phospho-D-glucono-1,5-lactone were formed when sepharose-immobilized N(6)-CM-NAD was recycled by T. maritima glycerol dehydrogenase, indicating that N(6)-immobilized NAD is suitable for use by a variety of different dehydrogenases. Observations of the enzyme active sites suggest that steric hindrance plays a greater role in limiting or allowing activity with the modified cofactor than do polarity and charge of the residues surrounding the N(6)-amine group on NAD.

  12. Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

    PubMed

    Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

    2015-11-15

    The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes.

  13. Consideration of diurnal variations in human blood NAD and NADP concentrations.

    PubMed

    Fukuwatari, Tsutomu; Shibata, Katsumi

    2009-06-01

    The sum of the urinary excretion of nicotinamide and its catabolites, which are metabolites of NAD and NADP, were observed to have clear diurnal variations in human urine. Then, we examined whether NAD and NADP in blood also showed the diurnal variation. All subjects were housed in the same facility and given the same diet during the experiment. In addition, we examined whether diurnal variations were affected by the intakes of dietary nicotinamide or not. As a result, neither the NAD nor the NADP content of the blood shows the diurnal variation regardless of the administered amount of nicotinamide. The concentrations of NAD and NADP did not increase according to the intake of nicotinamide. The existence of a mechanism by which NAD and the NADP levels of the blood are constantly maintained by the adjustment of the amount of excretion to the urinary bladder, was suggested.

  14. NADS: A Web Applet for Manipulation and Graphical Viewing of Nuclear Data

    SciTech Connect

    McKinley, M S; Beck, B R; McNabb, D P

    2004-11-30

    We have developed a program called NADS (Nuclear and Atomic Data System) which provides a web-based, user-friendly interface for viewing nuclear data. NADS uses a client/server model. The client is a Java applet that runs in a web browser. The server is a Python code that delivers pointwise data to the applet per user request and then plots the data. The data is also stored in tables for viewing and modifying. NADS can display 2-D, 3-D and 4-D (time sliced) data in a powerful, user-friendly environment. Currently, evaluated nuclear data are available from ENDF/B-V, ENDF/B-VI, JENDL, JEF and Lawrence Livermore National Laboratory's ENDL databases. LLNL's ENDL database has data for neutron, gamma and charged particles as projectiles. In addition to displaying and saving data, NADS has the capability to perform computations with the data. NADS is accessible over the Internet at http://nuclear.llnl.gov/.

  15. Hepatic NAD salvage pathway is enhanced in mice on a high-fat diet.

    PubMed

    Penke, Melanie; Larsen, Per S; Schuster, Susanne; Dall, Morten; Jensen, Benjamin A H; Gorski, Theresa; Meusel, Andrej; Richter, Sandy; Vienberg, Sara G; Treebak, Jonas T; Kiess, Wieland; Garten, Antje

    2015-09-01

    Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme for NAD salvage and the abundance of Nampt has been shown to be altered in non-alcoholic fatty liver disease. It is, however, unknown how hepatic Nampt is regulated in response to accumulation of lipids in the liver of mice fed a high-fat diet (HFD). HFD mice gained more weight, stored more hepatic lipids and had an impaired glucose tolerance compared with control mice. NAD levels as well as Nampt mRNA expression, protein abundance and activity were significantly increased in HFD mice. Enhanced NAD levels were associated with deacetylation of p53 and Nfκb indicating increased activation of Sirt1. Despite impaired glucose tolerance and increased hepatic lipid levels in HFD mice, NAD metabolism was significantly enhanced. Thus, improved NAD metabolism may be a compensatory mechanism to protect against negative impact of hepatic lipid accumulation.

  16. Solid acid-catalyzed depolymerization of barley straw driven by ball milling.

    PubMed

    Schneider, Laura; Haverinen, Jasmiina; Jaakkola, Mari; Lassi, Ulla

    2016-04-01

    This study describes a time and energy saving, solvent-free procedure for the conversion of lignocellulosic barley straw into reducing sugars by mechanocatalytical pretreatment. The catalytic conversion efficiency of several solid acids was tested which revealed oxalic acid dihydrate as a potential catalyst with high conversion rate. Samples were mechanically treated by ball milling and subsequently hydrolyzed at different temperatures. The parameters of the mechanical treatment were optimized in order to obtain sufficient amount of total reducing sugar (TRS) which was determined following the DNS assay. Additionally, capillary electrophoresis (CE) and Fourier transform infrared spectrometry (FT-IR) were carried out. Under optimal conditions TRS 42% was released using oxalic acid dihydrate as a catalyst. This study revealed that the acid strength plays an important role in the depolymerization of barley straw and in addition, showed, that the oxalic acid-catalyzed reaction generates low level of the degradation product 5-hydroxymethylfurfural (HMF). PMID:26859328

  17. Deletion or overexpression of mitochondrial NAD+ carriers in Saccharomyces cerevisiae alters cellular NAD and ATP contents and affects mitochondrial metabolism and the rate of glycolysis.

    PubMed

    Agrimi, Gennaro; Brambilla, Luca; Frascotti, Gianni; Pisano, Isabella; Porro, Danilo; Vai, Marina; Palmieri, Luigi

    2011-04-01

    The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.

  18. Transmission electron microscopic identification of silicon-containing particles in synovial fluid: potential confusion with calcium pyrophosphate dihydrate and apatite crystals.

    PubMed Central

    Bardin, T; Schumacher, H R; Lansaman, J; Rothfuss, S; Dryll, A

    1984-01-01

    Silicon-containing particles were identified by transmission electron microscopy (TEM) in thin sections of two synovial fluids, which also contained calcium pyrophosphate dihydrate (CPPD) crystals, aspirated during acute attacks of pseudogout. Such particles, which are interpreted as probably being artefacts from glassware, were electron dense and similar in appearance to some CPPD or hydroxyapatite crystals. Images PMID:6476921

  19. Effect of NADH on hypoxanthine hydroxylation by native NAD+-dependent xanthine oxidoreductase of rat liver, and the possible biological role of this effect.

    PubMed Central

    Kamiński, Z W; Jezewska, M M

    1981-01-01

    The course of the reaction sequence hypoxanthine leads to xanthine leads to uric acid, catalysed by the NAD+-dependent activity of xanthine oxidoreductase, was investigated under conditions either of immediate oxidation of the NADH formed or of NADH accumulation. The enzymic preparation was obtained from rat liver, and purified 75-fold (as compared with the 25000 g supernatant) on a 5'-AMP-Sepharose 4B column; in this preparation the NAD+-dependent activity accounted for 100% of total xanthine oxidoreductase activity. A spectrophotometric method was developed for continuous measurements of changes in the concentrations of the three purines involved. The time course as well as the effects of the concentrations of enzyme and of hypoxanthine were examined. NADH produced by the enzyme lowered its activity by 50%, resulting in xanthine accumulation and in decreases of uric acid formation and of hypoxanthine utilization. The inhibition of the Xanthine oxidoreductase NAD+-dependent activity by NADH is discussed as a possible factor in the regulation of IMP biosynthesis by the 'de novo' pathway or (from unchanged hypoxanthine) by ther salvage pathway. PMID:6952874

  20. Neuroprotective effect of ethyl pyruvate against Zn(2+) toxicity via NAD replenishment and direct Zn(2+) chelation.

    PubMed

    Kim, Seung-Woo; Lee, Hye-Kyung; Kim, Hyun-Ji; Yoon, Sung-Hwa; Lee, Ja-Kyeong

    2016-06-01

    Ethyl pyruvate (EP) is a simple aliphatic ester of pyruvic acid and has been shown to have robust protective effect in various pathological conditions. A variety of mechanisms have been reported to underlie the protective effects of EP, which include anti-inflammatory, anti-oxidative, and anti-apoptotic functions. Recently, we reported that EP suppressed high mobility group box 1 (HMGB1) release from primary microglial cells via direct Ca(2+) chelation. In the present study, we investigated whether and how EP chelates Zn(2+) in neurons when it is present at toxic levels. In cortical neurons treated with 40 μM of Zn(2+) for 24 h, both EP and pyruvate significantly suppressed neuronal cell death, although the potency of pyruvate was greater than that of EP, and that NAD replenishment contributed to the neuroprotective effects of both pyruvate and EP. However, when cortical neurons were exposed to acute treatment of Zn(2+) (400 μM, 15 min), EP, but not pyruvate, significantly suppressed neuronal death, despite the fact that NAD replenishment by EP was weaker than that by pyruvate. Spectrophotometric studies revealed that EP directly chelates Zn(2+), and this was confirmed in physiological contexts, such as, NMDA-treated primary cortical cultures and OGD-subjected hippocampal slice cultures, in which EP suppressed intracellular Zn(2+) elevation and neuronal cell death. In addition, EP markedly reduced the expressions of PARP-1 and of the NADPH oxidase subunit in Zn(2+)-treated primary cortical neurons, well known Zn(2+)-induced downstream processes. Together, these results show EP suppresses Zn(2+) induced neurotoxicity via dual functions, chelating Zn(2+) and promoting NAD replenishment.

  1. Molecular characterization of NAD+-dependent DNA ligase from Wolbachia endosymbiont of lymphatic filarial parasite Brugia malayi.

    PubMed

    Shrivastava, Nidhi; Nag, Jeetendra Kumar; Misra-Bhattacharya, Shailja

    2012-01-01

    The lymphatic filarial parasite, Brugia malayi contains Wolbachia endobacteria that are essential for development, viability and fertility of the parasite. Therefore, wolbachial proteins have been currently seen as the potential antifilarial drug targets. NAD(+)-dependent DNA ligase is characterized as a promising drug target in several organisms due to its crucial, indispensable role in DNA replication, recombination and DNA repair. We report here the cloning, expression and purification of NAD(+)-dependent DNA ligase of Wolbachia endosymbiont of B. malayi (wBm-LigA) for its molecular characterization. wBm-LigA has all the domains that are present in nearly all the eubacterial NAD(+)-dependent DNA ligases such as N-terminal adenylation domain, OB fold, helix-hairpin-helix (HhH) and BRCT domain except zinc-binding tetracysteine domain. The purified recombinant protein (683-amino acid) was found to be biochemically active and was present in its native form as revealed by the circular dichroism and fluorescence spectra. The purified recombinant enzyme was able to catalyze intramolecular strand joining on a nicked DNA as well as intermolecular joining of the cohesive ends of BstEII restricted lamda DNA in an in vitro assay. The enzyme was localized in the various life-stages of B. malayi parasites by immunoblotting and high enzyme expression was observed in Wolbachia within B. malayi microfilariae and female adult parasites along the hypodermal chords and in the gravid portion as evident by the confocal microscopy. Ours is the first report on this enzyme of Wolbachia and these findings would assist in validating the antifilarial drug target potential of wBm-LigA in future studies. PMID:22815933

  2. NAMPT-Mediated Salvage Synthesis of NAD+ Controls Morphofunctional Changes of Macrophages

    PubMed Central

    Venter, Gerda; Oerlemans, Frank T. J. J.; Willemse, Marieke; Wijers, Mietske; Fransen, Jack A. M.; Wieringa, Bé

    2014-01-01

    Functional morphodynamic behavior of differentiated macrophages is strongly controlled by actin cytoskeleton rearrangements, a process in which also metabolic cofactors ATP and NAD(H) (i.e. NAD+ and NADH) and NADP(H) (i.e. NADP+ and NADPH) play an essential role. Whereas the link to intracellular ATP availability has been studied extensively, much less is known about the relationship between actin cytoskeleton dynamics and intracellular redox state and NAD+-supply. Here, we focus on the role of nicotinamide phosphoribosyltransferase (NAMPT), found in extracellular form as a cytokine and growth factor, and in intracellular form as one of the key enzymes for the production of NAD+ in macrophages. Inhibition of NAD+ salvage synthesis by the NAMPT-specific drug FK866 caused a decrease in cytosolic NAD+ levels in RAW 264.7 and Maf-DKO macrophages and led to significant downregulation of the glycolytic flux without directly affecting cell viability, proliferation, ATP production capacity or mitochondrial respiratory activity. Concomitant with these differential metabolic changes, the capacity for phagocytic ingestion of particles and also substrate adhesion of macrophages were altered. Depletion of cytoplasmic NAD+ induced cell-morphological changes and impaired early adhesion in phagocytosis of zymosan particles as well as spreading performance. Restoration of NAD+ levels by NAD+, NMN, or NADP+ supplementation reversed the inhibitory effects of FK866. We conclude that direct coupling to local, actin-based, cytoskeletal dynamics is an important aspect of NAD+’s cytosolic role in the regulation of morphofunctional characteristics of macrophages. PMID:24824795

  3. NTP toxicity studies of sodium dichromate dihydrate (CAS No. 7789-12-0) administered in drinking water to male and female F344/N rats and B6C3F1 mice and male BALB/c and am3-C57BL/6 mice.

    PubMed

    Bucher, John R

    2007-01-01

    mg/L or greater and male and female mice exposed to 125 mg/L or greater was generally less than that by the control groups, and decreases in urine volume and increases in urine specific gravity in rats were related to reduced water consumption. Exposure to sodium dichromate dihydrate caused a microcytic hypochromic anemia in rats and mice, but the severity was less in mice. Serum cholesterol and triglyceride concentrations were decreased in rats. Increased bile acid concentrations in exposed groups of rats may have been due to altered hepatic function. The incidences of histiocytic cellular infiltration were generally significantly increased in the duodenum of rats and mice, the liver of female rats, and the mesenteric lymph node of mice exposed to 125 mg/L or greater. Significantly increased nonneoplastic lesions (focal ulceration, regenerative epithelial hyperplasia, and squamous epithelial metaplasia) occurred in the glandular stomach of male and female rats exposed to 1,000 mg/L. Incidences of epithelial hyperplasia of the duodenum were significantly increased in all exposed groups of mice. In study 2, sodium dichromate dihydrate was administered in drinking water to groups of 10 male B6C3F1, 10 male BALB/c, and five male am3-C57BL/6 mice for 3 months at exposure concentrations of 0, 62.5, 125, or 250 mg/L (equivalent to average daily doses of approximately 8, 15, or 25 mg/kg sodium dichromate dihydrate or 2.8, 5.2, or 8.7 mg/kg chromium to B6C3F1, BALB/c, and am3-C57BL/6 mice). All mice in study 2 survived until study termination. Mean body weights of 125 and 250 mg/L B6C3F1 and BALB/c mice and all exposed groups of am3-C57BL/6 mice were less than those of the control groups. Mice exposed to 250 mg/L consumed less water than the control groups. Exposure concentration-related decreases in mean red cell volumes and mean red cell hemoglobin values were observed in all three mouse strains. Erythrocyte counts were increased in exposed B6C3F1 and BALB/c mice but not

  4. NTP toxicity studies of sodium dichromate dihydrate (CAS No. 7789-12-0) administered in drinking water to male and female F344/N rats and B6C3F1 mice and male BALB/c and am3-C57BL/6 mice.

    PubMed

    Bucher, John R

    2007-01-01

    mg/L or greater and male and female mice exposed to 125 mg/L or greater was generally less than that by the control groups, and decreases in urine volume and increases in urine specific gravity in rats were related to reduced water consumption. Exposure to sodium dichromate dihydrate caused a microcytic hypochromic anemia in rats and mice, but the severity was less in mice. Serum cholesterol and triglyceride concentrations were decreased in rats. Increased bile acid concentrations in exposed groups of rats may have been due to altered hepatic function. The incidences of histiocytic cellular infiltration were generally significantly increased in the duodenum of rats and mice, the liver of female rats, and the mesenteric lymph node of mice exposed to 125 mg/L or greater. Significantly increased nonneoplastic lesions (focal ulceration, regenerative epithelial hyperplasia, and squamous epithelial metaplasia) occurred in the glandular stomach of male and female rats exposed to 1,000 mg/L. Incidences of epithelial hyperplasia of the duodenum were significantly increased in all exposed groups of mice. In study 2, sodium dichromate dihydrate was administered in drinking water to groups of 10 male B6C3F1, 10 male BALB/c, and five male am3-C57BL/6 mice for 3 months at exposure concentrations of 0, 62.5, 125, or 250 mg/L (equivalent to average daily doses of approximately 8, 15, or 25 mg/kg sodium dichromate dihydrate or 2.8, 5.2, or 8.7 mg/kg chromium to B6C3F1, BALB/c, and am3-C57BL/6 mice). All mice in study 2 survived until study termination. Mean body weights of 125 and 250 mg/L B6C3F1 and BALB/c mice and all exposed groups of am3-C57BL/6 mice were less than those of the control groups. Mice exposed to 250 mg/L consumed less water than the control groups. Exposure concentration-related decreases in mean red cell volumes and mean red cell hemoglobin values were observed in all three mouse strains. Erythrocyte counts were increased in exposed B6C3F1 and BALB/c mice but not

  5. Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD(+) , NADPH, or NAD(+) /10-Oxogeranial: Reaction Mechanisms.

    PubMed

    Hu, Yumei; Liu, Weidong; Malwal, Satish R; Zheng, Yingying; Feng, Xinxin; Ko, Tzu-Ping; Chen, Chun-Chi; Xu, Zhongxia; Liu, Meixia; Han, Xu; Gao, Jian; Oldfield, Eric; Guo, Rey-Ting

    2015-12-14

    Structures of the iridoid synthase nepetalactol synthase in the presence of NAD(+) , NADPH or NAD(+) /10-oxogeranial were solved. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form the byproduct S-10-oxo-citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1-C2 to form the cisoid isomer, leading to formation of the cis-enolate, together with rotation about C4-C5, which enables cyclization and lactol production. The structure is similar to that of progesterone-5β-reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10-oxogeranial structure also serves as a model for β-face hydride attack in progesterone 5β-reductases and is of general interest in the context of asymmetric synthesis. PMID:26768532

  6. Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7

    PubMed Central

    Chen, Hao; Babino, Darwin; Schoenbichler, Stefan A.; Arkhipova, Valeryia; Töchterle, Sonja; Martin, Fabian; Huck, Christian W.; von Lintig, Johannes; Meyer, Dirk

    2015-01-01

    Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation. PMID:26618989

  7. Upcoming replacements for NAD83, NAVD88 and IGLD85

    NASA Astrophysics Data System (ADS)

    Smith, D. A.; Snay, R.

    2009-05-01

    The National Geodetic Survey (NGS), part of the National Oceanic and Atmospheric Administration (NOAA) is responsible for defining, maintaining and providing access to the National Spatial Reference System (NSRS) for the United States. The NSRS is the official system to which all civil federal mapping agencies should refer, and contains, amongst other things, the official geopotential (historically "vertical") datum of NAVD 88, the 3-D geometric reference system (historically "horizontal datum") of NAD 83 and great lakes datum (IGLD 85). Although part of the United States NSRS, all three of these datums have been created through international partnerships across North America. Unfortunately, time has shown both the systematic errors existent within these datums, as well as the inherent weaknesses of relying exclusively on passive monuments to define and provide access to these datums. In recognition of these issues, the National Geodetic Survey has issued a "10 year plan", available online, which outlines steps which will be taken to update NAD 83, NAVD 88 and IGLD 85 concurrently around the year 2018. The primary source of success will be in the refinement of the CORS network and the upcoming execution of the GRAV-D project (Gravity for the Re-definition of the American Vertical Datum). Conversations are ongoing with colleagues in Canada, Mexico, Central America and the Caribbean in order to coordinate all of these efforts across the entire continent. The largest changes expected to occur are the removal of over 2 meters of non-geocentricity in NAD 83; the removal of decimeters of bias and over a meter of tilt in NAVD 88; the addition of the ability to track crustal motions (subsidence, tectonics, etc) in the datums; the removal of leveling as a tool for long-line height differencing; the use of a "best" geoid as the orthometric height reference surface; the addition of datum velocities (motions of the 3-D geometric reference system origin and motions of the geoid

  8. Efficient whole-cell biocatalyst for acetoin production with NAD+ regeneration system through homologous co-expression of 2,3-butanediol dehydrogenase and NADH oxidase in engineered Bacillus subtilis.

    PubMed

    Bao, Teng; Zhang, Xian; Rao, Zhiming; Zhao, Xiaojing; Zhang, Rongzhen; Yang, Taowei; Xu, Zhenghong; Yang, Shangtian

    2014-01-01

    Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD+ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD+ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD+ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD+ regeneration may also be a useful way for improving the productivity of NAD+-dependent chemistry-based products. PMID:25036158

  9. Efficient Whole-Cell Biocatalyst for Acetoin Production with NAD+ Regeneration System through Homologous Co-Expression of 2,3-Butanediol Dehydrogenase and NADH Oxidase in Engineered Bacillus subtilis

    PubMed Central

    Rao, Zhiming; Zhao, Xiaojing; Zhang, Rongzhen; Yang, Taowei; Xu, Zhenghong; Yang, Shangtian

    2014-01-01

    Acetoin (3-hydroxy-2-butanone), an extensively-used food spice and bio-based platform chemical, is usually produced by chemical synthesis methods. With increasingly requirement of food security and environmental protection, bio-fermentation of acetoin by microorganisms has a great promising market. However, through metabolic engineering strategies, the mixed acid-butanediol fermentation metabolizes a certain portion of substrate to the by-products of organic acids such as lactic acid and acetic acid, which causes energy cost and increases the difficulty of product purification in downstream processes. In this work, due to the high efficiency of enzymatic reaction and excellent selectivity, a strategy for efficiently converting 2,3-butandiol to acetoin using whole-cell biocatalyst by engineered Bacillus subtilis is proposed. In this process, NAD+ plays a significant role on 2,3-butanediol and acetoin distribution, so the NADH oxidase and 2,3-butanediol dehydrogenase both from B. subtilis are co-expressed in B. subtilis 168 to construct an NAD+ regeneration system, which forces dramatic decrease of the intracellular NADH concentration (1.6 fold) and NADH/NAD+ ratio (2.2 fold). By optimization of the enzymatic reaction and applying repeated batch conversion, the whole-cell biocatalyst efficiently produced 91.8 g/L acetoin with a productivity of 2.30 g/(L·h), which was the highest record ever reported by biocatalysis. This work indicated that manipulation of the intracellular cofactor levels was more effective than the strategy of enhancing enzyme activity, and the bioprocess for NAD+ regeneration may also be a useful way for improving the productivity of NAD+-dependent chemistry-based products. PMID:25036158

  10. N-arylazido-. beta. -alanyl-NAD sup + , a new NAD sup + photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase

    SciTech Connect

    Deng, P.S.K.; Chen, S. ); Hatefi, Y. )

    1990-01-30

    N-Arylaziod-{beta}-alanyl-NAD{sup +}(N3{prime}-0-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)NAD{sup +}) has been prepared by alkaline phosphatase treatment of arylaziod-{beta}-alanyl-NADP{sup +} (N3{prime}-O-(3-(N-(4-azido-2-nitrophenyl)amino)propionyl)NADP{sup +}). This NAD{sup +} analogue was found to be a potent competitive inhibitor with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase. The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-{beta}-alanyl-NAD{sup +} bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the M{sub r} = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylaziod-{beta}-alanyl-NAD{sup +} is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the M{sub r} = 51,000 subunit contain the NADH binding site. Results are also presented to show that N-arylazido-{beta}-alanyl-NAD{sup +} binds the dehydrogenase in a more effective manner than A-arylazido-{beta}-alanyl-NAD{sup +}.

  11. Monocyte-derived extracellular Nampt-dependent biosynthesis of NAD+ protects the heart against pressure overload

    PubMed Central

    Yano, Masamichi; Akazawa, Hiroshi; Oka, Toru; Yabumoto, Chizuru; Kudo-Sakamoto, Yoko; Kamo, Takehiro; Shimizu, Yu; Yagi, Hiroki; Naito, Atsuhiko T.; Lee, Jong-Kook; Suzuki, Jun-ichi; Sakata, Yasushi; Komuro, Issei

    2015-01-01

    Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step in the salvage pathway for nicotinamide adenine dinucleotide (NAD+) biosynthesis, and thereby regulates the deacetylase activity of sirtuins. Here we show accommodative regulation of myocardial NAD+ by monocyte-derived extracellular Nampt (eNampt), which is essential for hemodynamic compensation to pressure overload. Although intracellular Nampt (iNampt) expression was decreased in pressure-overloaded hearts, myocardial NAD+ concentration and Sirt1 activity were preserved. In contrast, iNampt was up-regulated in spleen and monocytes, and circulating eNampt protein and nicotinamide mononucleotide (NMN), a key precursor of NAD+, were significantly increased. Pharmacological inhibition of Nampt by FK866 or depletion of monocytes/macrophages by clodronate liposomes disrupted the homeostatic mechanism of myocardial NAD+ levels and NAD+-dependent Sirt1 activity, leading to susceptibility to cardiomyocyte apoptosis and cardiac decompensation in pressure-overloaded mice. These biochemical and hemodynamic defects were prevented by systemic administration of NMN. Our studies uncover a crucial role of monocyte-derived eNampt in myocardial adaptation to pressure overload, and highlight a potential intervention controlling myocardial NAD+ against heart failure. PMID:26522369

  12. Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity*

    PubMed Central

    Lay, Fung T.; Mills, Grant D.; Poon, Ivan K. H.; Cowieson, Nathan P.; Kirby, Nigel; Baxter, Amy A.; van der Weerden, Nicole L.; Dogovski, Con; Perugini, Matthew A.; Anderson, Marilyn A.; Kvansakul, Marc; Hulett, Mark D.

    2012-01-01

    The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys4 as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys4 that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys4 mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens. PMID:22511788

  13. Human monocytes/macrophages are a target of Neisseria meningitidis Adhesin A (NadA).

    PubMed

    Franzoso, Susanna; Mazzon, Cristina; Sztukowska, Maryta; Cecchini, Paola; Kasic, Tihana; Capecchi, Barbara; Tavano, Regina; Papini, Emanuele

    2008-05-01

    Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA(Delta351-405), a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.

  14. NAD+ metabolism, a therapeutic target for age-related metabolic disease

    PubMed Central

    Auwerx, Johan

    2013-01-01

    Nicotinamide adenine dinucleotide (NAD) is a central metabolic cofactor by virtue of its redox capacity, and as such regulates a wealth of metabolic transformations. However, the identification of the longevity protein Sir2, the founding member of the sirtuin protein family, as being NAD+-dependent reignited interest in this metabolite. The sirtuins (SIRT1-7 in mammals) utilize NAD+ to deacetylate proteins in different subcellular compartments with a variety of functions, but with a strong convergence on optimizing mitochondrial function. Since cellular NAD+ levels are limiting for sirtuin activity, boosting its levels is a powerful means to activate sirtuins as a potential therapy for mitochondrial, often age-related, diseases. Indeed, supplying excess precursors, or blocking its utilization by PARP enzymes or CD38/CD157, boosts NAD+ levels, activates sirtuins and promotes healthy aging. Here, we discuss the current state of knowledge of NAD+ metabolism, primarily in relation to sirtuin function. We highlight how NAD+ levels change in diverse physiological conditions, and how this can be employed as a pharmacological strategy. PMID:23742622

  15. NAMPT-Mediated NAD(+) Biosynthesis in Adipocytes Regulates Adipose Tissue Function and Multi-organ Insulin Sensitivity in Mice.

    PubMed

    Stromsdorfer, Kelly L; Yamaguchi, Shintaro; Yoon, Myeong Jin; Moseley, Anna C; Franczyk, Michael P; Kelly, Shannon C; Qi, Nathan; Imai, Shin-Ichiro; Yoshino, Jun

    2016-08-16

    Obesity is associated with adipose tissue dysfunction and multi-organ insulin resistance. However, the mechanisms of such obesity-associated systemic metabolic complications are not clear. Here, we characterized mice with adipocyte-specific deletion of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting NAD(+) biosynthetic enzyme known to decrease in adipose tissue of obese and aged rodents and people. We found that adipocyte-specific Nampt knockout mice had severe insulin resistance in adipose tissue, liver, and skeletal muscle and adipose tissue dysfunction, manifested by increased plasma free fatty acid concentrations and decreased plasma concentrations of a major insulin-sensitizing adipokine, adiponectin. Loss of Nampt increased phosphorylation of CDK5 and PPARγ (serine-273) and decreased gene expression of obesity-linked phosphorylated PPARγ targets in adipose tissue. These deleterious alterations were normalized by administering rosiglitazone or a key NAD(+) intermediate, nicotinamide mononucleotide (NMN). Collectively, our results provide important mechanistic and therapeutic insights into obesity-associated systemic metabolic derangements, particularly multi-organ insulin resistance.

  16. Crystallization and preliminary crystallographic analysis of NAD{sup +}-preferring aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum

    SciTech Connect

    Yasutake, Yoshiaki; Nishiya, Yoshiaki; Tamura, Noriko; Tamura, Tomohiro

    2006-06-01

    NAD{sup +}-preferring aldohexose dehydrogenase from the thermoacidophilic archaeon T. acidophilum was crystallized using the hanging-drop vapour-diffusion technique and X-ray diffraction data were collected to a resolution of 2.8 Å. The aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a 28 kDa molecular-weight enzyme that catalyzes the oxidation of various aldohexoses, with a preference for NAD{sup +} rather than NADP{sup +} as a cofactor. The recombinant AldT was crystallized using the hanging-drop vapour-diffusion technique at 293 K under several acidic conditions with polyethylene glycol (PEG) and ammonium sulfate as precipitants. Optimization of the initial crystallizations conditions yielded single crystals in solution containing 0.1 M sodium acetate pH 4.6, 18%(w/v) PEG 4000, 0.2 M ammonium sulfate and 15%(v/v) glycerol. An X-ray diffraction data set was collected to a resolution of 2.8 Å.

  17. NAMPT-mediated NAD+ biosynthesis in adipocytes regulates adipose tissue function and multi-organ insulin sensitivity in mice

    PubMed Central

    Stromsdorfer, Kelly L.; Yamaguchi, Shintaro; Yoon, Myeong Jin; Moseley, Anna C.; Franczyk, Michael P.; Kelly, Shannon C.; Qi, Nathan; Imai, Shin-ichiro; Yoshino, Jun

    2016-01-01

    SUMMARY Obesity is associated with adipose tissue dysfunction and multi-organ insulin resistance. However, the mechanisms of such obesity-associated systemic metabolic complications are not clear. Here, we characterized mice with adipocyte-specific deletion of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting NAD+ biosynthetic enzyme known to decrease in adipose tissue of obese and aged rodents and people. We found that adipocyte-specific Nampt knockout mice had severe insulin resistance in adipose tissue, liver, and skeletal muscle, and adipose tissue dysfunction, manifested by increased plasma free fatty acids concentrations and decreased plasma concentrations of a major insulin-sensitizing adipokine, adiponectin. Loss of Nampt increased phosphorylation of CDK5 and PPARγ (serine-273) and decreased gene expression of obesity-linked phosphorylated PPARγ targets in adipose tissue. Remarkably, these deleterious alterations were normalized by administering rosiglitazone or a key NAD+ intermediate, nicotinamide mononucleotide (NMN). Collectively, our results provide important mechanistic and therapeutic insights into obesity-associated systemic metabolic derangements, particularly multi-organ insulin resistance. PMID:27498863

  18. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    PubMed

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.

  19. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    PubMed

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. PMID:25280628

  20. Male rats fed methyl- and folate-deficient diets with or without niacin develop hepatic carcinomas associated with decreased tissue NAD concentrations and altered poly(ADP-ribose) polymerase activity.

    PubMed

    Henning, S M; Swendseid, M E; Coulson, W F

    1997-01-01

    Folate is an essential cofactor in the generation of endogenous methionine, and there is evidence that folate deficiency exacerbates the effects of a diet low in choline and methionine, including alterations in poly(ADP-ribose) polymerase (PARP) activity, an enzyme associated with DNA replication and repair. Because PARP requires NAD as its substrate, we postulated that a deficiency of both folate and niacin would enhance the development of liver cancer in rats fed a diet deficient in methionine and choline. In two experiments, rats were fed choline- and folate-deficient, low methionine diets containing either 12 or 8% casein (12% MCFD, 8% MCFD) or 6% casein and 6% gelatin with niacin (MCFD) or without niacin (MCFND) and were compared with folate-supplemented controls. Liver NAD concentrations were lower in all methyl-deficient rats after 2-17 mo. At 17 mo, NAD concentrations in other tissues of rats fed these diets were also lower than in controls. Compared with control values, liver PARP activity was enhanced in rats fed the 12% MCFD diet but was lower in MCFND-fed rats following a further reduction in liver NAD concentration. These changes in PARP activity associated with lower NAD concentrations may slow DNA repair and enhance DNA damage. Only rats fed the MCFD and MCFND diets developed hepatocarcinomas after 12-17 mo. In Experiment 2, hepatocarcinomas were found in 100% of rats fed the MCFD and MCFND diets. These preliminary results indicate that folic acid deficiency enhances tumor development. Because tumors developed in 100% of the MCFD-fed rats and because tissue concentrations of NAD in these animals were also low, further studies are needed to clearly define the role of niacin in methyl-deficient rats.

  1. NAD+-Carrying Mesoporous Silica Nanoparticles Can Prevent Oxidative Stress-Induced Energy Failures of Both Rodent Astrocytes and PC12 Cells

    PubMed Central

    Chen, Heyu; Wang, Yao; Zhang, Jixi; Ma, Yingxin; Wang, Caixia; Zhou, Ying; Gu, Hongchen; Ying, Weihai

    2013-01-01

    Aim To test the hypothesis that NAD+-carrying mesoporous silica nanoparticles (M-MSNs@NAD+) can effectively deliver NAD+ into cells to produce cytoprotective effects. Methods & Materials NAD+ was incorporated into M-MSNs. Primary rat astrocyte cultures and PC12 cells were treated with H2O2, followed by post-treatment with M-MSNs@NAD+. After various durations of the post-treatment, intracellular NAD+ levels, intracellular ATP levels and lactate dehydrogenase (LDH) release were determined. Results & Discussion M-MSNs can be effectively loaded with NAD+. The M-MSNs@NAD+ can significantly attenuate H2O2-induced NAD+ and ATP decreases in both astrocyte cultures and PC12 cells. M-MSNs@NAD+ can also partially prevent the H2O2-induced LDH release from both astrocyte cultures and PC12 cells. In contrast, the NAD+ that is spontaneously released from the M-MSNs@NAD+ is insufficient to prevent the H2O2-induced damage. Conclusions Our study has suggested the first approach that can effectively deliver NAD+ into cells, which provides an important basis both for elucidating the roles of intracellular NAD+ in biological functions and for therapeutic applications of NAD+. Our study has also provided the first direct evidence demonstrating a key role of NAD+ depletion in oxidative stress-induced ATP decreases. PMID:24040179

  2. The MTCY428.08 Gene of Mycobacterium tuberculosis Codes for NAD+ Synthetase

    PubMed Central

    Cantoni, Rita; Branzoni, Manuela; Labò, Monica; Rizzi, Menico; Riccardi, Giovanna

    1998-01-01

    The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD+ synthetases. The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene of M. tuberculosis is the structural gene for NAD+ synthetase. PMID:9620974

  3. Growth, structural, thermal, dielectric, mechanical and optical characterization of 2, 3-Dimethoxy-10-oxostrychnidinium hydrogen oxalate dihydrate single crystal

    NASA Astrophysics Data System (ADS)

    Krishnan, P.; Gayathri, K.; Jayasakthi, M.; Gunasekaran, S.; Anbalagan, G.

    2013-11-01

    Single crystal of 2, 3-Dimethoxy-10-oxostrychnidinium hydrogen oxalate dihydrate has been grown by slow evaporation solution growth technique (SEST) using ethanol-water solution at room temperature. It crystallizes in the orthorhombic system with space group of P212121. The crystalline perfection of the grown single crystal has been examined by high resolution X-ray diffraction analysis (HRXRD). The optical absorption studies show that the crystal is transparent in the visible region with a lower cut-off wavelength of 342 nm and the optical energy band gap Eg is found to be 3.52 eV. The electrical properties have been assessed by dielectric measurement at different temperatures. Hardness values measured using Vickers hardness indenter show considerable anisotropy. Laser damage threshold study is also carried out for the grown crystal.

  4. Water in Crystalline Fibers of Dihydrate β-Chitin Results in Unexpected Absence of Intramolecular Hydrogen Bonding

    PubMed Central

    Sawada, Daisuke; Nishiyama, Yoshiharu; Langan, Paul; Forsyth, V. Trevor; Kimura, Satoshi; Wada, Masahisa

    2012-01-01

    The complete crystal structure (including hydrogen) of dihydrate β-chitin, a homopolymer of N-acetylglucosamine hydrate, was determined using high-resolution X-ray and neutron fiber diffraction data collected from bathophilous tubeworm Lamellibrachia satsuma. Two water molecules per N-acetylglucosamine residue are clearly localized in the structure and these participate in most of the hydrogen bonds. The conformation of the labile acetamide groups and hydroxymethyl groups are similar to those found in anhydrous β-chitin, but more relaxed. Unexpectedly, the intrachain O3-H…O5 hydrogen bond typically observed for crystalline β,1–4 glycans is absent, providing important insights into its relative importance and its relationship to solvation. PMID:22724007

  5. Development of an enhanced anticaries efficacy dual component dentifrice containing sodium fluoride and dicalcium phosphate dihydrate.

    PubMed

    Sullivan, R J; Masters, J; Cantore, R; Roberson, A; Petrou, I; Stranick, M; Goldman, H; Guggenheim, B; Gaffar, A

    2001-05-01

    A dual-chamber dentifrice, which contains sodium fluoride (NaF) in one component and dicalcium phosphate dihydrate (dical) in the other, has been developed. The dentifrice is packaged in a dual-chamber tube and is formulated to deliver 1100 ppm F. A series of studies consisting of in vitro fluoride uptake, in vivo calcium labeling, intraoral remineralization-demineralization, and animal caries studies were performed to support the improved anticaries efficacy of this product in comparison to a sodium fluoride/silica dentifrice (NaF/silica). An in vitro fluoride uptake study comparing 1100 ppm F NaF/dical dentifrice to 1100 ppm F NaF/silica showed that NaF/dical delivered significantly more fluoride than NaF/silica, 3.72 +/- 0.36 micrograms/cm2 versus 2.41 +/- 0.10 micrograms/cm2. A 6-day in vivo brushing study with a 44Ca labeled NaF/dical dentifrice showed that calcium from dical penetrated demineralized enamel and was present in plaque up to 18 hrs since the last brushing. An intra-oral remineralization-demineralization study was performed to evaluate NaF/dical's ability to promote remineralization in comparison to three silica-based dentifrices containing 0, 250, and 1100 ppm F as NaF. The percent mineral changes after treatment were +20.44 +/- 17.14 for NaF/dical, +9.27 +/- 19.53 for 1100 ppm NaF/silica, -1.43 +/- 20.57 for 250 ppm NaF/silica, and -12.36 +/- 32.76 for 0 ppm F/silica. A statistical analysis showed that the dual-chamber NaF/dical dentifrice was significantly more effective than the 1100 ppm NaF/silica dentifrice at promoting remineralization. A rat caries study was performed to evaluate NaF/dical ability to prevent caries in comparison to 1100 ppm F NaF/silica, 250 ppm F NaF/silica, silica, and dical dentifrices. The mean smooth surface caries scores were 1.6 +/- 2.8 for NaF/dical, 5.5 +/- 6.2 for 1100 ppm F NaF/silica, 10.6 +/- 6.2 for 250 ppm F NaF/silica, 13.7 +/- 4.7 for 0 ppm F/silica, and 9.5 +/- 7.8 0 ppm F/dical. A statistical analysis

  6. Three-dimensional hydrogen-bonded framework structures in flunarizinium nicotinate and flunarizinediium bis(4-toluenesulfonate) dihydrate.

    PubMed

    Kavitha, Channappa N; Yathirajan, Hemmige S; Kaur, Manpreet; Hosten, Eric C; Betz, Richard; Glidewell, Christopher

    2014-08-01

    The structures of two salts of flunarizine, namely 1-bis[(4-fluorophenyl)methyl]-4-[(2E)-3-phenylprop-2-en-1-yl]piperazine, C26H26F2N2, are reported. In flunarizinium nicotinate {systematic name: 4-bis[(4-fluorophenyl)methyl]-1-[(2E)-3-phenylprop-2-en-1-yl]piperazin-1-ium pyridine-3-carboxylate}, C26H27F2N2(+)·C6H4NO2(-), (I), the two ionic components are linked by a short charge-assisted N-H...O hydrogen bond. The ion pairs are linked into a three-dimensional framework structure by three independent C-H...O hydrogen bonds, augmented by C-H...π(arene) hydrogen bonds and an aromatic π-π stacking interaction. In flunarizinediium bis(4-toluenesulfonate) dihydrate {systematic name: 1-[bis(4-fluorophenyl)methyl]-4-[(2E)-3-phenylprop-2-en-1-yl]piperazine-1,4-diium bis(4-methylbenzenesulfonate) dihydrate}, C26H28F2N2(2+)·2C7H7O3S(-)·2H2O, (II), one of the anions is disordered over two sites with occupancies of 0.832 (6) and 0.168 (6). The five independent components are linked into ribbons by two independent N-H...O hydrogen bonds and four independent O-H...O hydrogen bonds, and these ribbons are linked to form a three-dimensional framework by two independent C-H...O hydrogen bonds, but C-H...π(arene) hydrogen bonds and aromatic π-π stacking interactions are absent from the structure of (II). Comparisons are made with some related structures.

  7. Structure of the Francisella tularensis enoyl-acyl carrier protein reductase (FabI) in complex with NAD[superscript +] and triclosan

    SciTech Connect

    Mehboob, Shahila; Truong, Kent; Santarsiero, Bernard D.; Johnson, Michael E.

    2010-11-19

    Enoyl-acyl carrier protein reductase (FabI) catalyzes the last rate-limiting step in the elongation cycle of the fatty-acid biosynthesis pathway and has been validated as a potential antimicrobial drug target in Francisella tularensis. The development of new antibiotic therapies is important both to combat potential drug-resistant bioweapons and to address the broader societal problem of increasing antibiotic resistance among many pathogenic bacteria. The crystal structure of FabI from F. tularensis (FtuFabI) in complex with the inhibitor triclosan and the cofactor NAD{sup +} has been solved to a resolution of 2.1 {angstrom}. Triclosan is known to effectively inhibit FabI from different organisms. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. Comparison of our structure with the previously determined FtuFabI structure (PDB code 2jjy) which is bound to only NAD{sup +} reveals the conformation of the substrate-binding loop, electron density for which was missing in the earlier structure, and demonstrates a shift in the conformation of the NAD{sup +} cofactor. This shift in the position of the phosphate groups allows more room in the active site for substrate or inhibitor to bind and be better accommodated. This information will be crucial for virtual screening studies to identify novel scaffolds for development into new active inhibitors.

  8. Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol.

    PubMed

    Sanai, Masaharu; Endo, Satoshi; Matsunaga, Toshiyuki; Ishikura, Shuhei; Tajima, Kazuo; El-Kabbani, Ossama; Hara, Akira

    2007-08-01

    Mammalian 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3alpha-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD(+)-dependent 3alpha-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3alpha-hydroxysteroids and bile acids using NAD(+) as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3alpha-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD(+)-dependent type of cytosolic 3alpha-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3alpha-HSD resulted in a switch in favor of NADP(+) specificity, suggesting their key roles in coenzyme specificity.

  9. Discovery of a Nitric Acid Trihydrate (NAT) PSC Belt Around Antarctica by MIPAS on Envisat

    NASA Astrophysics Data System (ADS)

    Hopfner, M.; Larsen, N.; Spang, R.; Luo, B.; Ma, J.; Svendsen, S. H.; Eckermann, S. D.; Massoli, P.; Cairo, F.; Snels, M.; di Donfrancesco, G.; Knudsen, B.; Biermann, U.; Stiller, G.; von Clarmann, T.; Fischer, H.

    2005-12-01

    Due to a globally changing climate with decreasing stratospheric temperatures, polar stratospheric cloud (PSC) occurrence might intensify in future. This could counteract the recovery of the ozone layer due to a decreasing stratospheric chlorine loading as consequence of the Montreal protocol and its amendments. To improve models of future ozone loss it is necessary to answer still open questions regarding nucleation processes of nitric acid containing solid cloud particles. The mid-infrared limb-emission sounder Michelson Interferometer for Passive Atmospheric Sounding (MIPAS) on Envisat is the first instrument able to monitor continuously the development of PSC appearance and composition with full coverage of the polar regions even during polar night. We have analyzed MIPAS observations during the Antarctic winter 2003 with respect to PSC composition. Coincident lidar observations from McMurdo were used for comparison. By application of new refractive index data we could prove that a distinct spectral signature at around 820~cm-1 as observed by MIPAS is due to a composition of β-NAT. This has been the first evidence for the existence of NAT PSCs on a large scale. We could not find any spectroscopic evidence for the presence of nitric acid dihydrate (NAD) from any MIPAS PSC observation. During the initial phase of PSC development over Antarctica the MIPAS measurements reveal the formation of a belt of NAT PSCs in mid-June 2003. By mesoscale microphysical simulations we have shown that this sudden onset of NAT was caused by heterogeneous nucleation on ice in the cooling phases of large-amplitude stratospheric mountain waves over the Antarctic Peninsula and the Ellsworth Mountains. This suggests a more significant role for mountain waves in Antarctic PSC formation than has heretofore been appreciated. MIPAS observations of PSCs in a period of three weeks before this event show no indication for the presence of NAT clouds, but are consistent with supercooled

  10. Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria.

    PubMed

    Doelle, H W

    1971-12-01

    Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.

  11. NAD Malic Enzyme and the Control of Carbohydrate Metabolism in Potato Tubers1

    PubMed Central

    Jenner, Helen L.; Winning, Brenda M.; Millar, A. Harvey; Tomlinson, Kim L.; Leaver, Christopher J.; Hill, Steven A.

    2001-01-01

    Potato (Solanum tuberosum) plants were transformed with a cDNA encoding the 59-kD subunit of the potato tuber NAD-dependent malic enzyme (NADME) in the antisense orientation. Measurements of the maximum catalytic activity of NADME in tubers revealed a range of reductions in the activity of this enzyme down to 40% of wild-type activity. There were no detrimental effects on plant growth or tuber yield. Biochemical analyses of developing tubers indicated that a reduction in NADME activity had no detectable effects on flux through the tricarboxylic acid cycle. However, there was an effect on glycolytic metabolism with significant increases in the concentration of 3-phosphoglycerate and phosphoenolpyruvate. These results suggest that alterations in the levels of intermediates toward the end of the glycolytic pathway may allow respiratory flux to continue at wild-type rates despite the reduction in NADME. There was also a statistically significant negative correlation between NADME activity and tuber starch content, with tubers containing reduced NADME having an increased starch content. The effect on plastid metabolism may result from the observed glycolytic perturbations. PMID:11457964

  12. NAD(P)H:quinone oxidoreductase 1 inducer activity of some novel anilinoquinazoline derivatives

    PubMed Central

    Ghorab, Mostafa M; Alsaid, Mansour S; Higgins, Maureen; Dinkova-Kostova, Albena T; Shahat, Abdelaaty A; Elghazawy, Nehal H; Arafa, Reem K

    2016-01-01

    The Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements pathway enables cells to survive oxidative stress conditions through regulating the expression of cytoprotective enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1). This work presents the design and synthesis of novel anilinoquinazoline derivatives (2–16a) and evaluation of their NQO1 inducer activity in murine cells. Molecular docking of the new compounds was performed to assess their ability to inhibit Keap1–Nrf2 protein–protein interaction through occupying the Keap1–Nrf2-binding domain, which leads to Nrf2 accumulation and enhanced gene expression of NQO1. Docking results showed that all compounds can potentially interact with Keap1; however, 1,5-dimethyl-2-phenyl-4-(2-phenylquinazolin-4-ylamino)-1,2-dihydropyrazol-3-one (9), the most potent inducer, showed the largest number of interactions with key amino acids in the binding pocket (Arg483, Tyr525, and Phe478) compared to the native ligand or any other compound in this series. PMID:27540279

  13. Two Very Precisely Instrumentally Documented Meteorite Falls: Zdar nad Sazavou and Stubenberg - Prediction and Reality

    NASA Astrophysics Data System (ADS)

    Spurny, P.; Borovicka, J.; Haloda, J.; Shrbeny, L.; Heinlein, D.

    2016-08-01

    Recently two bright bolides Zdar nad Sazavou (Czechia) and Stubenberg (Germany) were recorded by the cameras of the Czech Fireball Network. Basic data and comparison of the predicted impact areas and real positions of the meteorites will be presented.

  14. 78 FR 36571 - North American Datum of 1983 (NAD 83) Outer Continental Shelf (OCS) Provisional Official...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-18

    ... Bureau of Ocean Energy Management North American Datum of 1983 (NAD 83) Outer Continental Shelf (OCS) Provisional Official Protraction Diagram (OPDs) AGENCY: Bureau of Ocean Energy Management (BOEM), Interior... Ocean Energy Management. BILLING CODE 4310-MR-P...

  15. Stereospecificity of NAD+/NADH Reactions: A Project Experiment for Advanced Undergraduates.

    ERIC Educational Resources Information Center

    Lowrey, Jonathan S.; And Others

    1981-01-01

    Presents background information, materials needed, and experimental procedures to study enzymes dependent on pyridine nucleotide coenzymes (NAD/NADH). The experiments, suitable for advanced organic or biochemistry courses, require approximately 10-15 hours to complete. (SK)

  16. Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

    SciTech Connect

    Moonen, Harald J.J. . E-mail: h.moonen@grat.unimaas.nl; Geraets, Liesbeth; Vaarhorst, Anika; Bast, Aalt; Wouters, Emiel F.M.; Hageman, Geja J.

    2005-12-30

    Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD{sup +}, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD{sup +} pool, and of NAD{sup +}-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD{sup +} levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzyme inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.

  17. Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves.

    PubMed

    Ahmad-Sohdi, Nor-Ain-Shahajar; Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Hassan, Maizom

    2015-01-01

    Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control. PMID:26600471

  18. Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves

    PubMed Central

    Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura

    2015-01-01

    Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control. PMID:26600471

  19. Specific binding of nucleotides and NAD+ to Clostridium difficile toxin A.

    PubMed

    Lobban, M D; Borriello, S P

    1992-02-24

    Binding of nucleotides, a tetrapolyphosphate, and NAD+ to purified toxin A of Clostridium difficile was determined by monitoring changes in intrinsic fluorescence following excitation at 280 nm, and recording emissions at 340 nm. Binding was specific for concentrations over the range 5 to 100 microM for ATP, GTP, and their respective non-hydrolysable analogues AMP-PNP and Gpp(NH)p, tetrapolyphosphate and NAD+. PMID:1544441

  20. NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells

    NASA Astrophysics Data System (ADS)

    Yamada, Shigeru; Kotake, Yaichiro; Demizu, Yosuke; Kurihara, Masaaki; Sekino, Yuko; Kanda, Yasunari

    2014-08-01

    Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

  1. A regulatory role of NAD redox status on flavin cofactor homeostasis in S. cerevisiae mitochondria.

    PubMed

    Giancaspero, Teresa Anna; Locato, Vittoria; Barile, Maria

    2013-01-01

    Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two redox cofactors of pivotal importance for mitochondrial functionality and cellular redox balance. Despite their relevance, the mechanism by which intramitochondrial NAD(H) and FAD levels are maintained remains quite unclear in Saccharomyces cerevisiae. We investigated here the ability of isolated mitochondria to degrade externally added FAD and NAD (in both its reduced and oxidized forms). A set of kinetic experiments demonstrated that mitochondrial FAD and NAD(H) destroying enzymes are different from each other and from the already characterized NUDIX hydrolases. We studied here, in some detail, FAD pyrophosphatase (EC 3.6.1.18), which is inhibited by NAD(+) and NADH according to a noncompetitive inhibition, with Ki values that differ from each other by an order of magnitude. These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation in S. cerevisiae. PMID:24078860

  2. The simultaneous determination of NAD(H) and NADP(H) utilization by glutamate dehydrogenase.

    PubMed

    Treberg, Jason R; Brosnan, Margaret E; Brosnan, John T

    2010-11-01

    Glutamate dehydrogenase (GDH) from vertebrates is unusual among NAD(P)H-dependent dehydrogenases in that it can use either NAD(H) or NADP(H) as cofactor. In this study, we measure the rate of cofactor utilization by bovine GDH when both cofactors are present. Methods for both reaction directions were developed, and for the first time, to our knowledge, the GDH activity has been simultaneously studied in the presence of both NAD(H) and NADP(H). Our data indicate that NADP(H) has inhibitory effects on the rate of NAD(H) utilization by GDH, a characteristic of GDH not previously recognized. The response of GDH to allosteric activators in the presence of NAD(H) and NADP(H) suggests that ADP and leucine moderate much of the inhibitory effect of NADP(H) on the utilization of NAD(H). These results illustrate that simple assumptions of cofactor preference by mammalian GDH are incomplete without an appreciation of allosteric effects when both cofactors are simultaneously present.

  3. NAMPT-Mediated NAD(+) Biosynthesis Is Essential for Vision In Mice.

    PubMed

    Lin, Jonathan B; Kubota, Shunsuke; Ban, Norimitsu; Yoshida, Mitsukuni; Santeford, Andrea; Sene, Abdoulaye; Nakamura, Rei; Zapata, Nicole; Kubota, Miyuki; Tsubota, Kazuo; Yoshino, Jun; Imai, Shin-Ichiro; Apte, Rajendra S

    2016-09-27

    Photoreceptor death is the endpoint of many blinding diseases. Identifying unifying pathogenic mechanisms in these diseases may offer global approaches for facilitating photoreceptor survival. We found that rod or cone photoreceptor-specific deletion of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the major NAD(+) biosynthetic pathway beginning with nicotinamide, caused retinal degeneration. In both cases, we could rescue vision with nicotinamide mononucleotide (NMN). Significantly, retinal NAD(+) deficiency was an early feature of multiple mouse models of retinal dysfunction, including light-induced degeneration, streptozotocin-induced diabetic retinopathy, and age-associated dysfunction. Mechanistically, NAD(+) deficiency caused metabolic dysfunction and consequent photoreceptor death. We further demonstrate that the NAD(+)-dependent mitochondrial deacylases SIRT3 and SIRT5 play important roles in retinal homeostasis and that NAD(+) deficiency causes SIRT3 dysfunction. These findings demonstrate that NAD(+) biosynthesis is essential for vision, provide a foundation for future work to further clarify the mechanisms involved, and identify a unifying therapeutic target for diverse blinding diseases. PMID:27681422

  4. A Regulatory Role of NAD Redox Status on Flavin Cofactor Homeostasis in S. cerevisiae Mitochondria

    PubMed Central

    Giancaspero, Teresa Anna; Barile, Maria

    2013-01-01

    Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two redox cofactors of pivotal importance for mitochondrial functionality and cellular redox balance. Despite their relevance, the mechanism by which intramitochondrial NAD(H) and FAD levels are maintained remains quite unclear in Saccharomyces cerevisiae. We investigated here the ability of isolated mitochondria to degrade externally added FAD and NAD (in both its reduced and oxidized forms). A set of kinetic experiments demonstrated that mitochondrial FAD and NAD(H) destroying enzymes are different from each other and from the already characterized NUDIX hydrolases. We studied here, in some detail, FAD pyrophosphatase (EC 3.6.1.18), which is inhibited by NAD+ and NADH according to a noncompetitive inhibition, with Ki values that differ from each other by an order of magnitude. These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation in S. cerevisiae. PMID:24078860

  5. The UDP-glucose dehydrogenase of Escherichia coli K-12 displays substrate inhibition by NAD that is relieved by nucleotide triphosphates.

    PubMed

    Mainprize, Iain L; Bean, Jordan D; Bouwman, Catrien; Kimber, Matthew S; Whitfield, Chris

    2013-08-01

    UDP-glucose dehydrogenase (Ugd) generates UDP-glucuronic acid, an important precursor for the production of many hexuronic acid-containing bacterial surface glycostructures. In Escherichia coli K-12, Ugd is important for biosynthesis of the environmentally regulated exopolysaccharide known as colanic acid, whereas in other E. coli isolates, the same enzyme is required for production of the constitutive group 1 capsular polysaccharides, which act as virulence determinants. Recent studies have implicated tyrosine phosphorylation in the activation of Ugd from E. coli K-12, although it is not known if this is a feature shared by bacterial Ugd proteins. The activities of Ugd from E. coli K-12 and from the group 1 capsule prototype (serotype K30) were compared. Surprisingly, for both enzymes, site-directed Tyr → Phe mutants affecting the previously proposed phosphorylation site retained similar kinetic properties to the wild-type protein. Purified Ugd from E. coli K-12 had significant levels of NAD substrate inhibition, which could be alleviated by the addition of ATP and several other nucleotide triphosphates. Mutations in a previously identified UDP-glucuronic acid allosteric binding site decreased the binding affinity of the nucleotide triphosphate. Ugd from E. coli serotype K30 was not inhibited by NAD, but its activity still increased in the presence of ATP.

  6. Bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex a new apoptotic agent through Flk-1 down regulation, caspase-3 activation and oligonucleosomes DNA fragmentation.

    PubMed

    Azab, Hassan A; Hussein, Belal H M; El-Azab, Mona F; Gomaa, Mohamed; El-Falouji, Abdullah I

    2013-01-01

    New bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex was synthesized and characterized. In vivo anti-angiogenic activities of bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex against Ehrlich ascites carcinoma (EAC) cells are described. The newly synthesized complex resulted in inhibition of proliferation of EAC cells and ascites formation. The anti-tumor effect was found to be through anti-angiogenic activity as evident by the reduction of microvessel density in EAC solid tumors. The anti-angiogenic effect is mediated through down-regulation of VEGF receptor type-2 (Flk-1). The complex was also found to significantly increase the level of caspase-3 in laboratory animals compared to the acridine ligand and to the control group. This was also consistent with the DNA fragmentation detected by capillary electrophoresis that proved the apoptotic effect of the new complex. Our complex exhibited anti-angiogenic and apoptotic activity in vivo, a thing that makes it a potential effective chemotherapeutic agent. The interaction of calf thymus DNA (ct-DNA) with bis(acridine-9-carboxylate)-nitro-europium(III) dihydrate complex has been investigated using fluorescence technique. A competitive experiment of the europium(III)-acridine complex with ethidium bromide (EB) to bind DNA revealed that interaction between the europium(III)-acridine and DNA was via intercalation. The interaction of the synthesized complex with tyrosine kinases was also studied using molecular docking simulation to further substantiate its mode of action.

  7. NAD(P)H oxidase-dependent intracellular and extracellular O2·- production in coronary arterial myocytes from CD38 knockout mice

    PubMed Central

    Xu, Ming; Zhang, Yang; Xia, Min; Li, Xiao-Xue; Ritter, Joseph K; Zhang, Fan; Li, Pin-Lan

    2011-01-01

    Activation of NAD(P)H oxidase has been reported to produce superoxide (O2 ·-) extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. However, it remains unknown how the activity of NAD(P)H oxidase is regulated in arterial myocytes. Recently, CD38-associated ADP-ribosylcyclase has been reported to use NAD(P)H oxidase product, NAD+ or NADP+ to produce cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), which mediates intracellular Ca2+ signaling. The present study was designed to test a hypothesis that CD38/cADPR pathway as a downstream event exerts feedback regulatory action on the NAD(P)H oxidase activity in production of extra- or intracellular O2 ·-in mouse coronary arterial myocytes (CAMs). By fluorescent microscopic imaging, we simultaneously monitored extra- and intracellular O2 ·-production in wild-type (CD38+/+) and CD38 knockout (CD38-/-) CAMs in response to oxotremorine (OXO), a muscarinic type 1 (M1) receptor agonist. It was found that CD38 deficiency prevented OXO-induced intracellular but not extracellular O2 ·-production in CAMs. Consistently, the OXO-induced intracellular O2 ·-production was markedly inhibited by CD38 shRNA or CD38 inhibitor nicotinamide in CD38+/+ CAMs. Further, Nox4 siRNA inhibited OXO-induced intracellular but not extracellular O2 ·- production, whereas Nox1 siRNA attenuated both intracellular and extracellular O2 ·-production in CD38+/+ CAMs. Direct delivery of exogenous cADPR into CAMs markedly elevated intracellular Ca2+ concentration and restored intracellular O2 ·-production in CD38-/- CAMs. Functionally, CD38 deficiency or Nox1 siRNA and Nox4 siRNA prevented OXO-induced contraction in isolated perfused coronary arteries in CD38 WT mice. These results provide direct evidence that CD38/cADPR pathway importantly controls Nox4-mediated intracellular O2 ·-production and that CD38-dependent intracellular O2

  8. Metabolic Response to NAD Depletion across Cell Lines Is Highly Variable

    PubMed Central

    Xiao, Yang; Kwong, Mandy; Daemen, Anneleen; Belvin, Marcia; Liang, Xiaorong; Hatzivassiliou, Georgia

    2016-01-01

    Nicotinamide adenine dinucleotide (NAD) is a cofactor involved in a wide range of cellular metabolic processes and is a key metabolite required for tumor growth. NAMPT, nicotinamide phosphoribosyltransferase, which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN), the immediate precursor of NAD, is an attractive therapeutic target as inhibition of NAMPT reduces cellular NAD levels and inhibits tumor growth in vivo. However, there is limited understanding of the metabolic response to NAD depletion across cancer cell lines and whether all cell lines respond in a uniform manner. To explore this we selected two non-small cell lung carcinoma cell lines that are sensitive to the NAMPT inhibitor GNE-617 (A549, NCI-H1334), one that shows intermediate sensitivity (NCI-H441), and one that is insensitive (LC-KJ). Even though NAD was reduced in all cell lines there was surprising heterogeneity in their metabolic response. Both sensitive cell lines reduced glycolysis and levels of di- and tri-nucleotides and modestly increased oxidative phosphorylation, but they differed in their ability to combat oxidative stress. H1334 cells activated the stress kinase AMPK, whereas A549 cells were unable to activate AMPK as they contain a mutation in LKB1, which prevents activation of AMPK. However, A549 cells increased utilization of the Pentose Phosphate pathway (PPP) and had lower reactive oxygen species (ROS) levels than H1334 cells, indicating that A549 cells are better able to modulate an increase in oxidative stress. Inherent resistance of LC-KJ cells is associated with higher baseline levels of NADPH and a delayed reduction of NAD upon NAMPT inhibition. Our data reveals that cell lines show heterogeneous response to NAD depletion and that the underlying molecular and genetic framework in cells can influence the metabolic response to NAMPT inhibition. PMID:27711204

  9. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences.

    PubMed

    Ying, Weihai

    2008-02-01

    Accumulating evidence has suggested that NAD (including NAD+ and NADH) and NADP (including NADP+ and NADPH) could belong to the fundamental common mediators of various biological processes, including energy metabolism, mitochondrial functions, calcium homeostasis, antioxidation/generation of oxidative stress, gene expression, immunological functions, aging, and cell death: First, it is established that NAD mediates energy metabolism and mitochondrial functions; second, NADPH is a key component in cellular antioxidation systems; and NADH-dependent reactive oxygen species (ROS) generation from mitochondria and NADPH oxidase-dependent ROS generation are two critical mechanisms of ROS generation; third, cyclic ADP-ribose and several other molecules that are generated from NAD and NADP could mediate calcium homeostasis; fourth, NAD and NADP modulate multiple key factors in cell death, such as mitochondrial permeability transition, energy state, poly(ADP-ribose) polymerase-1, and apoptosis-inducing factor; and fifth, NAD and NADP profoundly affect aging-influencing factors such as oxidative stress and mitochondrial activities, and NAD-dependent sirtuins also mediate the aging process. Moreover, many recent studies have suggested novel paradigms of NAD and NADP metabolism. Future investigation into the metabolism and biological functions of NAD and NADP may expose fundamental properties of life, and suggest new strategies for treating diseases and slowing the aging process.

  10. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus

    PubMed Central

    Sharma, Onkar; O’Seaghdha, Maghnus; Velarde, Jorge J.; Wessels, Michael R.

    2016-01-01

    A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. PMID:26938870

  11. Structural Basis for NADH/NAD+ Redox Sensing by a Rex Family Repressor

    SciTech Connect

    McLaughlin, K.J.; Soares, A.; Strain-Damerell, C. M.; Xie, K.; Brekasis, D.; Pagent, M. S. B.; Kielkopf, C. L.

    2010-05-28

    Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD{sup +} is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD{sup +} ratio. Here we investigate the molecular mechanism for NADH/NAD{sup +} sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD{sup +}, (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40{sup o} closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.

  12. Insights into the Sirtuin Mechanism from Ternary Complexes Containing NAD[superscript +] and Acetylated Peptide

    SciTech Connect

    Hoff, Kevin G.; Avalos, Jose L.; Sens, Kristin; Wolberger, Cynthia

    2010-07-22

    Sirtuin proteins comprise a unique class of NAD{sup +}-dependent protein deacetylases. Although several structures of sirtuins have been determined, the mechanism by which NAD{sup +} cleavage occurs has remained unclear. We report the structures of ternary complexes containing NAD{sup +} and acetylated peptide bound to the bacterial sirtuin Sir2Tm and to a catalytic mutant (Sir2Tm{sup H116Y}). NAD{sup +} in these structures binds in a conformation different from that seen in previous structures, exposing the {alpha} face of the nicotinamide ribose to the carbonyl oxygen of the acetyl lysine substrate. The NAD{sup +} conformation is identical in both structures, suggesting that proper coenzyme orientation is not dependent on contacts with the catalytic histidine. We also present the structure of Sir2Tm{sup H116A} bound to deacteylated peptide and 3{prime}-O-acetyl ADP ribose. Taken together, these structures suggest a mechanism for nicotinamide cleavage in which an invariant phenylalanine plays a central role in promoting formation of the O-alkylamidate reaction intermediate and preventing nicotinamide exchange.

  13. Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody.

    PubMed

    Malito, Enrico; Biancucci, Marco; Faleri, Agnese; Ferlenghi, Ilaria; Scarselli, Maria; Maruggi, Giulietta; Lo Surdo, Paola; Veggi, Daniele; Liguori, Alessia; Santini, Laura; Bertoldi, Isabella; Petracca, Roberto; Marchi, Sara; Romagnoli, Giacomo; Cartocci, Elena; Vercellino, Irene; Savino, Silvana; Spraggon, Glen; Norais, Nathalie; Pizza, Mariagrazia; Rappuoli, Rino; Masignani, Vega; Bottomley, Matthew James

    2014-12-01

    Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.

  14. Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody

    PubMed Central

    Malito, Enrico; Biancucci, Marco; Faleri, Agnese; Ferlenghi, Ilaria; Scarselli, Maria; Maruggi, Giulietta; Lo Surdo, Paola; Veggi, Daniele; Liguori, Alessia; Santini, Laura; Bertoldi, Isabella; Petracca, Roberto; Marchi, Sara; Romagnoli, Giacomo; Cartocci, Elena; Vercellino, Irene; Savino, Silvana; Spraggon, Glen; Norais, Nathalie; Pizza, Mariagrazia; Rappuoli, Rino; Masignani, Vega; Bottomley, Matthew James

    2014-01-01

    Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5–15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA. PMID:25404323

  15. Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

    PubMed Central

    Sporty, Jennifer L.; Kabir, Md. Mohiuddin; Turteltaub, Kenneth W.; Ognibene, Ted; Lin, Su-Ju; Bench, Graham

    2009-01-01

    A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotin-amide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH3CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV–Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures. PMID:18763242

  16. N′-[(E)-(3-Fluoro­pyridin-2-yl)methyl­idene]pyridine-3-carbohydrazide dihydrate

    PubMed Central

    Nair, Yamuna; Sithambaresan, M.; Nair, S. Muraleedharan; Kurup, M. R. Prathapachandra

    2014-01-01

    The organic molecule in the title dihydrate, C12H9FN4O·2H2O, exists in the E conformation with respect to the azo­methane C=N double bond. The mol­ecule is approximately planar, with a maximum deviation of 0.117 (1) Å for the carbonyl O atom from the mean plane of the mol­ecule. Both pyridine rings are essentially coplanar with the central C(=O)N2C unit [dihedral angles = 1.99 (7) and 5.71 (8)°], exhibiting a significant difference in dihedral angles from its benzohydrazide analogue. The crystal packing features N—H⋯O, O—H⋯N and O—H⋯O hydrogen-bond inter­actions, which lead to the formation of a chain along the c-axis direction through one of the water mol­ecules present, and these chains are stacked one over the other by means of π–π inter­actions [with centroid–centroid distances of 3.7099 (10) and 3.6322 (10) Å] between the aromatic rings in neighbouring anti­parallel mol­ecules, building a three-dimensional supra­molecular network. PMID:24826177

  17. Crystal growth, structural, thermal and mechanical behavior of L-arginine 4-nitrophenolate 4-nitrophenol dihydrate (LAPP) single crystals

    NASA Astrophysics Data System (ADS)

    Mahadevan, M.; Ramachandran, K.; Anandan, P.; Arivanandhan, M.; Bhagavannarayana, G.; Hayakawa, Y.

    2014-12-01

    Single crystals of L-arginine 4-nitrophenolate 4-nitrophenol dihydrate (LAPP) have been grown successfully from the solution of L-arginine and 4-nitrophenol. Slow evaporation of solvent technique was adopted to grow the bulk single crystals. Single crystal X-ray diffraction analysis confirms the grown crystal has monoclinic crystal system with space group of P21. Powder X-ray diffraction analysis shows the good crystalline nature. The crystalline perfection of the grown single crystals was analyzed by HRXRD by employing a multicrystal X-ray diffractometer. The functional groups were identified from proton NMR spectroscopic analysis. Linear and nonlinear optical properties were determined by UV-Vis spectrophotometer and Kurtz powder technique respectively. It is found that the grown crystal has no absorption in the green wavelength region and the SHG efficiency was found to be 2.66 times that of the standard KDP. The Thermal stability of the crystal was found by obtaining TG/DTA curve. The mechanical behavior of the grown crystal has been studied by Vicker's microhardness method.

  18. Crystal growth and the nonlinear optical properties of 4-nitrophenol sodium salt dihydrate and its deuterated material

    NASA Astrophysics Data System (ADS)

    Minemoto, Hisashi; Ozaki, Yusuke; Sonoda, Nobuo; Sasaki, Takatomo

    1994-10-01

    Large organic ionic crystals on the order of a few centimeters of 4-nitrophenol sodium(:Na) salt dihydrate (NPNa) and DNPNa, in which the water of crystallization of NPNa was deuteraged, were grown for second-harmonic generation (SHG). The Vickers hardness and thermal conductivities of NPNa were about two times larger than those of conventional organic molecular crystals. The effective nonlinear optical constants of NPNa and DNPNa were estimated to be 5.0 and 5.5 pm/V, respectively. The optical loss coefficients, measured by spectrophotometry, of NPNa and DNPNa were 1.8 and 0.6 dB/cm at 1064 nm, respectively. The substitution of D2O for H2O in NPNa crystal is very effective in reducing the optical loss coefficient. High power of 4.4 mW green SH light was obtained using a 1.5-mm-thick DNPNa crystal as the intracavity SHG device of a diode-pumped Nd:YVO4 laser.

  19. Structure of 1-methyl-6-oxyquinolinium betaine dihydrate studied by X-ray diffraction, DFT calculations, vibrational and NMR spectra

    NASA Astrophysics Data System (ADS)

    Barczyński, P.; Ratajczak-Sitarz, M.; Katrusiak, A.; Szafran, M.

    2010-07-01

    The crystals of 1-methyl-6-oxyquinolinium betaine dihydrate, 6QB·2H 2O, are triclinic, space group P1¯. The oxygen atom of 6QB exhibits an extremely rare capability of accepting four hydrogen bonds. It is engaged in four hydrogen bonds to water molecules of the 2.823, 2.825, 2.833 and 2.849 Å; each water molecule interacts with two neighbouring 6QB molecules linking them into infinite sheets. Differences in geometrical parameters between the X-ray and calculated molecules reflect changes in their structures between zwitterion and quinonoid forms. The probable assignments of the experimental FTIR solid spectrum have been made on the basis of B3LYP/6-311G(d,p) calculated frequencies in vacuum. Both 1H and 13C chemical shifts are solvent dependent. Linear correlations between the experimental 1H and 13C NMR chemical shifts of 6QB·2H 2O in solutions and the GIAO/B3LYP/6-311G(d,p) calculated magnetic isotropic shielding tensors ( σcal) using the screening solvation model, δexp = a + bσcal, are reported.

  20. The effect of autoclaving on the physical and biological properties of dicalcium phosphate dihydrate bioceramics: brushite vs. monetite.

    PubMed

    Tamimi, Faleh; Le Nihouannen, Damien; Eimar, Hazem; Sheikh, Zeeshan; Komarova, Svetlana; Barralet, Jake

    2012-08-01

    Dicalcium phosphate dihydrate (brushite) is an osteoconductive biomaterial with great potential as a bioresorbable cement for bone regeneration. Preset brushite cement can be dehydrated into dicalcium phosphate anhydrous (monetite) bioceramics by autoclaving. This heat treatment results in changes in the physical characteristics of the material, improving in vivo bioresorption. This property is a great advantage in bone regeneration; however, it is not known how autoclaving brushite preset cement might improve its capacity to regenerate bone. This study was designed to compare brushite bioceramics with monetite bioceramics in terms of physical characteristics in vitro, and in vivo performance upon bone implantation. In this study we observed that monetite bioceramics prepared by autoclaving preset brushite cements had higher porosity, interconnected porosity and specific surface area than their brushite precursors. In vitro cell culture experiments revealed that bone marrow cells expressed higher levels of osteogenic genes Runx2, Opn, and Alp when the cells were cultured on monetite ceramics rather than on brushite ones. In vivo experiments revealed that monetite bioceramics resorbed faster than brushite ones and were more infiltrated with newly formed bone. In summary, autoclaving preset brushite cements results in a material with improved properties for bone regeneration procedures. PMID:22522010

  1. Growth and characterisation of crystals of a new organic complex of thiourea with quinine sulphate dihydrate: an NLO material.

    PubMed

    Nair, Lekshmi P; Bijini, B R; Prasanna, S; Nair, C M K; Deepa, M; Babu, K Rajendra

    2014-01-01

    An organic complex of thiourea and quinine sulphate dihydrate (TQS) has been grown for the first time by gel method. The structure determination was done by the single crystal XRD technique. The crystal belongs to monoclinic system, P21 space group with cell dimensions a=6.228 (3) Å, b=20.4051 (4) Å, c=11.0600 (6) Å, β=101.9811(2)°. The crystal structure is stabilized by the hydrogen bonding. The functional groups present in the complex were analysed by the Fourier Transform Infrared spectroscopic method. The stoichiometry of the complex was confirmed by the elemental analysis. Thermal properties of the complex were determined by TGA and DTA methods and the complex melts at 222.53°C. The optical transparency of the crystal was studied using UV-Visible absorption spectra. The optical band gap is found to be 2.5 eV. The SHG conversion efficiency of TQS was investigated using Kurtz and Perry method and found to be higher than that of the reference material, potassium dihydrogen phosphate (KDP).

  2. Surfactant-assisted intercalation of high molecular weight poly(ethylene oxide) into vanadyl phosphate di-hydrate

    SciTech Connect

    Ferreira, Joao Paulo L.; Oliveira, Herenilton P.

    2012-03-15

    Graphical abstract: CuK{sub {alpha}} X-ray diffraction patterns of the VOPO{sub 4}/PEO (A) e VOPO{sub 4}/CTA (B) and VOPO{sub 4}/CTA/PEO (C). Highlights: Black-Right-Pointing-Pointer VOPO{sub 4}/PEO has been synthesized by using CTAB, thereby improving PEO intercalation. Black-Right-Pointing-Pointer The d-spacing increase from 1.30 nm (VOPO{sub 4}/PEO) to 2.94 nm (VOPO{sub 4}/CTA/PEO). Black-Right-Pointing-Pointer This strategy was viable for intercalation of PEO with high molecular weight. -- Abstract: A high molecular weight poly(ethylene oxide)/layered vanadyl phosphate di-hydrate intercalation compound was synthesized via the surfactant-assisted approach. Results confirmed that surfactant molecules were replaced with the polymer, while the lamellar structure of the matrix was retained, and that the material presents high specific surface area. In addition, intercalation produced a more thermally stable polymer as evidenced by thermal analysis.

  3. Changes in NAD(P)H fluorescence and membrane current produced by glutamate uptake into salamander Müller cells.

    PubMed Central

    Barbour, B; Magnus, C; Szatkowski, M; Gray, P T; Attwell, D

    1993-01-01

    1. Glutamate uptake into isolated, whole-cell patch-clamped glial cells was studied by monitoring the increase of cell fluorescence generated as glutamate and NAD(P) were converted into alpha-ketoglutarate and NAD(P)H by glutamate dehydrogenase. The current generated by the glutamate uptake carrier was recorded simultaneously. 2. L-Glutamate evoked an increase of cell fluorescence and an inward uptake current. L- and D-aspartate generated an uptake current but no fluorescence response, consistent with the amino acid specificity of glutamate dehydrogenase. 3. In the absence of external sodium the glutamate-evoked fluorescence response and uptake current were abolished, showing that there is no sodium-independent glutamate uptake across the cell membrane. 4. Varying the glutamate concentration altered both the fluorescence response and the uptake current. The fluorescence response saturated at a lower glutamate concentration than the uptake current, and depended in a Michaelis-Menten fashion on the uptake current. 5. The fluorescence response and the uptake current were reduced by membrane depolarization, and also by removal of intracellular potassium. 6. The dependence of the fluorescence response on uptake current when membrane potential was altered or intracellular potassium was removed was the same as that seen when the external glutamate concentration was altered. 7. These fluorescence studies show that glutamate uptake is inhibited by depolarization and by removal of intracellular potassium, consistent with the conclusion of earlier work in which uptake was monitored solely as a membrane current. The data are consistent with high-affinity electrogenic sodium- and potassium-dependent glutamate uptake with fixed stoichiometry being the only significant influx route for glutamate. Other possible interpretations of the data are also discussed. PMID:8105078

  4. Mitochondrial NAD+-dependent malic enzyme from Anopheles stephensi: a possible novel target for malaria mosquito control

    PubMed Central

    2011-01-01

    Background Anopheles stephensi mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs' cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Therefore, the identification of ME in mitochondria from immortalized A. stephensi (ASE) cells and the investigation of the stereoselectivity of malate analogues are relevant in understanding the physiological role of ME in cells of this important malaria parasite vector and its potential as a possible novel target for insecticide development. Methods To characterize the mitochondrial ME from immortalized ASE cells (Mos. 43; ASE), mass spectrometry analyses of trypsin fragments of ME, genomic sequence analysis and biochemical assays were performed to identify the enzyme and evaluate its activity in terms of cofactor dependency and inhibitor preference. Results The encoding gene sequence and primary sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from Anopheles gambiae (98%) and 59% homologous to the mitochondrial NADP+-dependent ME isoform from Homo sapiens. Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that (i) Vmax with NAD+ was 3-fold higher than that with NADP+, (ii) addition of Mg2+ or Mn2+ increased the Vmax by 9- to 21-fold, with Mn2+ 2.3-fold more effective than Mg2+, (iii) succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, (iv) among the analogs of L-malate tested as inhibitors of the NAD+-dependent ME catalyzed reaction, small (2- to 3-carbons) organic diacids carrying a 2-hydroxyl/keto group behaved as the most potent inhibitors of ME activity (e.g., oxaloacetate, tartronic acid and oxalate). Conclusions The biochemical characterization of Anopheles stephensi ME is of critical relevance given its important role in bioenergetics, suggesting

  5. Intracellular NAD+ levels are associated with LPS-induced TNF-α release in pro-inflammatory macrophages

    PubMed Central

    Al-Shabany, Abbas Jawad; Moody, Alan John; Foey, Andrew David; Billington, Richard Andrew

    2016-01-01

    Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD+ has previously been shown to regulate tumour necrosis factor-α (TNF-α) synthesis and TNF-α has been shown to regulate NAD+ homoeostasis providing a link between a pro-inflammatory response and redox status. In the present study, we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD+ levels and expression levels of NAD+ homoeostasis enzymes. Challenge with bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD+ levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD+ synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRNA levels of some NAD+ homoeostasis enzymes in M1-like cells. Surprisingly, despite M2-like cells not releasing TNF-α or changing NAD+ levels in response to lipopolysaccharide, they showed similar mRNA changes compared with M1-like cells. These data further strengthen the link between pro-inflammatory responses in macrophages and NAD+. The agonist-induced rise in NAD+ shows striking parallels to well-known second messengers and raises the possibility that NAD+ is acting in a similar manner in this model. PMID:26764408

  6. Association of hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD) variants and colorectal cancer risk.

    PubMed

    Frank, Bernd; Hoeft, Birgit; Hoffmeister, Michael; Linseisen, Jakob; Breitling, Lutz P; Chang-Claude, Jenny; Brenner, Hermann; Nieters, Alexandra

    2011-02-01

    A recent study examined associations of tagging single nucleotide polymorphisms (tagSNPs) in 43 fatty acid metabolism-related genes and risk of colorectal cancer (CRC), showing rs8752, rs2612656 and a haplotype [comprising both of the single nucleotide polymorphisms (SNPs)] in the hydroxyprostaglandin dehydrogenase 15-(NAD) (HPGD) gene to be positively associated with CRC risk. In the present study, we attempted to replicate these single marker and haplotype associations, using 1795 CRC cases and 1805 controls from the German Darmkrebs: Chancen der Verhütung durch Screening study (DACHS). In addition to rs8752 and rs2612656, HPGD tagSNPs rs9312555, rs17360144 and rs7349744 were genotyped for haplotype analyses. Except for a marginally significant inverse association of HPGD rs8752 with CRC risk [odds ratio (OR) = 0.85; 95% confidence interval (CI) = 0.74, 0.98; P = 0.03], none of the analyzed tagSNPs showed any association with CRC. Subset analyses for colon and rectal cancers yielded similar, yet non-significant risk estimates at all five loci. Also, none of the haplotypes was found to be associated with CRC, colon or rectal cancers. However, rs8752 was significantly associated with a decreased risk of CRC among individuals with a body mass index < 30 (OR = 0.82, 95% CI = 0.70, 0.95, P = 0.01) as well as among smokers (OR = 0.74, 95% CI = 0.61, 0.90, P = 0.003). Yet, our data do not support the previously reported associations of HPGD tagSNPs and risk of CRC. PMID:21047993

  7. Inhibition of de novo NAD(+) synthesis by oncogenic URI causes liver tumorigenesis through DNA damage.

    PubMed

    Tummala, Krishna S; Gomes, Ana L; Yilmaz, Mahmut; Graña, Osvaldo; Bakiri, Latifa; Ruppen, Isabel; Ximénez-Embún, Pilar; Sheshappanavar, Vinayata; Rodriguez-Justo, Manuel; Pisano, David G; Wagner, Erwin F; Djouder, Nabil

    2014-12-01

    Molecular mechanisms responsible for hepatocellular carcinoma (HCC) remain largely unknown. Using genetically engineered mouse models, we show that hepatocyte-specific expression of unconventional prefoldin RPB5 interactor (URI) leads to a multistep process of HCC development, whereas its genetic reduction in hepatocytes protects against diethylnitrosamine (DEN)-induced HCC. URI inhibits aryl hydrocarbon (AhR)- and estrogen receptor (ER)-mediated transcription of enzymes implicated in L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD(+)) metabolism, thereby causing DNA damage at early stages of tumorigenesis. Restoring NAD(+) pools with nicotinamide riboside (NR) prevents DNA damage and tumor formation. Consistently, URI expression in human HCC is associated with poor survival and correlates negatively with L-tryptophan catabolism pathway. Our results suggest that boosting NAD(+) can be prophylactic or therapeutic in HCC. PMID:25453901

  8. Structural Basis of Inhibition of the Human NAD+ -Dependent Deacetylase SIRT5 by Suramin

    SciTech Connect

    Schuetz,A.; Min, J.; Antoshenko, T.; Wang, C.; Allali-Hassani, A.; Dong, A.; Loppnau, P.; vedadi, M.; Bochkarev, A.; et al.

    2007-01-01

    Sirtuins are NAD+-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date, several sirtuin inhibitors and activators have been identified, but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD+-dependent deacetylase activity with an IC50 value of 22 {mu}M. To provide insights into how sirtuin function is altered by inhibitors, we determined two crystal structures of SIRT5, one in complex with ADP-ribose, the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD+, the product, and the substrate-binding site. Finally, our structures may enable the rational design of more potent inhibitors.

  9. Human NAD(P)H:quinone oxidoreductase2. Gene structure, activity, and tissue-specific expression.

    PubMed

    Jaiswal, A K

    1994-05-20

    Human NAD(P)H:quinone oxidoreductase2 (NQO2) gene, 1336 base pairs (bp) of the 5'-flanking region and 165 bp of the 3'-flanking region, have been sequenced. NQO2 gene is 20 kilobase pairs in length and have seven exons interrupted by six introns as compared to the previously cloned NQO1 gene which contains six exons. 187 bp of the first exon in the NQO2 gene are noncoding and are absent in the NQO1 gene. 92 bp of the second exon in the NQO2 gene corresponded to the first exon of the NQO1 gene and so on. The sizes and nucleotide sequences of exons 3-6 are highly conserved between NQO2 and NQO1 genes. The last exon in the NQO2 gene is 1603 bp shorter than the last exon of the NQO1 gene and encodes for 58 amino acids as compared to 101 amino acids encoded by the NQO1 gene. This makes NQO2 protein 43 amino acids shorter than the NQO1 protein. The high degree of conservation between NQO2 and NQO1 gene organization and sequence confirmed that NQO2 gene encodes for a second member of the NQO gene family in human. Nucleotide sequence analysis of the 5'-flanking region of the NQO2 gene revealed presence of four SP1 binding sites at positions -214, -170, -106, and -75, a single copy of the antioxidant response element (ARE) at nucleotide -936, and three copies of xenobiotic response element (XRE) at positions -708, -557, and -51. ARE and XRE elements have previously been found in the promoters of the NQO1 and glutathione S-transferase Ya subunit genes and mediate increases in their expression in response to polycyclic aromatic compounds, phenolic antioxidants, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively. The NQO2 cDNA-derived protein in monkey kidney COS1 cells efficiently catalyzed nitroreduction of anti-tumor compound CB10-200, an analog of nitrophenylaziridine. Northern blot analysis indicates that NQO2 gene is expressed in human heart, brain, lung, liver, and skeletal muscle but does not express in placenta. In contrast, the NQO1 gene was expressed in

  10. Ca2+ controls slow NAD(P)H oscillations in glucose-stimulated mouse pancreatic islets

    PubMed Central

    Luciani, Dan S; Misler, Stanley; Polonsky, Kenneth S

    2006-01-01

    Exposure of pancreatic islets of Langerhans to physiological concentrations of glucose leads to secretion of insulin in an oscillatory pattern. The oscillations in insulin secretion are associated with oscillations in cytosolic Ca2+ concentration ([Ca2+]c). Evidence suggests that the oscillations in [Ca2+]c and secretion are driven by oscillations in metabolism, but it is unclear whether metabolic oscillations are intrinsic to metabolism or require Ca2+ feedback. To address this question we explored the interaction of Ca2+ concentration and islet metabolism using simultaneous recordings of NAD(P)H autofluorescence and [Ca2+]c, in parallel with measurements of mitochondrial membrane potential (ΔΨm). All three parameters responded to 10 mm glucose with multiphasic dynamics culminating in slow oscillations with a period of ∼5 min. This was observed in ∼90% of islets examined from various mouse strains. NAD(P)H oscillations preceded those of [Ca2+]c, but their upstroke was often accelerated during the increase in [Ca2+]c, and Ca2+ influx was a prerequisite for their generation. Prolonged elevations of [Ca2+]c augmented NAD(P)H autofluorescence of islets in the presence of 3 mm glucose, but often lowered NAD(P)H autofluorescence of islets exposed to 10 mm glucose. Comparable rises in [Ca2+]c depolarized ΔΨm. The NAD(P)H lowering effect of an elevation of [Ca2+]c was reversed during inhibition of mitochondrial electron transport. These findings reveal the existence of slow oscillations in NAD(P)H autofluorescence in intact pancreatic islets, and suggest that they are shaped by Ca2+ concentration in a dynamic balance between activation of NADH-generating mitochondrial dehydrogenases and a Ca2+-induced decrease in NADH. We propose that a component of the latter reflects mitochondrial depolarization by Ca2+, which reduces respiratory control and consequently accelerates oxidation of NADH. PMID:16455690

  11. Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

    PubMed

    Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

    2015-06-01

    Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic

  12. Axon Self-Destruction: New Links among SARM1, MAPKs, and NAD+ Metabolism.

    PubMed

    Gerdts, Josiah; Summers, Daniel W; Milbrandt, Jeffrey; DiAntonio, Aaron

    2016-02-01

    Wallerian axon degeneration is a form of programmed subcellular death that promotes axon breakdown in disease and injury. Active degeneration requires SARM1 and MAP kinases, including DLK, while the NAD+ synthetic enzyme NMNAT2 prevents degeneration. New studies reveal that these pathways cooperate in a locally mediated axon destruction program, with NAD+ metabolism playing a central role. Here, we review the biology of Wallerian-type axon degeneration and discuss the most recent findings, with special emphasis on critical signaling events and their potential as therapeutic targets for axonopathy. PMID:26844829

  13. NAD(+)-dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease.

    PubMed

    Cerutti, Raffaele; Pirinen, Eija; Lamperti, Costanza; Marchet, Silvia; Sauve, Anthony A; Li, Wei; Leoni, Valerio; Schon, Eric A; Dantzer, Françoise; Auwerx, Johan; Viscomi, Carlo; Zeviani, Massimo

    2014-06-01

    Mitochondrial disorders are highly heterogeneous conditions characterized by defects of the mitochondrial respiratory chain. Pharmacological activation of mitochondrial biogenesis has been proposed as an effective means to correct the biochemical defects and ameliorate the clinical phenotype in these severely disabling, often fatal, disorders. Pathways related to mitochondrial biogenesis are targets of Sirtuin1, a NAD(+)-dependent protein deacetylase. As NAD(+) boosts the activity of Sirtuin1 and other sirtuins, intracellular levels of NAD(+) play a key role in the homeostatic control of mitochondrial function by the metabolic status of the cell. We show here that supplementation with nicotinamide riboside, a natural NAD(+) precursor, or reduction of NAD(+) consumption by inhibiting the poly(ADP-ribose) polymerases, leads to marked improvement of the respiratory chain defect and exercise intolerance of the Sco2 knockout/knockin mouse, a mitochondrial disease model characterized by impaired cytochrome c oxidase biogenesis. This strategy is potentially translatable into therapy of mitochondrial disorders in humans. PMID:24814483

  14. Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis.

    PubMed

    Goody, Michelle F; Kelly, Meghan W; Lessard, Kevin N; Khalil, Andre; Henry, Clarissa A

    2010-08-15

    Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not beta-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn plays roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis. PMID:20566368

  15. Crystal structure of a ternary complex of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei, NAD+ and 2-oxoisocaproate at 1.9 A resolution.

    PubMed

    Dengler, U; Niefind, K; Kiess, M; Schomburg, D

    1997-04-01

    D-2-hydroxyisocaproate dehydrogenase (D-HicDH) from Lactobacillus casei is a homodimer with 333 amino acids and a molecular mass of 37 kDa per subunit. The enzyme belongs to the protein family of NAD+-dependent D-2-hydroxycarboxylate dehydrogenases and within this family to the subgroup of D-lactate dehydrogenases (D-LDHs). Compared with other D-LDHs D-HicDH is characterized by a very low specificity regarding size and chemical constitution of the accepted D-2-hydroxycarboxylates. Hexagonal crystals of recombinant D-HicDH in the presence of NAD+ and 2-oxoisocaproate (4-methyl-2-oxopentanoate) were grown with ammonium sulphate as precipitating agent. The structure of these crystals was solved by molecular replacement and refined to a final R-factor of 19.6% for all measured X-ray reflections in the resolution range (infinity to 1.86 A). Both NAD+ and 2-oxoisocaproate were identified in the electron density map; binding of the latter in the active site, however, competes with a sulphate ion, which is also defined by electron density. Additionally the final model contains 182 water molecules and a second sulphate ion. The binding of both an in vitro substrate and the natural cosubstrate in the active site provides substantial insight into the catalytic mechanism and allows us to assess previously published active site models for this enzyme family, in particular the two most controversial points, the role of the conserved Arg234 and substrate binding. Furthermore the overall topology and details of the D-HicDH structure are described, discussed against the background of homologous structures and compared with one closely and one distantly related protein.

  16. An Electron Paramagnetic Resonance Spectroscopic Study of Copper Hopping in Doped Bis(L-histidinato)cadmium Dihydrate

    PubMed Central

    Colaneri, Michael J.; Vitali, Jacqueline; Kirschbaum, Kristin

    2013-01-01

    Electron Paramagnetic Resonance (EPR) spectroscopy was used to study Cu(II) dynamic behavior in a doped biological model crystal; bis(L-histidinato)cadmium dihydrate, in order to gain better insight into copper site stability in metalloproteins. Temperature dependent changes in the low temperature X-band EPR spectra became visible around 100 K and continued up to room temperature. The measured 298 K g-tensor (principal values: 2.17, 2.16, 2.07) and copper hyperfine coupling tensor (principal values: −260, − 190, −37 MHz) were similar to the average of the 77 K tensor values pertaining to two neighboring histidine binding sites. The observed temperature dependence was interpreted using Anderson’s theory of motional narrowing, where the magnetic parameters for the different states are averaged as the copper rapidly hops between sites. The EPR pattern was also found to undergo a sharp sigmoidal-shaped, temperature dependent conversion between two species with a critical temperature Tc ≈ 160 K. The species below Tc hops between the two low temperature site patterns, and the one above Tc represents an average of the molecular spin Hamiltonian coupling tensors of the two 77 K sites. In addition, the low and high temperature species hop between one another, contributing to the dynamic averaging. Spectral simulations using this 4-state model determined a hop rate between the two low temperature sites νh4 = 4.5 × 108 s−1 and between the low and high temperature states νh2 = 1.7 × 108 s−1 at 160 K. An Arrhenius relationship of hop rate and temperature gave energy barriers of ΔE4 = 389 cm−1 and ΔE2 = 656 cm−1 between the two low temperature sites, and between the low and high temperature states, respectively. PMID:23530765

  17. The prevalence of chondrocalcinosis of the symphysis pubis on CT scan and correlation with calcium pyrophosphate dihydrate crystal deposition disease.

    PubMed

    Patel, Trusha; Ryan, Lawrence; Dubois, Melissa; Carrera, Guillermo; Baynes, Keith; Mannem, Rajeev; Mulkerin, Jennifer; Visotcky, Alexis

    2016-03-01

    Calcium pyrophosphate dihydrate (CPP) crystal deposition in the articular cartilage can often be seen radiographically as chondrocalcinosis (CC). CPP crystals preferentially deposit in fibrocartilages such as the knee menisci and symphysis pubis (SP). We sought to determine the prevalence of CC in the SP on computed tomography (CT) of the abdomen and pelvis. This retrospective study involved readings on 1070 consecutive CTs of the abdomen and pelvis performed over 3 months in patients over 65 years of age. Medical records of 226 patients found to have CC were reviewed to determine age, gender, documentation of CPPD on problem lists or in medical histories, and whether radiology readings of the CTs mentioned CC. SP CC was identified in 21.1 % (226/1070) of consecutive CT scans with the mean age of CT+ patients being 78.6. Of the 226 patients with SP CC, the observation of CC was documented in only 5.3 % (12/226) of the radiology reports. Of the 12 instances in which the radiology reports mentioned CC, this observation was never (0/12) transmitted to the medical history or problem list. The prevalence of SP CC in patients older than 65 was 21.1 %. Since the majority of CTs of the abdomen and pelvis are not ordered for evaluation of musculoskeletal conditions, this is likely a true prevalence without selection bias. When CC of the SP was present on images, radiologists routinely overlooked or chose not to report CC. Even in the rare instances when it was reported, that information was not added to the medical history or problem list. There are several clinical situations (e.g., acute monoarthritis or atypical osteoarthritis) in which recognizing that a patient has CPP deposition would be useful. Taking the time to review images may yield clinically important findings that are not mentioned anywhere on the patient chart.

  18. Crystal structure of 2-methyl-1H-imidazol-3-ium hydrogen oxalate dihydrate.

    PubMed

    Diop, Mouhamadou Birame; Diop, Libasse; Plasseraud, Laurent; Cattey, Hélène

    2016-08-01

    Single crystals of the title mol-ecular salt, C4H7N2 (+)·HC2O4 (-)·2H2O, were isolated from the reaction of 2-methyl-1H-imidazole and oxalic acid in a 1:1 molar ratio in water. In the crystal, the cations and anions are positioned alternately along an infinite [010] ribbon and linked together through bifurcated N-H⋯(O,O) hydrogen bonds. The water mol-ecules of crystallization link the chains into (10-1) bilayers, with the methyl groups of the cations organized in an isotactic manner. PMID:27536393

  19. Crystal structure of 2-methyl-1H-imidazol-3-ium hydrogen oxalate dihydrate

    PubMed Central

    Diop, Mouhamadou Birame; Diop, Libasse; Plasseraud, Laurent; Cattey, Hélène

    2016-01-01

    Single crystals of the title mol­ecular salt, C4H7N2 +·HC2O4 −·2H2O, were isolated from the reaction of 2-methyl-1H-imidazole and oxalic acid in a 1:1 molar ratio in water. In the crystal, the cations and anions are positioned alternately along an infinite [010] ribbon and linked together through bifurcated N—H⋯(O,O) hydrogen bonds. The water mol­ecules of crystallization link the chains into (10-1) bilayers, with the methyl groups of the cations organized in an isotactic manner. PMID:27536393

  20. Isolation and characterization of a Chinese hamster ovary cell line deficient in fatty alcohol:NAD+ oxidoreductase activity.

    PubMed Central

    James, P F; Rizzo, W B; Lee, J; Zoeller, R A

    1990-01-01

    We have isolated a mutant Chinese hamster ovary cell line that is defective in long-chain fatty alcohol oxidation. The ability of the mutant cells to convert labeled hexadecanol to the corresponding fatty acid in vivo was reduced to 5% of the parent strain. Whole-cell homogenates from the mutant strain, FAA.1, were deficient in long-chain fatty alcohol:NAD+ oxidoreductase (FAO; EC 1.1.1.192) activity, which catalyzes the oxidation of hexadecanol to hexadecanoic acid, although the intermediate fatty aldehyde was formed normally. A direct measurement of fatty aldehyde dehydrogenase showed that the FAA.1 strain was defective in this component of FAO activity. FAA.1 is a two-stage mutant that was selected from a previously described parent strain, ZR-82, which is defective in ether lipid biosynthesis and peroxisome assembly. Because of combined defects in ether lipid biosynthesis and fatty alcohol oxidation, the ability of the FAA.1 cells to incorporate hexadecanol into complex lipids was greatly impaired, resulting in a 60-fold increase in cellular fatty alcohol levels. As the FAO deficiency in FAA.1 cells appears to be identical to the defect associated with the human genetic disorder Sjögren-Larsson syndrome, the FAA.1 cell line may be useful in studying this disease. Images PMID:2201021

  1. Benzofuran-, benzothiophene-, indazole- and benzisoxazole- quinones: excellent substrates for NAD(P)H:quinone oxidoreductase 1

    PubMed Central

    Newsome, Jeffery J.; Hassani, Mary; Swann, Elizabeth; Bibby, Jane M.; Beall, Howard D.; Moody, Christopher J.

    2013-01-01

    A series of heterocyclic quinones based on benzofuran, benzothiophene, indazole and benzisoxazole has been synthesized, and evaluated for their ability to function as substrates for recombinant human NAD(P)H:quinone oxidoreductase (NQO1), a two-electron reductase upregulated in tumor cells. Overall, the quinones are excellent substrates for NQO1, approaching the reduction rates observed for menadione PMID:23635904

  2. NAD+-Metabolizing Ectoenzymes in Remodeling Tumor–Host Interactions: The Human Myeloma Model

    PubMed Central

    Horenstein, Alberto L.; Chillemi, Antonella; Quarona, Valeria; Zito, Andrea; Roato, Ilaria; Morandi, Fabio; Marimpietri, Danilo; Bolzoni, Marina; Toscani, Denise; Oldham, Robert J.; Cuccioloni, Massimiliano; Sasser, A. Kate; Pistoia, Vito; Giuliani, Nicola; Malavasi, Fabio

    2015-01-01

    Nicotinamide adenine dinucleotide (NAD+) is an essential co-enzyme reported to operate both intra- and extracellularly. In the extracellular space, NAD+ can elicit signals by binding purinergic P2 receptors or it can serve as the substrate for a chain of ectoenzymes. As a substrate, it is converted to adenosine (ADO) and then taken up by the cells, where it is transformed and reincorporated into the intracellular nucleotide pool. Nucleotide-nucleoside conversion is regulated by membrane-bound ectoenzymes. CD38, the main mammalian enzyme that hydrolyzes NAD+, belongs to the ectoenzymatic network generating intracellular Ca2+-active metabolites. Within this general framework, the extracellular conversion of NAD+ can vary significantly according to the tissue environment or pathological conditions. Accumulating evidence suggests that tumor cells exploit such a network for migrating and homing to protected areas and, even more importantly, for evading the immune response. We report on the experience of this lab to exploit human multiple myeloma (MM), a neoplastic expansion of plasma cells, as a model to investigate these issues. MM cells express high levels of surface CD38 and grow in an environment prevalently represented by closed niches hosted in the bone marrow (BM). An original approach of this study derives from the recent use of the clinical availability of therapeutic anti-CD38 monoclonal antibodies (mAbs) in perturbing tumor viability and enzymatic functions in conditions mimicking what happens in vivo. PMID:26393653

  3. Protective Pleiotropic Effect of Flavonoids on NAD+ Levels in Endothelial Cells Exposed to High Glucose

    PubMed Central

    Boesten, Daniëlle M. P. H. J.; von Ungern-Sternberg, Saskia N. I.; den Hartog, Gertjan J. M.; Bast, Aalt

    2015-01-01

    NAD+ is important for oxidative metabolism by serving as an electron transporter. Hyperglycemia decreases NAD+ levels by activation of the polyol pathway and by overactivation of poly(ADP-ribose)-polymerase (PARP). We examined the protective role of three structurally related flavonoids (rutin, quercetin, and flavone) during high glucose conditions in an in vitro model using human umbilical vein endothelial cells (HUVECs). Additionally we assessed the ability of these flavonoids to inhibit aldose reductase enzyme activity. We have previously shown that flavonoids can inhibit PARP activation. Extending these studies, we here provide evidence that flavonoids are also able to protect endothelial cells against a high glucose induced decrease in NAD+. In addition, we established that flavonoids are able to inhibit aldose reductase, the key enzyme in the polyol pathway. We conclude that this protective effect of flavonoids on NAD+ levels is a combination of the flavonoids ability to inhibit both PARP activation and aldose reductase enzyme activity. This study shows that flavonoids, by a combination of effects, maintain the redox state of the cell during hyperglycemia. This mode of action enables flavonoids to ameliorate diabetic complications. PMID:26180598

  4. Catastrophic NAD+ depletion in activated T lymphocytes through Nampt inhibition reduces demyelination and disability in EAE.

    PubMed

    Bruzzone, Santina; Fruscione, Floriana; Morando, Sara; Ferrando, Tiziana; Poggi, Alessandro; Garuti, Anna; D'Urso, Agustina; Selmo, Martina; Benvenuto, Federica; Cea, Michele; Zoppoli, Gabriele; Moran, Eva; Soncini, Debora; Ballestrero, Alberto; Sordat, Bernard; Patrone, Franco; Mostoslavsky, Raul; Uccelli, Antonio; Nencioni, Alessio

    2009-11-19

    Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD(+) synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD(+) depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-gamma and TNF-alpha production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD(+)-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD(+) depletion. In addition, we relate defective IFN-gamma and TNF-alpha production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders.

  5. Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

    PubMed Central

    Ferrando, Tiziana; Poggi, Alessandro; Garuti, Anna; D'Urso, Agustina; Selmo, Martina; Benvenuto, Federica; Cea, Michele; Zoppoli, Gabriele; Moran, Eva; Soncini, Debora; Ballestrero, Alberto; Sordat, Bernard; Patrone, Franco; Mostoslavsky, Raul; Uccelli, Antonio; Nencioni, Alessio

    2009-01-01

    Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders. PMID:19936064

  6. Augmentation of NAD+ by NQO1 attenuates cisplatin-mediated hearing impairment

    PubMed Central

    Kim, H-J; Oh, G-S; Shen, A; Lee, S-B; Choe, S-K; Kwon, K-B; Lee, S; Seo, K-S; Kwak, T H; Park, R; So, H-S

    2014-01-01

    Cisplatin (cis-diaminedichloroplatinum-II) is an extensively used chemotherapeutic agent, and one of its most adverse effects is ototoxicity. A number of studies have demonstrated that these effects are related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of cellular energy metabolism and homeostasis. Here, we demonstrate for the first time that, in cisplatin-mediated ototoxicity, the levels and activities of SIRT1 are suppressed by the reduction of intracellular NAD+ levels. We provide evidence that the decrease in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) transferase (PARP)-1 activation and microRNA-34a through p53 activation aggravates cisplatin-mediated ototoxicity. Moreover, we show that the induction of cellular NAD+ levels using β-lapachone (β-Lap), whose intracellular target is NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD+ levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity. PMID:24922076

  7. Crosstalk of Signaling and Metabolism Mediated by the NAD(+)/NADH Redox State in Brain Cells.

    PubMed

    Winkler, Ulrike; Hirrlinger, Johannes

    2015-12-01

    The energy metabolism of the brain has to be precisely adjusted to activity to cope with the organ's energy demand, implying that signaling regulates metabolism and metabolic states feedback to signaling. The NAD(+)/NADH redox state constitutes a metabolic node well suited for integration of metabolic and signaling events. It is affected by flux through metabolic pathways within a cell, but also by the metabolic state of neighboring cells, for example by lactate transferred between cells. Furthermore, signaling events both in neurons and astrocytes have been reported to change the NAD(+)/NADH redox state. Vice versa, a number of signaling events like astroglial Ca(2+) signals, neuronal NMDA-receptors as well as the activity of transcription factors are modulated by the NAD(+)/NADH redox state. In this short review, this bidirectional interdependence of signaling and metabolism involving the NAD(+)/NADH redox state as well as its potential relevance for the physiology of the brain and the whole organism in respect to blood glucose regulation and body weight control are discussed.

  8. Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

    NASA Technical Reports Server (NTRS)

    Warner, R. L.; Huffaker, R. C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

  9. Isolation of a high malic and low acetic acid-producing sake yeast Saccharomyces cerevisiae strain screened from respiratory inhibitor 2,4-dinitrophenol (DNP)-resistant strains.

    PubMed

    Kosugi, Shingo; Kiyoshi, Keiji; Oba, Takahiro; Kusumoto, Kenichi; Kadokura, Toshimori; Nakazato, Atsumi; Nakayama, Shunichi

    2014-01-01

    We isolated 2,4-dinitrophenol (DNP)-resistant sake yeast strains by UV mutagenesis. Among the DNP-resistant mutants, we focused on strains exhibiting high malic acid and low acetic acid production. The improved organic acid composition is unlikely to be under the control of enzyme activities related to malic and acetic acid synthesis pathways. Instead, low mitochondrial activity was observed in DNP-resistant mutants, indicating that the excess pyruvic acid generated during glycolysis is not metabolized in the mitochondria but converted to malic acid in the cytosol. In addition, the NADH/NAD(+) ratio of the DNP-resistant strains was higher than that of the parental strain K901. These results suggest that the increased NADH/NAD(+) ratio together with the low mitochondrial activity alter the organic acid composition because malic acid synthesis requires NADH, while acetic acid uses NAD(+).

  10. Presence of extracellular NAD(+) and NADH in cultures of wood-degrading fungi.

    PubMed

    Kido, Ryuta; Takeeda, Midori; Manabe, Mitsuhiro; Miyagawa, Yutaka; Itakura, Shuji; Tanaka, Hiromi

    2015-01-01

    Our previous studies indicated that extracellular glycoproteins produced by some white-rot and brown-rot basidiomycetous fungi reduce Fe(III) to Fe(II) and O2 to H2O2 and produce hydroxyl radicals. The continuous generation of hydroxyl radicals requires a constant supply of O2 and an electron donor for the reduction of oxidized forms of the glycoproteins to the reduced forms. However, electron donors for this reaction, such as NADH, have not been identified. In this study, the amounts of the extracellular pyridine coenzymes, NAD(+) and NADH, were measured in agar cultures of four white-rot fungi, one brown-rot fungus, and three soft-rot fungi. The sums of NAD(+) and NADH detected in wood-containing cultures of all five basidiomycetes were greater than those in glucose cultures. The amounts of NAD(+) were higher than those of NADH in all wood-containing cultures except that of Irpex lacteus, and NAD(+) was greater than NADH in all glucose cultures except that of Fomitopsis palustris. Significant amounts of pyridine coenzymes were present in glucose and wood-containing cultures of the three soft-rot fungi. The non-wood-degrading fungus, Penicillium funiculosum, did not produce NAD(+) or NADH in either glucose or wood-containing cultures. The extracellular pyridine coenzyme levels were relatively high compared to the rates of extracellular hydroxyl radical generation in wood-degrading fungal cultures. Thus, white-, brown-, and soft-rot fungi produce pyridine coenzymes that could serve as electron donors for the production of hydroxyl radicals during wood degradation.

  11. Decline in NAD(P)H autofluorescence precedes apoptotic cell death from chemotherapy

    NASA Astrophysics Data System (ADS)

    Toms, Steven A.; Muhammad, Osman; Jackson, Heather; Lin, Wei-Chiang

    2005-11-01

    OBJECTIVE: Optical spectroscopic tools exist that allow open surgical and minimally invasive assays of intrinsic tissue optics. Optical detection of cellular and tissue viability may offer a minimally invasive way to assess tumor responsiveness to chemotherapies. We report on an optical spectroscopic change that precedes apoptotic cell death and appears related to NAD(P)H autofluorescence. METHODS: The cell lines SW 480 and U87-MG were grown in culture and treated with cisplatin 100 μg/ml and tamoxifen 10 μM, respectively. Fluorescence spectroscopy at 355 nm excitation and 460 nm emission were collected. MTS assays were used to determine cell viability. Cell lysates were analyzed for NAD(P)H concentrations by mass spectroscopy. RESULTS: Autoflourescence at 355 nm excitation and 460 nm emission declines markedly despite normalization for cell number and total protein concentration after treatment with tamoxifen or cisplatin. The autofluorescence drop precedes the loss of cell viability as measured by MTS assay. For example, the relative viability of the U87-MG cell treated with tamoxifen at hours 0, 8, 12 and 24 of treatment was 100 +/- 6, 85 +/- 6, 53 +/- 9 and 0 +/- 3. The relative fluorescence at the same time points were 100 +/- 2, 57 +/- 6, 47 +/- 3, and 0 +/- 1. TUNNEL assays confirm that cell death is via apoptosis. The key cellular fluorophore at these wavelengths is NAD(P)H. Mass spectroscopic analysis of cell lysates at these time points reveals a drop in NAD(P)H concentrations that is parallel to the loss of fluorescence signal. CONCLUSIONS: NAD(P)H autofluoresence decline precedes apoptotic cell death. This may allow the design of minimally invasive spectroscopic tools to monitor chemotherapeutic response.

  12. The evolving role of the NAD+/nicotinamide metabolome in skin homeostasis, cellular bioenergetics, and aging.

    PubMed

    Oblong, John E

    2014-11-01

    Human skin is exposed to daily environmental insults, particularly solar radiation, that triggers a range of molecular responses. These perturbations to the normal homeostatic state can lead to cellular dysfunction and, ultimately, impacts tissue integrity and accelerates skin aging (photoaging). One of the responses is increased oxidative stress which has been shown to disrupt cellular bioenergetics. This can be detected by depletion of the nucleotide energy metabolites NAD+ and ATP as both an acute transient decrease and, over time, a more permanent chronic reduction due in part to cumulative damage of mitochondria. NAD+ and its primary precursor nicotinamide have been known for some time to impact skin homeostasis based on linkages to dietary requirements, treatment of various inflammatory conditions, photoaging, and prevention of cancer. Cellular NAD+ pools are known to be lower in aged skin and treatment with nicotinamide is hypothesized to restore these levels, thereby mitigating cellular bioenergetics dysfunction. In dermal fibroblasts, nicotinamide is able to protect against oxidative stress to glycolysis, oxidative phosphorylation as well as increase mitochondrial efficiency via sirtuin-dependent selective mitophagy. Recent research has found that NAD+ cellular pools are more dynamic than previously thought, oscillating in tandem with free nicotinamide, and serves as a regulatory point and feedback loop in cellular metabolism regulation, maintenance of mitochondrial efficiency, and circadian rhythmicity. Since UV-induced oxidative stress in skin can disrupt these processes, continued molecular understanding of the role of NAD+ and nicotinamide in skin biology is important to identify interventions that would help maintain its normal homeostatic functions and efficient cellular bioenergetics.

  13. The evolving role of the NAD+/nicotinamide metabolome in skin homeostasis, cellular bioenergetics, and aging.

    PubMed

    Oblong, John E

    2014-11-01

    Human skin is exposed to daily environmental insults, particularly solar radiation, that triggers a range of molecular responses. These perturbations to the normal homeostatic state can lead to cellular dysfunction and, ultimately, impacts tissue integrity and accelerates skin aging (photoaging). One of the responses is increased oxidative stress which has been shown to disrupt cellular bioenergetics. This can be detected by depletion of the nucleotide energy metabolites NAD+ and ATP as both an acute transient decrease and, over time, a more permanent chronic reduction due in part to cumulative damage of mitochondria. NAD+ and its primary precursor nicotinamide have been known for some time to impact skin homeostasis based on linkages to dietary requirements, treatment of various inflammatory conditions, photoaging, and prevention of cancer. Cellular NAD+ pools are known to be lower in aged skin and treatment with nicotinamide is hypothesized to restore these levels, thereby mitigating cellular bioenergetics dysfunction. In dermal fibroblasts, nicotinamide is able to protect against oxidative stress to glycolysis, oxidative phosphorylation as well as increase mitochondrial efficiency via sirtuin-dependent selective mitophagy. Recent research has found that NAD+ cellular pools are more dynamic than previously thought, oscillating in tandem with free nicotinamide, and serves as a regulatory point and feedback loop in cellular metabolism regulation, maintenance of mitochondrial efficiency, and circadian rhythmicity. Since UV-induced oxidative stress in skin can disrupt these processes, continued molecular understanding of the role of NAD+ and nicotinamide in skin biology is important to identify interventions that would help maintain its normal homeostatic functions and efficient cellular bioenergetics. PMID:24794404

  14. Activation of endothelial NAD(P)H oxidase accelerates early glomerular injury in diabetic mice

    PubMed Central

    Nagasu, Hajime; Satoh, Minoru; Kiyokage, Emi; Kidokoro, Kengo; Toida, Kazunori; Channon, Keith M; Kanwar, Yashpal S; Sasaki, Tamaki; Kashihara, Naoki

    2016-01-01

    Increased generation of reactive oxygen species (ROS) is a common denominative pathogenic mechanism underlying vascular and renal complications in diabetes mellitus. Endothelial NAD(P)H oxidase is a major source of vascular ROS, and it has an important role in endothelial dysfunction. We hypothesized that activation of endothelial NAD(P)H oxidase initiates and worsens the progression of diabetic nephropathy, particularly in the development of albuminuria. We used transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NAD(P)H oxidase, Nox2 (NOX2TG). NOX2TG mice were crossed with Akita insulin-dependent diabetic (Akita) mice that develop progressive hyperglycemia. We compared the progression of diabetic nephropathy in Akita versus NOX2TG-Akita mice. NOX2TG-Akita mice and Akita mice developed significant albuminuria above the baseline at 6 and 10 weeks of age, respectively. Compared with Akita mice, NOX2TG-Akita mice exhibited higher levels of NAD(P)H oxidase activity in glomeruli, developed glomerular endothelial perturbations, and attenuated expression of glomerular glycocalyx. Moreover, in contrast to Akita mice, the NOX2TG-Akita mice had numerous endothelial microparticles (blebs), as detected by scanning electron microscopy, and increased glomerular permeability. Furthermore, NOX2TG-Akita mice exhibited distinct phenotypic changes in glomerular mesangial cells expressing α-smooth muscle actin, and in podocytes expressing increased levels of desmin, whereas the glomeruli generated increased levels of ROS. In conclusion, activation of endothelial NAD(P)H oxidase in the presence of hyperglycemia initiated and exacerbated diabetic nephropathy characterized by the development of albuminuria. Moreover, ROS generated in the endothelium compounded glomerular dysfunctions by altering the phenotypes of mesangial cells and compromising the integrity of the podocytes. PMID:26552047

  15. Atomic-Resolution Structures of Horse Liver Alcohol Dehydrogenase with NAD[superscript +] and Fluoroalcohols Define Strained Michaelis Complexes

    SciTech Connect

    Plapp, Bryce V.; Ramaswamy, S.

    2013-01-16

    Structures of horse liver alcohol dehydrogenase complexed with NAD{sup +} and unreactive substrate analogues, 2,2,2-trifluoroethanol or 2,3,4,5,6-pentafluorobenzyl alcohol, were determined at 100 K at 1.12 or 1.14 {angstrom} resolution, providing estimates of atomic positions with overall errors of 0.02 {angstrom}, the geometry of ligand binding, descriptions of alternative conformations of amino acid residues and waters, and evidence of a strained nicotinamide ring. The four independent subunits from the two homodimeric structures differ only slightly in the peptide backbone conformation. Alternative conformations for amino acid side chains were identified for 50 of the 748 residues in each complex, and Leu-57 and Leu-116 adopt different conformations to accommodate the different alcohols at the active site. Each fluoroalcohol occupies one position, and the fluorines of the alcohols are well-resolved. These structures closely resemble the expected Michaelis complexes with the pro-R hydrogens of the methylene carbons of the alcohols directed toward the re face of C4N of the nicotinamide rings with a C-C distance of 3.40 {angstrom}. The oxygens of the alcohols are ligated to the catalytic zinc at a distance expected for a zinc alkoxide (1.96 {angstrom}) and participate in a low-barrier hydrogen bond (2.52 {angstrom}) with the hydroxyl group of Ser-48 in a proton relay system. As determined by X-ray refinement with no restraints on bond distances and planarity, the nicotinamide rings in the two complexes are slightly puckered (quasi-boat conformation, with torsion angles of 5.9{sup o} for C4N and 4.8{sup o} for N1N relative to the plane of the other atoms) and have bond distances that are somewhat different compared to those found for NAD(P){sup +}. It appears that the nicotinamide ring is strained toward the transition state on the path to alcohol oxidation.

  16. Loss of NADH Oxidase Activity in Streptococcus mutans Leads to Rex-Mediated Overcompensation in NAD+ Regeneration by Lactate Dehydrogenase

    PubMed Central

    Baker, J. L.; Derr, A. M.; Faustoferri, R. C.

    2015-01-01

    ABSTRACT Previous studies of the oral pathogen Streptococcus mutans have determined that this Gram-positive facultative anaerobe mounts robust responses to both acid and oxidative stresses. The water-forming NADH oxidase (Nox; encoded by nox) is thought to be critical for the regeneration of NAD+, for use in glycolysis, and for the reduction of oxygen, thereby preventing the formation of damaging reactive oxygen species. In this study, the free NAD+/NADH ratio in a nox deletion strain (Δnox) was discovered to be remarkably higher than that in the parent strain, UA159, when the strains were grown in continuous culture. This unanticipated result was explained by significantly elevated lactate dehydrogenase (Ldh; encoded by ldh) activity and ldh transcription in the Δnox strain, which was mediated in part by the redox-sensing regulator Rex. cDNA microarray analysis of S. mutans cultures exposed to simultaneous acid stress (growth at a low pH) and oxidative stress (generated through the deletion of nox or the addition of exogenous oxygen) revealed a stress response synergistically heightened over that with either stress alone. In the Δnox strain, this elevated stress response included increased glucose phosphoenolpyruvate phosphotransferase system (PTS) activity, which appeared to be due to elevated manL transcription, mediated in part, like elevated ldh transcription, by Rex. While the Δnox strain does possess a membrane composition different from that of the parent strain, it did not appear to have defects in either membrane permeability or ATPase activity. However, the altered transcriptome and metabolome of the Δnox strain were sufficient to impair its ability to compete with commensal peroxigenic oral streptococci during growth under aerobic conditions. IMPORTANCE Streptococcus mutans is an oral pathogen whose ability to outcompete commensal oral streptococci is strongly linked to the formation of dental caries. Previous work has demonstrated that the S

  17. The evolution of substrate specificity-associated residues and Ca(2+) -binding motifs in EF-hand-containing type II NAD(P)H dehydrogenases.

    PubMed

    Hao, Meng-Shu; Rasmusson, Allan G

    2016-07-01

    Most eukaryotic organisms, except some animal clades, have mitochondrial alternative electron transport enzymes that allow respiration to bypass the energy coupling in oxidative phosphorylation. The energy bypass enzymes in plants include the external type II NAD(P)H dehydrogenases (DHs) of the NDB family, which are characterized by an EF-hand domain for Ca(2+) binding. Here we investigate these plant enzymes by combining molecular modeling with evolutionary analysis. Molecular modeling of the Arabidopsis thaliana AtNDB1 with the yeast ScNDI1 as template revealed distinct similarities in the core catalytic parts, and highlighted the interaction between the pyridine nucleotide and residues correlating with NAD(P)H substrate specificity. The EF-hand domain of AtNDB1 has no counterpart in ScNDI1, and was instead modeled with Ca(2+) -binding signal transducer proteins. Combined models displayed a proximity of the AtNDB1 EF-hand domain to the substrate entrance side of the catalytic part. Evolutionary analysis of the eukaryotic NDB-type proteins revealed ancient and recent reversions between the motif observed in proteins specific for NADH (acidic type) and NADPH (non-acidic type), and that the clade of enzymes with acidic motifs in angiosperms derives from non-acidic-motif NDB-type proteins present in basal plants, fungi and protists. The results suggest that Ca(2+) -dependent external NADPH oxidation is an ancient process, indicating that it has a fundamental importance for eukaryotic cellular redox metabolism. In contrast, the external NADH DHs in plants are products of a recent expansion, mirroring the expansion of the alternative oxidase family.

  18. Novel assay for simultaneous measurement of pyridine mononucleotides synthesizing activities allows dissection of the NAD(+) biosynthetic machinery in mammalian cells.

    PubMed

    Zamporlini, Federica; Ruggieri, Silverio; Mazzola, Francesca; Amici, Adolfo; Orsomando, Giuseppe; Raffaelli, Nadia

    2014-11-01

    The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma. PMID:25223558

  19. Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

    SciTech Connect

    Puzon, Geoffrey J.; Huang, Yan C.; Dohnalkova, Alice; Xun, Luying

    2008-06-01

    Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)3 precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD+)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD+-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD+-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD+ with a doubling time of 17 h, and even more slowly on the NAD+-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD+ dissociated from the NAD+-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.

  20. Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

    PubMed

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA.

  1. Conservation of the organization of the mitochondrial nad3 and rps12 genes in evolutionarily distant angiosperms.

    PubMed

    Perrotta, G; Regina, T M; Ceci, L R; Quagliariello, C

    1996-06-12

    The organization of the genes nad3 and rps12 has been investigated in the mitochondrial genome of two dicotyledonous plants - Helianthus and Magnolia - and one monocotyledonous plant (Allium). These plants all contain a complete rps12 gene downstream of the nad3 gene. This arrangement is thus highly conserved within angiosperms. The two genes are co-transcribed and the transcript is modified at several positions by RNA editing of the C to U-type, thus confirming that both genes encode functional proteins. Some 26, 35 and 27 editing events have been identified in the PCR-derived nad3-rps12 cDNA population from sunflower, Magnolia and onion, respectively. Editing of the nad3-rps12 transcript is thus more extensive in Magnolia than in the other angiosperms so far investigated and radically changes the genomically encoded polypeptide sequence. A novel species-specific codon modification was observed in Magnolia. Several homologous sites show differences in editing pattern among plant species. A C-to-U alteration is also found in the non-coding region separating the nad3 and rps12 genes in sunflower. The PCR-derived cDNA populations from the nad3-rps12 loci analysed were found to be differently edited. In addition the plant species show marked variations in the completeness of RNA editing, with only the Magnolia nad3 mRNA being edited fully. PMID:8676875

  2. Amperometric lactate biosensor for flow injection analysis based on a screen-printed carbon electrode containing Meldola's Blue-Reinecke salt, coated with lactate dehydrogenase and NAD+.

    PubMed

    Piano, M; Serban, S; Pittson, R; Drago, G A; Hart, J P

    2010-06-30

    A biosensor for the measurement of lactate in serum has been developed, which is based on a screen-printed carbon electrode, modified with Meldola's Blue-Reinecke Salt (MBRS-SPCE), coated with the enzyme lactate dehydrogenase NAD(+) dependent (from Porcine heart), and NAD(+). A cellulose acetate layer was deposited on the top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V vs. Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer pH 10 containing 0.1 M potassium chloride solution; a flow rate of 0.8 ml min(-1) was used throughout the investigation. The biosensor response was linear over the range 0.55-10 mM lactate; the former represents the detection limit. The precision of the system was determined by carrying out 10 repeat injections of 10 mM l(+)lactic acid standard; the calculated coefficient of variation was 4.28%. It was demonstrated that this biosensor system could be applied to the direct measurement of lactate in serum without pre-treatment; therefore, this would allow high throughput-analysis, at low cost, for this clinically important analyte. PMID:20685431

  3. Crystal and molecular structure of the dihydrate of the artificial sweetener lactitol: 4-O-β- D-galactopyranosyl- D-glucitol.2H 2O

    NASA Astrophysics Data System (ADS)

    Kanter, Jan A.; Schouten, Arie; van Bommel, Mark

    1990-10-01

    Crystallization of lactitol from aqueous ethanol readily yields crystals of the monohydrate, the structure of which has recently been reported. Slow evaporation of very concentrated aqueous syrups results in the crystalline dihydrate. The space group is P4 32 12 with a = 8.762(2), c = 45.508(8) Å, V = 3493.8(13) Å 3, Z = 8, Dc = 1.446 g cm -3, R = 0.037 for 2017 unique observed reflections and 310 variables. The galactopyranosyl ring has the 4C1 chair conformation and the carbon chain of the glucitol fragment has a non-planar, bent MAA conformation. The conformations about the glycosidic C(1)O(1) and O(1)C(14) bonds are different from those observed in the monohydrate: the torsion angles O(5)C(1)O(1)C(14) and C(1)O(1)C(14)C(13) differ by 29.6° and 15.0°, respectively. The orientations of the terminal C(11)O(11) bonds with respect to the carbon-atom chain of the glucitol fragment also differ appreciably: in the dihydrate the pertinent torsion angle is -47.3(3)° and in the monohydrate 75.5(2)°. All hydroxyl groups are involved in a complex three-dimensional system of hydrogen bonds, in which the two water molecules constitute an important cohesive element

  4. Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine.

    PubMed

    Wilson, B A; Reich, K A; Weinstein, B R; Collier, R J

    1990-09-18

    Glutamic acid-148, an active-site residue of diphtheria toxin identified by photoaffinity labeling with NAD, was replaced with aspartic acid, glutamine, or serine by directed mutagenesis of the F2 fragment of the toxin gene. Wild-type and mutant F2 proteins were synthesized in Escherichia coli, and the corresponding enzymic fragment A moieties (DTA) were derived, purified, and characterized. The Glu----Asp (E148D), Glu----Gln (E148Q), and Glu----Ser (E148S) mutations caused reductions in NAD:EF-2 ADP-ribosyltransferase activity of ca. 100-, 250-, and 300-fold, respectively, while causing only minimal changes in substrate affinity. The effects of the mutations on NAD-glycohydrolase activity were considerably different; only a 10-fold reduction in activity was observed for E148S, and the E148D and E148Q mutants actually exhibited a small but reproducible increase in NAD-glycohydrolytic activity. Photolabeling by nicotinamide-radiolabeled NAD was diminished ca. 8-fold in the E148D mutant and was undetectable in the other mutants. The results confirm that Glu-148 plays a crucial role in the ADP-ribosylation of EF-2 and imply an important function for the side-chain carboxyl group in catalysis. The carboxyl group is also important for photochemical labeling by NAD but not for NAD-glycohydrolase activity. The pH dependence of the catalytic parameters for the ADP-ribosyltransferase reaction revealed a group in DTA-wt that titrates with an apparent pKa of 6.2-6.3 and is in the protonated state in the rate-determining step.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. Infrared optical constants of H2O ice, amorphous nitric acid solutions, and nitric acid hydrates

    NASA Technical Reports Server (NTRS)

    Toon, Owen B.; Koehler, Birgit G.; Middlebrook, Ann M.; Tolbert, Margaret A.; Jordon, Joseph

    1994-01-01

    We determined the infrared optical constants of nitric acid trihydrate, nitric acid dihydrate, nitric acid monohydrate, and solid amorphous nitric acid solutions which crystallize to form these hydrates. We have also found the infrared optical constants of H2O ice. We measured the transmission of infrared light throught thin films of varying thickness over the frequency range from about 7000 to 500/cm at temperatures below 200 K. We developed a theory for the transmission of light through a substrate that has thin films on both sides. We used an iterative Kramers-Kronig technique to determine the optical constants which gave the best match between measured transmission spectra and those calculated for a variety of films of different thickness. These optical constants should be useful for calculations of the infrared spectrum of polar stratospheric clouds.

  6. Acidic Deep Eutectic Solvents As Hydrolytic Media for Cellulose Nanocrystal Production.

    PubMed

    Sirviö, Juho Antti; Visanko, Miikka; Liimatainen, Henrikki

    2016-09-12

    In this study, a new method to fabricate cellulose nanocrystals (CNCs) based on DES pretreatment of wood cellulose fibers with choline chloride and organic acids are reported. Oxalic acid (anhydrous and dihydrate), p-toluenesulfonic acid monohydrate, and levulinic acid were studied as acid components of DESs. DESs were formed at elevated temperatures (60-100 °C) by combining choline chloride with organic acids and were then used to hydrolyze less ordered amorphous regions of cellulose. All the DES treatments resulted in degradation of wood fibers into microsized fibers and after mechanically disintegrating, CNCs were successfully obtained from choline chloride/oxalic acid dihydrate-treated fibers, whereas no liberation of CNCs was observed with other DESs. The DES-produced CNCs had a width and length of 9-17 and 310-410 nm, respectively. The crystallinity indexes (CrIs) and carboxylic acid content of the CNCs were 66-71% and 0.20-0.28 mmol/g, respectively. CNCs exhibited good thermal stabilities (the onset thermal degradation temperatures ranged from 275-293 °C). The demonstrated acidic DES method exhibits certain advantages over previously reported CNC productions, namely, milder processing conditions and easily obtainable and relatively inexpensive biodegradable solvents with low toxicity (compared, e.g., to ILs).

  7. Acidic Deep Eutectic Solvents As Hydrolytic Media for Cellulose Nanocrystal Production.

    PubMed

    Sirviö, Juho Antti; Visanko, Miikka; Liimatainen, Henrikki

    2016-09-12

    In this study, a new method to fabricate cellulose nanocrystals (CNCs) based on DES pretreatment of wood cellulose fibers with choline chloride and organic acids are reported. Oxalic acid (anhydrous and dihydrate), p-toluenesulfonic acid monohydrate, and levulinic acid were studied as acid components of DESs. DESs were formed at elevated temperatures (60-100 °C) by combining choline chloride with organic acids and were then used to hydrolyze less ordered amorphous regions of cellulose. All the DES treatments resulted in degradation of wood fibers into microsized fibers and after mechanically disintegrating, CNCs were successfully obtained from choline chloride/oxalic acid dihydrate-treated fibers, whereas no liberation of CNCs was observed with other DESs. The DES-produced CNCs had a width and length of 9-17 and 310-410 nm, respectively. The crystallinity indexes (CrIs) and carboxylic acid content of the CNCs were 66-71% and 0.20-0.28 mmol/g, respectively. CNCs exhibited good thermal stabilities (the onset thermal degradation temperatures ranged from 275-293 °C). The demonstrated acidic DES method exhibits certain advantages over previously reported CNC productions, namely, milder processing conditions and easily obtainable and relatively inexpensive biodegradable solvents with low toxicity (compared, e.g., to ILs). PMID:27478001

  8. Crystal structure of tetra­methyl­tetra­thia­fulvalenium (1S)-camphor-10-sulfonate dihydrate

    PubMed Central

    Sommer, Mathieu; Allain, Magali; Mézière, Cécile; Pop, Flavia; Giffard, Michel

    2015-01-01

    Electro-oxidation of tetra­methyl­tetra­thia­fulvalene (TMTTF) in the presence of the chiral anion (1S)-camphor-10-sulfonate (S-camphSO3 −) in tetra­hydro­furan/water medium afforded a 1/1 salt formulated as TMTTF·S-camphSO3·2H2O or 2-(4,5-dimethyl-1,3-di­thiol-2-yl­idene)-4,5-dimethyl-1,3-di­thiole radical ion (1+) [(1S)-7,7-dimethyl-2-oxobi­cyclo­[2.2.1]heptan-1-yl]methane­sulfonate dihydrate, C10H12S4 +·C10H15O4S−·2H2O. In this salt, two independent TMTTF units are present but, in both cases, the observed bond lengths and especially the central C=C distance [1.392 (6) and 1.378 (6) Å] are in agreement with a complete oxidation of TMTTF which is thus present as TMTTF. + radical cations. These cations form one-dimensional stacks in which they are associated two by two, forming dimers with short [3.472 (1) to 3.554 (2) Å] S⋯S contacts. The two S-camphSO3 anions present also form stacks and are connected with each other via the water mol­ecules with many O—H⋯O hydrogen bonds ranging from 1.86 (3) to 2.15 (4) Å; the O—H⋯O hydrogen-bonding network can be described as being constituted of C 2 2(6) chains bearing R 3 3(11) lateral rings. On the other hand, the columns of cations and anions are connected through C—H⋯O hydrogen bonds, forming a system expanding in three directions; finally, the result is a three-dimensional network of O—H⋯O and C—H⋯O hydrogen bonds. PMID:26279858

  9. Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases.

    PubMed

    Tynan, J; Forde, J; McMahon, M; Mulcahy, P

    2000-12-01

    This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked

  10. Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer.

    PubMed

    Chen, Xianzhong; Fang, Huiying; Rao, Zhiming; Shen, Wei; Zhuge, Bin; Wang, Zhengxiang; Zhuge, Jian

    2008-08-01

    The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD(+)-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1Delta and gpd1Delta/gpd2Delta osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536.

  11. Direct detection of formaldehyde in air by a novel NAD+- and glutathione-independent formaldehyde dehydrogenase-based biosensor.

    PubMed

    Achmann, S; Hermann, M; Hilbrig, F; Jérôme, V; Hämmerle, M; Freitag, R; Moos, R

    2008-05-15

    An amperometric enzyme-based sensor-system for the direct detection of formaldehyde in air is under investigation. The biosensor is based on a native bacterial NAD(+)- and glutathione-independent formaldehyde dehydrogenase as biorecognition element. The enzyme was isolated from Hyphomicrobium zavarzinii strain ZV 580, grown on methylamine hydrochloride in a fed-batch process. The sensor depends on the enzymatic conversion of the analyte to formic acid. Released electrons are detected in an amperometric measurement at 0.2V vs. Ag/AgCl reference electrode by means of a redox-mediator. To optimize the sensing device, Ca(2+) and pyrroloquinoline quinone (PQQ) were added to the buffer solution as reconstitutional substances. At this stage, the sensor shows linear response in the tested ppm-range with a sensitivity of 0.39 microA/ppm. The signal is highly reproducible with respect to sensitivity and base line signal. Reproducibility of sensitivity is more than 90% within the same bacterial batch and even when enzyme of different bacterial batches is used. PMID:18585147

  12. Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

    PubMed Central

    Nakatsuma, Akira; Kaneda, Mugiho; Kodama, Hiromi; Morikawa, Mika; Watabe, Satoshi; Nakaishi, Kazunari; Yamashita, Masakane; Yoshimura, Teruki; Miura, Toshiaki; Ninomiya, Masaki; Ito, Etsuro

    2015-01-01

    To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA. PMID:26098695

  13. Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD.

    PubMed

    Djavadi-Ohaniance, L; Hatefi, H

    1975-12-25

    Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees

  14. Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction.

    PubMed

    Fang, Evandro Fei; Scheibye-Knudsen, Morten; Brace, Lear E; Kassahun, Henok; SenGupta, Tanima; Nilsen, Hilde; Mitchell, James R; Croteau, Deborah L; Bohr, Vilhelm A

    2014-05-01

    Mitochondrial dysfunction is a common feature in neurodegeneration and aging. We identify mitochondrial dysfunction in xeroderma pigmentosum group A (XPA), a nucleotide excision DNA repair disorder with severe neurodegeneration, in silico and in vivo. XPA-deficient cells show defective mitophagy with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD(+)-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP-1. This phenotype is rescued by PARP-1 inhibition or by supplementation with NAD(+) precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration. Our findings reveal a nuclear-mitochondrial crosstalk that is critical for the maintenance of mitochondrial health.

  15. Defective Mitophagy in XPA via PARP1 Hyperactivation and NAD+/SIRT1 Reduction

    PubMed Central

    Brace, Lear E.; Kassahun, Henok; SenGupta, Tanima; Nilsen, Hilde; Mitchell, James R.; Croteau, Deborah L.; Bohr, Vilhelm A.

    2015-01-01

    SUMMARY Mitochondrial dysfunction is a common feature in neurodegeneration and aging. We identify mitochondrial dysfunction in xeroderma pigmentosum group A (XPA), a nucleotide excision DNA repair disorder with severe neurodegeneration, in silico and in vivo. XPA deficient cells show defective mitophagy with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD+-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP1. This phenotype is rescued by PARP1 inhibition or by supplementation with NAD+ precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration. Our findings reveal a novel nuclear-mitochondrial cross-talk that is critical for the maintenance of mitochondrial health. PMID:24813611

  16. Purification and characterization of an NAD(P)H:quinone oxidoreductase from Glycine max seedlings.

    PubMed

    Rescigno, A; Sollai, F; Masala, S; Porcu, M C; Sanjust, E; Rinaldi, A C; Curreli, N; Grifi, D; Rinaldi, A

    1995-01-01

    An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.

  17. An NAD(P)H-Dependent Artificial Transfer Hydrogenase for Multienzymatic Cascades.

    PubMed

    Okamoto, Yasunori; Köhler, Valentin; Ward, Thomas R

    2016-05-11

    Enzymes typically depend on either NAD(P)H or FADH2 as hydride source for reduction purposes. In contrast, organometallic catalysts most often rely on isopropanol or formate to generate the reactive hydride moiety. Here we show that incorporation of a Cp*Ir cofactor possessing a biotin moiety and 4,7-dihydroxy-1,10-phenanthroline into streptavidin yields an NAD(P)H-dependent artificial transfer hydrogenase (ATHase). This ATHase (0.1 mol%) catalyzes imine reduction with 1 mM NADPH (2 mol%), which can be concurrently regenerated by a glucose dehydrogenase (GDH) using only 1.2 equiv of glucose. A four-enzyme cascade consisting of the ATHase, the GDH, a monoamine oxidase, and a catalase leads to the production of enantiopure amines. PMID:27100673

  18. Structure of adenosine-5'-mononicotinate (AMN) trihydrate: an analog of NAD for testing intramolecular stacking.

    PubMed

    Ramasubbu, N; Parthasarathy, R

    1988-12-15

    C16H16N6O5.3H2O, Mr = 426.4, monoclinic, P21, a = 9.535 (2), b = 13.932 (2), c = 7.138 (2) A, beta = 93.13 (2) degrees, V = 946.85 A3, Z = 2, Dx = 1.495 g cm-3, lambda (Cu K alpha) = 1.5418 A, mu = 9.93 cm-1, F(000) = 428, T = 294 K, R = 0.045 and wR = 0.059 for 1460 observed reflections [I greater than 3 sigma (I)]. The AMN molecules, unlike NAD or other model structures of NAD, are not charged and exhibit intra- as well as intermolecular stacking of pyridine ring over adenine ring. There is extensive hydrogen bonding in the crystal involving the pyridine and adenine rings and the three water molecules. Rather surprisingly, the ester carbonyl O atom is not involved in the hydrogen bonding.

  19. Divergent intron conservation in the mitochondrial nad2 gene: signatures for the three bryophyte classes (mosses, liverworts, and hornworts) and the lycophytes.

    PubMed

    Pruchner, Dagmar; Beckert, Susanne; Muhle, Hermann; Knoop, Volker

    2002-09-01

    The slow-evolving mitochondrial DNAs of plants have potentially conserved information on the phylogenetic branching of the earliest land plants. We present the nad2 gene structures in hornworts and liverworts and in the presumptive earliest-branching vascular land plant clade, the Lycopodiopsida. Taken together with the recently obtained nad2 data for mosses, each class of bryophytes presents another pattern of angiosperm-type introns conserved in nad2: intron nad2i1 in mosses; intron nad2i3 in liverworts; and both introns, nad2i3 and nad2i4, in hornworts. The lycopods Isoetes and Lycopodium show diverging intron conservation and feature a unique novel intron, termed nad2i3b. Hence, mitochondrial introns in general are positionally stable in the bryophytes and provide significant intraclade phylogenetic information, but the nad2 introns, in particular, cannot resolve the interclade relationships of the bryophyte classes and to the tracheophytes. The necessity for RNA editing to reconstitute conserved codon entities in nad2 is obvious for all clades except the marchantiid liverworts. Finally, we find that particularly small group II introns appear as a general feature of the Isoetes chondriome. Plant mitochondrial peculiarities such as RNA editing frequency, U-to-C type of RNA editing, and small group II introns appear to be genus-specific rather than gene-specific features.

  20. Infrared optical constants of crystalline sodium chloride dihydrate: application to study the crystallization of aqueous sodium chloride solution droplets at low temperatures.

    PubMed

    Wagner, Robert; Möhler, Ottmar; Schnaiter, Martin

    2012-08-23

    Complex refractive indices of sodium chloride dihydrate, NaCl·2H(2)O, have been retrieved in the 6000-800 cm(-1) wavenumber regime from the infrared extinction spectra of crystallized aqueous NaCl solution droplets. The data set is valid in the temperature range from 235 to 216 K and was inferred from crystallization experiments with airborne particles performed in the large coolable aerosol and cloud chamber AIDA at the Karlsruhe Institute of Technology. The retrieval concept was based on the Kramers-Kronig relationship for a complex function of the optical constants n and k whose imaginary part is proportional to the optical depth of a small particle absorption spectrum in the Rayleigh approximation. The appropriate proportionality factor was inferred from a fitting algorithm applied to the extinction spectra of about 1 μm sized particles, which, apart from absorption, also featured a pronounced scattering contribution. NaCl·2H(2)O is the thermodynamically stable crystalline solid in the sodium chloride-water system below the peritectic at 273.3 K; above 273.3 K, the anhydrous NaCl is more stable. In contrast to anhydrous NaCl crystals, the dihydrate particles reveal prominent absorption signatures at mid-infrared wavelengths due to the hydration water molecules. Formation of NaCl·2H(2)O was only detected at temperatures clearly below the peritectic and was first evidenced in a crystallization experiment conducted at 235 K. We have employed the retrieved refractive indices of NaCl·2H(2)O to quantify the temperature dependent partitioning between anhydrous and dihydrate NaCl particles upon crystallization of aqueous NaCl solution droplets. It was found that the temperature range from 235 to 216 K represents the transition regime where the composition of the crystallized particle ensemble changes from almost only NaCl to almost only NaCl·2H(2)O particles. Compared to the findings on the NaCl/NaCl·2H(2)O partitioning from a recent study conducted with micron

  1. Infrared optical constants of crystalline sodium chloride dihydrate: application to study the crystallization of aqueous sodium chloride solution droplets at low temperatures.

    PubMed

    Wagner, Robert; Möhler, Ottmar; Schnaiter, Martin

    2012-08-23

    Complex refractive indices of sodium chloride dihydrate, NaCl·2H(2)O, have been retrieved in the 6000-800 cm(-1) wavenumber regime from the infrared extinction spectra of crystallized aqueous NaCl solution droplets. The data set is valid in the temperature range from 235 to 216 K and was inferred from crystallization experiments with airborne particles performed in the large coolable aerosol and cloud chamber AIDA at the Karlsruhe Institute of Technology. The retrieval concept was based on the Kramers-Kronig relationship for a complex function of the optical constants n and k whose imaginary part is proportional to the optical depth of a small particle absorption spectrum in the Rayleigh approximation. The appropriate proportionality factor was inferred from a fitting algorithm applied to the extinction spectra of about 1 μm sized particles, which, apart from absorption, also featured a pronounced scattering contribution. NaCl·2H(2)O is the thermodynamically stable crystalline solid in the sodium chloride-water system below the peritectic at 273.3 K; above 273.3 K, the anhydrous NaCl is more stable. In contrast to anhydrous NaCl crystals, the dihydrate particles reveal prominent absorption signatures at mid-infrared wavelengths due to the hydration water molecules. Formation of NaCl·2H(2)O was only detected at temperatures clearly below the peritectic and was first evidenced in a crystallization experiment conducted at 235 K. We have employed the retrieved refractive indices of NaCl·2H(2)O to quantify the temperature dependent partitioning between anhydrous and dihydrate NaCl particles upon crystallization of aqueous NaCl solution droplets. It was found that the temperature range from 235 to 216 K represents the transition regime where the composition of the crystallized particle ensemble changes from almost only NaCl to almost only NaCl·2H(2)O particles. Compared to the findings on the NaCl/NaCl·2H(2)O partitioning from a recent study conducted with micron

  2. [Subcellular localization of isozymes of NAD-dependent malate dehydrogenase in sugar beet Beta vulgaris L].

    PubMed

    Iudina, R S; Levites, E V

    2008-12-01

    Subcellular localization of isozymes of NAD-dependent malate dehydrogenase (MDH) in sugar beet was studied. Isozymes ss and 11 controlled by loci Mdh2 and Mdh3, respectively, were shown to locate in mitochondria, whereas isozyme pp controlled by locus Mdh1, in microbodies. All examined samples lack hybrid MDH isozymes, which could testify to the interaction between products of nonallelic Mdh genes. This can be explained by the localization of nonallelic isozymes in various compartments of the cell and organelles.

  3. Structure of Nampt/PBEF/visfatin, a mammalian NAD[superscript +]biosynthetic enzyme

    SciTech Connect

    Wang, Tao; Zhang, Xiangbin; Bheda, Poonam; Revollo, Javier R.; Imai, Shin-ichiro; Wolberger, Cynthia

    2010-07-22

    Nicotinamide phosphoribosyltransferase (Nampt) synthesizes nicotinamide mononucleotide (NMN) from nicotinamide in a mammalian NAD{sup +} biosynthetic pathway and is required for SirT1 activity in vivo. Nampt has also been presumed to be a cytokine (PBEF) or a hormone (visfatin). The crystal structure of Nampt in the presence and absence of NMN shows that Nampt is a dimeric type II phosphoribosyltransferase and provides insights into the enzymatic mechanism.

  4. The mechanism of RNA 5′ capping with NAD+, NADH and desphospho-CoA.

    PubMed

    Bird, Jeremy G; Zhang, Yu; Tian, Yuan; Panova, Natalya; Barvík, Ivan; Greene, Landon; Liu, Min; Buckley, Brian; Krásný, Libor; Lee, Jeehiun K; Kaplan, Craig D; Ebright, Richard H; Nickels, Bryce E

    2016-07-21

    The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency, and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms. PMID:27383794

  5. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+

    NASA Astrophysics Data System (ADS)

    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

    2016-09-01

    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP+ to NAD+. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD+, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

  6. Gentamicin differentially alters cellular metabolism of cochlear hair cells as revealed by NAD(P)H fluorescence lifetime imaging

    NASA Astrophysics Data System (ADS)

    Zholudeva, Lyandysha V.; Ward, Kristina G.; Nichols, Michael G.; Smith, Heather Jensen

    2015-05-01

    Aminoglycoside antibiotics are implicated as culprits of hearing loss in more than 120,000 individuals annually. Research has shown that the sensory cells, but not supporting cells, of the cochlea are readily damaged and/or lost after use of such antibiotics. High-frequency outer hair cells (OHCs) show a greater sensitivity to antibiotics than high- and low-frequency inner hair cells (IHCs). We hypothesize that variations in mitochondrial metabolism account for differences in susceptibility. Fluorescence lifetime microscopy was used to quantify changes in NAD(P)H in sensory and supporting cells from explanted murine cochleae exposed to mitochondrial uncouplers, inhibitors, and an ototoxic antibiotic, gentamicin (GM). Changes in metabolic state resulted in a redistribution of NAD(P)H between subcellular fluorescence lifetime pools. Supporting cells had a significantly longer lifetime than sensory cells. Pretreatment with GM increased NAD(P)H intensity in high-frequency sensory cells, as well as the NAD(P)H lifetime within IHCs. GM specifically increased NAD(P)H concentration in high-frequency OHCs, but not in IHCs or pillar cells. Variations in NAD(P)H intensity in response to mitochondrial toxins and GM were greatest in high-frequency OHCs. These results demonstrate that GM rapidly alters mitochondrial metabolism, differentially modulates cell metabolism, and provides evidence that GM-induced changes in metabolism are significant and greatest in high-frequency OHCs.

  7. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(.).

    PubMed

    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

    2016-01-01

    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP(+) to NAD(+). This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT. PMID:27587230

  8. The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity.

    PubMed

    Cantó, Carles; Houtkooper, Riekelt H; Pirinen, Eija; Youn, Dou Y; Oosterveer, Maaike H; Cen, Yana; Fernandez-Marcos, Pablo J; Yamamoto, Hiroyasu; Andreux, Pénélope A; Cettour-Rose, Philippe; Gademann, Karl; Rinsch, Chris; Schoonjans, Kristina; Sauve, Anthony A; Auwerx, Johan

    2012-06-01

    As NAD(+) is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38-both NAD(+) consumers-increases NAD(+) bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD(+) precursor with the ability to increase NAD(+) levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD(+) levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function. PMID:22682224

  9. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+

    PubMed Central

    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

    2016-01-01

    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP+ to NAD+. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD+, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT. PMID:27587230

  10. SIRT1-mediated eNAMPT secretion from adipose tissue regulates hypothalamic NAD+ and function in mice

    PubMed Central

    Yoon, Myeong Jin; Yoshida, Mitsukuni; Johnson, Sean; Takikawa, Akiko; Usui, Isao; Tobe, Kazuyuki; Nakagawa, Takashi; Yoshino, Jun; Imai, Shin-ichiro

    2015-01-01

    SUMMARY Nicotinamide phosphoribosyltransferase (NAMPT), the key NAD+ biosynthetic enzyme, has two different forms, intra- and extracellular (iNAMPT and eNAMPT), in mammals. However, the significance of eNAMPT secretion remains unclear. Here we demonstrate that deacetylation of iNAMPT by the mammalian NAD+-dependent deacetylase SIRT1 predisposes the protein to secretion in adipocytes. NAMPT mutants reveal that SIRT1 deacetylates lysine 53 and enhances eNAMPT activity and secretion. Adipose tissue-specific Nampt knockout and knockin (ANKO and ANKI) mice show reciprocal changes in circulating eNAMPT, affecting hypothalamic NAD+/SIRT1 signaling and physical activity accordingly. The defect in physical activity observed in ANKO mice is ameliorated by nicotinamide mononucleotide (NMN). Furthermore, administration of a NAMPT-neutralizing antibody decreases hypothalamic NAD+ production, and treating ex vivo hypothalamic explants with purified eNAMPT enhances NAD+, SIRT1 activity, and neural activation. Thus, our findings indicate a critical role of adipose tissue as a modulator for the regulation of NAD+ biosynthesis at a systemic level. PMID:25921090

  11. The Streptococcus pyogenes NAD+ Glycohydrolase Modulates Epithelial Cell PARylation and HMGB1 Release

    PubMed Central

    Chandrasekaran, Sukantha; Caparon, Michael G.

    2015-01-01

    Streptococcus pyogenes uses the cytolysin Streptolysin O (SLO) to translocate an enzyme, the S. pyogenes NAD+ glycohydrolase (SPN), into the host cell cytosol. However, the function of SPN in this compartment is not known. As a complication, many S. pyogenes strains express a SPN variant lacking NAD+ glycohydrolase (NADase) activity. Here, we show that SPN modifies several SLO- and NAD+-dependent host cell responses in patterns that correlate with NADase activity. SLO pore formation results in hyper-activation of the cellular enzyme Poly-ADP-ribose Polymerase-1 (PARP-1) and production of polymers of Poly-ADP-ribose (PAR). However, while SPN NADase activity moderates PARP-1 activation and blocks accumulation of PAR, these processes continued unabated in the presence of NADase-inactive SPN. Temporal analyses revealed that while PAR production is initially independent of NADase activity, PAR rapidly disappears in the presence of NADase-active SPN, host cell ATP is depleted and the pro-inflammatory mediator High-Mobility Group Box-1 (HMGB1) protein is released from the nucleus by a PARP-1 dependent mechanism. In contrast, HMGB1 is not released in response to NADase-inactive SPN and instead the cells release elevated levels of IL-8 and TNFα. Thus, SPN and SLO combine to induce cellular responses subsequently influenced by the presence or absence of NADase activity. PMID:25818652

  12. Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes.

    PubMed

    Cahn, J K B; Baumschlager, A; Brinkmann-Chen, S; Arnold, F H

    2016-01-01

    NAD(P)H-dependent enzymes are ubiquitous in metabolism and cellular processes and are also of great interest for pharmaceutical and industrial applications. Here, we present a structure-guided enzyme engineering strategy for improving catalytic properties of NAD(P)H-dependent enzymes toward native or native-like reactions using mutations to the enzyme's adenine-binding pocket, distal to the site of catalysis. Screening single-site saturation mutagenesis libraries identified mutations that increased catalytic efficiency up to 10-fold in 7 out of 10 enzymes. The enzymes improved in this study represent three different cofactor-binding folds (Rossmann, DHQS-like, and FAD/NAD binding) and utilize both NADH and NADPH. Structural and biochemical analyses show that the improved activities are accompanied by minimal changes in other properties (cooperativity, thermostability, pH optimum, uncoupling), and initial tests on two enzymes (ScADH6 and EcFucO) show improved functionality in Escherichia coli. PMID:26512129

  13. IcmQ in the Type 4b secretion system contains a novel NAD+ binding domain

    PubMed Central

    Farelli, Jeremiah D.; Gumbart, James C.; Akey, Ildiko V.; Hempstead, Andrew; Amyot, Whitney; Head, James F.; McKnight, C. James; Isberg, Ralph R.; Akey, Christopher W.

    2013-01-01

    A Type4b secretion system (T4bSS) is required for Legionella growth in alveolar macrophages. IcmQ associates with IcmR, binds to membranes and has a critical role in the T4bSS. We have now solved a crystal structure of IcmR-IcmQ to further our understanding of this complex. This structure revealed an amphipathic four-helix bundle, formed by IcmR and the N-terminal domain of IcmQ, which is linked to a novel C-terminal domain of IcmQ (Qc) by a linker helix. The Qc domain has structural homology with ADP ribosyltransferase domains in certain bacterial toxins and binds NAD+ with a Kd in the physiological range. Structural homology and molecular dynamics were used to identify an extended NAD+ binding site on Qc and the resulting model was tested by mutagenesis and binding assays. Based on the data, we suggest that IcmR-IcmQ binds to membranes where it may interact with or perhaps modify a protein in the T4bSS when NAD+ is bound. PMID:23850453

  14. Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

    SciTech Connect

    Han, Seungil; Chang, Jeanne S.; Griffor, Matt; Pfizer

    2010-09-17

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  15. Identification of the NAD(P)H binding site of eukaryotic UDP-galactopyranose mutase.

    PubMed

    Dhatwalia, Richa; Singh, Harkewal; Solano, Luis M; Oppenheimer, Michelle; Robinson, Reeder M; Ellerbrock, Jacob F; Sobrado, Pablo; Tanner, John J

    2012-10-31

    UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD(P)H. Kinetic protein crystallography was used to obtain structures of oxidized Aspergillus fumigatus UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP(+). NAD(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, and thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All 12 residues that contact NADP(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP(H) binding sites. The structures address the longstanding question of how UGM binds NAD(P)H and provide new opportunities for drug discovery. PMID:23036087

  16. The hidden life of NAD+-consuming ectoenzymes in the endocrine system.

    PubMed

    Malavasi, Fabio; Deaglio, Silvia; Zaccarello, Gianluca; Horenstein, Alberto L; Chillemi, Antonella; Audrito, Valentina; Serra, Sara; Gandione, Marina; Zitella, Andrea; Tizzani, Alessandro

    2010-10-01

    Ectoenzymes are a family of cell surface molecules whose catalytic domain lies in the extracellular region. A subset of this family, nucleotide-metabolizing ectoenzymes, are key components in the regulation of the extracellular balance between nucleotides (e.g. NAD(+) or ATP) and nucleosides (e.g. adenosine). Their substrates and products are signalling molecules that act by binding to specific receptors, triggering signals that regulate a variety of functions, ranging from the migration of immune cells, to synaptic transmission in the brain, to hormone/receptor interactions in the glands. Almost two decades of accumulated data indicate that these regulatory processes significantly affect the endocrine system, a tightly controlled information signal complex with clear evidence of fine regulation. Functional models discussed in this review include insulin secretion, bone modelling and the association between hormones and behaviour. The emerging pattern is one of a system operating as a scale-free network that hinges around hubs of key molecules, such as NAD(+) or ATP. The underlying natural link between nucleotides, ectoenzymes and the endocrine system is far from being clearly demonstrated. However, the body of evidence supporting the existence of such connection is growing exponentially. This review will try to read the available evidence in a hypothesis-oriented perspective, starting from the description of NAD(+) and of ecto- and endoenzymes involved in its metabolism.

  17. On-line characterization of peroxidation intermediates of NAD{sup +} by CE-ESI-MS

    SciTech Connect

    Zhao, Zhongxi; Udseth, H.R.; Smith, R.D.

    1995-12-31

    Equine liver alcohol dehydrogenase (LADH) is one of the NAD-dependent enzymes with various physiological functions. LADH also exhibits several alternative catalytic activities. Among them, the most unusual one is the LADH-catalyzed degradation of NAD+ in the presence of hydrogen peroxide. A two-step reaction mechanism has been proposed for this enzymatic peroxidation. In the first (fast) step, NAD+ catalyzed by LADH is converted to a (so far) unidentified intermediate: {open_quotes}compound I{close_quotes}. The following slower step is enzyme independent and consists of at least two distinct processes; one of them leads to another unknown substance: {open_quotes}compound Y{close_quotes} which is the product of further reaction of {open_quotes}compound I{close_quotes} with excess H{sub 2}O{sub 2}; another process converts {open_quotes}compound I{close_quotes} to adenosine(5{prime})- diphospho(5)-{Beta}-D-ribose-NHCH=C(CHO)CONH{sub 2} (NADX) if catalase is added to destroy excess H{sub 2}O{sub 2} soon after the end of the faster enzymatic step. One of the advantages of capillary electrophoresis (CE) is its fast operation cycle, which could be utilized to monitor relatively slow reaction processes. Capillary electrophoresis-mass spectrometry (CE-MS) has the capability of high resolution separations coupled with highly sensitive and selective detection providing crucial molecular weight and structural information.

  18. Structural Basis of Streptococcus pyogenes Immunity to its NAD+ Glycohydrolase Toxin

    PubMed Central

    Smith, Craig L.; Ghosh, Joydeep; Elam, Jennifer Stine; Pinkner, Jerome S.; Hultgren, Scott J.; Caparon, Michael G.; Ellenberger, Tom

    2011-01-01

    SUMMARY The virulence of Gram-positive bacteria is enhanced by toxins like the Streptococcus pyogenes β-NAD+ glycohydrolase known as SPN. SPN-producing strains of S. pyogenes additionally express the protein immunity factor for SPN (IFS), which forms an inhibitory complex with SPN. We have determined crystal structures of the SPN-IFS complex and IFS alone, revealing that SPN is structurally related to ADP-ribosyl transferases but lacks the canonical binding site for protein substrates. SPN is instead a highly efficient glycohydrolase with the potential to deplete cellular levels of β-NAD+. The protective effect of IFS involves an extensive interaction with the SPN active site that blocks access to β-NAD+. The conformation of IFS changes upon binding to SPN, with repacking of an extended C-terminal α-helix into a compact shape. IFS is an attractive target for the development of novel bacteriocidal compounds functioning by blocking the bacterium's self-immunity to the SPN toxin. PMID:21300288

  19. Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

    PubMed Central

    Hsieh, Yi-Ching; Tedeschi, Philip; AdeBisi Lawal, Rialnat; Banerjee, Debabrata; Scotto, Kathleen; Kerrigan, John E.; Lee, Kuo-Chieh; Johnson-Farley, Nadine; Bertino, Joseph R.

    2013-01-01

    Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin’s lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment. PMID:23197646

  20. NAD(P)+-Malic Enzyme Mutants of Sinorhizobium sp. Strain NGR234, but Not Azorhizobium caulinodans ORS571, Maintain Symbiotic N2 Fixation Capabilities

    PubMed Central

    Zhang, Ye; Aono, Toshihiro; Poole, Phillip

    2012-01-01

    C4-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N2-fixing bacteria (bacteroids) within legume nodules. In Sinorhizobium meliloti bacteroids from alfalfa, NAD+-malic enzyme (DME) is required for N2 fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont Rhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that dme mutants of the broad-host-range Sinorhizobium sp. strain NGR234 formed nodules whose level of N2 fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single pckA and single dme mutants fixed N2 at reduced rates, a pckA dme double mutant had no N2-fixing activity (Fix−). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix− phenotype of S. meliloti dme mutants may be specific to the alfalfa-S. meliloti symbiosis. We therefore examined the ME-like genes azc3656 and azc0119 from Azorhizobium caulinodans, as azc3656 mutants were previously shown to form Fix− nodules on the tropical legume Sesbania rostrata. We found that purified AZC3656 protein is an NAD(P)+-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N2 fixation in A. caulinodans and S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s). PMID:22307295

  1. Crystal structure of 1,3-bis­(1,3-dioxoisoindolin-1-yl)urea dihydrate: a urea-based anion receptor

    PubMed Central

    Medrano, Felipe; Lujano, Sergio; Godoy-Alcántar, Carolina; Tlahuext, Hugo

    2014-01-01

    The whole mol­ecule of the title compound, C17H10N4O5·2H2O, is generated by twofold rotation symmetry and it crystallized as a dihydrate. The planes of the phthalimide moieties and the urea unit are almost normal to one another, with a dihedral angle of 78.62 (9)°. In the crystal, mol­ecules are linked by N—H⋯O and O—H⋯O hydrogen bonds, forming a three-dimensional framework structure. The crystal packing also features C—H⋯O hydrogen bonds and slipped parallel π–π inter­actions [centroid–centroid distance = 3.6746 (15) Å] involving the benzene rings of neighbouring phthalimide moieties. PMID:25484749

  2. CLearance of calcium pyrophosphate dihydrate crystals in vivo. II. Studies using triclinic crystals doubly labeled with 45Ca and 85Sr.

    PubMed

    McCarty, D J; Palmer, D W; James, C

    1979-10-01

    The clearance rate of isotopically labeled synthetic triclinic calcium pyrophosphate dihydrate (CPPD) crystals injection into rabbit joints was estimated by serial counting. Kinetic analysis using a four compartment model showed that half of the injected dose was cleared from 4 rabbit knee joints in 19.1 +/- 0.42 (SEM) days. Profound hypomagnesemia, produced in 2 rabbits with a low magnesium diet, did not affect the rate of crystal clearance detectably. Lavage of joints with solutions known to promote CPPD crystal solubility failed to remove detectable radioactivity. The previous finding of CPPD crystals in synovial phagocytes by electron microscopy, together with the finding of nuclide activity in the synovium and the failure to remove such activity by joint lavage, suggests that endocytosis by synovial cells is an important, effective mechanism controlling the synovial fluid concentration of crystals in patients with CPPD crystal deposition disease. PMID:226098

  3. Transferred multipolar atom model for 10β,17β-dihydroxy-17α-methylestr-4-en-3-one dihydrate obtained from the biotransformation of methyloestrenolone.

    PubMed

    Faroque, Muhammad Umer; Yousuf, Sammer; Zafar, Salman; Choudhary, M Iqbal; Ahmed, Maqsood

    2016-05-01

    Biotransformation is the structural modification of compounds using enzymes as the catalysts and it plays a key role in the synthesis of pharmaceutically important compounds. 10β,17β-Dihydroxy-17α-methylestr-4-en-3-one dihydrate, C19H28O3·2H2O, was obtained from the fungal biotransformation of methyloestrenolone. The structure was refined using the classical independent atom model (IAM) and a transferred multipolar atom model using the ELMAM2 database. The results from the two refinements have been compared. The ELMAM2 refinement has been found to be superior in terms of the refinement statistics. It has been shown that certain electron-density-derived properties can be calculated on the basis of the transferred parameters for crystals which diffract to ordinary resolution.

  4. Transferred multipolar atom model for 10β,17β-dihydroxy-17α-methylestr-4-en-3-one dihydrate obtained from the biotransformation of methyloestrenolone.

    PubMed

    Faroque, Muhammad Umer; Yousuf, Sammer; Zafar, Salman; Choudhary, M Iqbal; Ahmed, Maqsood

    2016-05-01

    Biotransformation is the structural modification of compounds using enzymes as the catalysts and it plays a key role in the synthesis of pharmaceutically important compounds. 10β,17β-Dihydroxy-17α-methylestr-4-en-3-one dihydrate, C19H28O3·2H2O, was obtained from the fungal biotransformation of methyloestrenolone. The structure was refined using the classical independent atom model (IAM) and a transferred multipolar atom model using the ELMAM2 database. The results from the two refinements have been compared. The ELMAM2 refinement has been found to be superior in terms of the refinement statistics. It has been shown that certain electron-density-derived properties can be calculated on the basis of the transferred parameters for crystals which diffract to ordinary resolution. PMID:27146568

  5. In Situ Investigation of a Self-Accelerated Cocrystal Formation by Grinding Pyrazinamide with Oxalic Acid.

    PubMed

    Kulla, Hannes; Greiser, Sebastian; Benemann, Sigrid; Rademann, Klaus; Emmerling, Franziska

    2016-01-01

    A new cocrystal of pyrazinamide with oxalic acid was prepared mechanochemically and characterized by PXRD, Raman spectroscopy, solid-state NMR spectroscopy, DTA-TG, and SEM. Based on powder X-ray diffraction data the structure was solved. The formation pathway of the reaction was studied in situ using combined synchrotron PXRD and Raman spectroscopy. Using oxalic acid dihydrate the initially neat grinding turned into a rapid self-accelerated liquid-assisted grinding process by the release of crystallization water. Under these conditions, the cocrystal was formed directly within two minutes. PMID:27428942

  6. Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+.

    PubMed

    Belenky, Peter; Racette, Frances G; Bogan, Katrina L; McClure, Julie M; Smith, Jeffrey S; Brenner, Charles

    2007-05-01

    Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function. PMID:17482543

  7. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

    PubMed

    Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

    2016-05-13

    Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS. PMID:27002158

  8. EPR and potentiometric studies of copper(II) binding to nicotinamide adenine dinucleotide (NAD+) in water solution.

    PubMed

    Hoffmann, Stanisław K; Goslar, Janina; Lijewski, Stefan; Basiński, Kamil; Gąsowska, Anna; Łomozik, Lechosław

    2012-06-01

    Coordination of Cu(II) by nicotinamide adenine dinucleotide (NAD(+)) molecule has been studied in water solutions of various pH by potentiometry and electron paramagnetic resonance (EPR) and electron spin echo (ESE) spectroscopy. Potentiometric results indicate Cu(II) coordination by protonated NAD(+) at low pH and by deprotonated NAD(+) at high pH. At medium pH value (around pH=7) NAD(+) is not able to coordinate Cu(II) ions effectively and mainly the Cu(H(2)O)(6) complexes exist in the studied solution. This has been confirmed by EPR results. Electronic structure of Cu(II)-NAD complex and coordination sites is determined from EPR and ESE measurements in frozen solutions (at 77K and 6K). EPR spectra exclude coordination with nitrogen atoms. Detailed analysis of EPR parameters (g(||)=2.420, g(perpendicular)==2.080, A(||)=-131×10(-4)cm(-1) and A(perpendicular)=8×10(-4)cm(-1)) performed in terms of molecular orbital (MO) theory shows that Cu(II)NAD complex has elongated axial octahedral symmetry with a relatively strong delocalization of unpaired electron density on in-plane and axial ligands. The distortion of octahedron is analyzed using A(||) vs. g(||) diagram for various CuO(x) complexes. Electron spin echo decay modulation excludes the coordination by oxygen atoms of phosphate groups. We postulate a coordination of Cu(II) by two hydroxyl oxygen atoms of two ribose moieties of the NAD molecules and four solvated water molecules both at low and high pH values with larger elongation of the octahedron at higher pH.

  9. Plastidial NAD-Dependent Malate Dehydrogenase Is Critical for Embryo Development and Heterotrophic Metabolism in Arabidopsis1[W][OPEN

    PubMed Central

    Beeler, Seraina; Liu, Hung-Chi; Stadler, Martha; Schreier, Tina; Eicke, Simona; Lue, Wei-Ling; Truernit, Elisabeth; Zeeman, Samuel C.; Chen, Jychian; Kötting, Oliver

    2014-01-01

    In illuminated chloroplasts, one mechanism involved in reduction-oxidation (redox) homeostasis is the malate-oxaloacetate (OAA) shuttle. Excess electrons from photosynthetic electron transport in the form of nicotinamide adenine dinucleotide phosphate, reduced are used by NADP-dependent malate dehydrogenase (MDH) to reduce OAA to malate, thus regenerating the electron acceptor NADP. NADP-MDH is a strictly redox-regulated, light-activated enzyme that is inactive in the dark. In the dark or in nonphotosynthetic tissues, the malate-OAA shuttle was proposed to be mediated by the constitutively active plastidial NAD-specific MDH isoform (pdNAD-MDH), but evidence is scarce. Here, we reveal the critical role of pdNAD-MDH in Arabidopsis (Arabidopsis thaliana) plants. A pdnad-mdh null mutation is embryo lethal. Plants with reduced pdNAD-MDH levels by means of artificial microRNA (miR-mdh-1) are viable, but dark metabolism is altered as reflected by increased nighttime malate, starch, and glutathione levels and a reduced respiration rate. In addition, miR-mdh-1 plants exhibit strong pleiotropic effects, including dwarfism, reductions in chlorophyll levels, photosynthetic rate, and daytime carbohydrate levels, and disordered chloroplast ultrastructure, particularly in developing leaves, compared with the wild type. pdNAD-MDH deficiency in miR-mdh-1 can be functionally complemented by expression of a microRNA-insensitive pdNAD-MDH but not NADP-MDH, confirming distinct roles for NAD- and NADP-linked redox homeostasis. PMID:24453164

  10. Dynamic changes in nicotinamide pyridine dinucleotide content in normal human epidermal keratinocytes and their effect on retinoic acid biosynthesis

    SciTech Connect

    Pinkas-Sarafova, Adriana . E-mail: apinkassaraf@notes.cc.sunysb.edu; Markova, N.G. . E-mail: nmarkova@notes.cc.sunysb.edu; Simon, M. . E-mail: marsimon@notes.cc.sunysb.edu

    2005-10-21

    The function of many enzymes that regulate metabolism and transcription depends critically on the nicotinamide pyridine dinucleotides. To understand the role of NAD(P)(H) in physiology and pathophysiology, it is imperative to estimate both their amount and ratios in a given cell type. In human epidermis and in cultured epidermal keratinocytes, we found that the total dinucleotide content is in the low millimolar range. The dinucleotide pattern changes during proliferation and maturation of keratinocytes in culture. Differences in the concentrations of NAD(P)(H) of 1.5- to 12-fold were observed. This resulted in alteration of the NAD(P)H/NAD(P) ratio, which could impact the differential regulation of both transcriptional and metabolic processes. In support of this notion, we provide evidence that the two-step oxidation of retinol to retinoic acid, a nuclear hormone critical for epidermal homeostasis, can be regulated by the relative physiological amounts of the pyridine dinucleotides.

  11. Improvement of NADPH bioavailability in Escherichia coli by replacing NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase GapA with NADP (+)-dependent GapB from Bacillus subtilis and addition of NAD kinase.

    PubMed

    Wang, Yipeng; San, Ka-Yiu; Bennett, George N

    2013-12-01

    Enzymatic synthesis of some industrially important compounds depends heavily on cofactor NADPH as the reducing agent. This is especially true in the synthesis of chiral compounds that are often used as pharmaceutical intermediates to generate the correct stereochemistry in bioactive products. The high cost and technical difficulty of cofactor regeneration often pose a challenge for such biocatalytic reactions. In this study, to increase NADPH bioavailability, the native NAD(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gapA gene in Escherichia coli was replaced with a NADP(+)-dependent gapB from Bacillus subtilis. To overcome the limitation of NADP(+) availability, E. coli NAD kinase, nadK was also coexpressed with gapB. The recombinant strains were then tested in three reporting systems: biosynthesis of lycopene, oxidation of cyclohexanone with cyclohexanone monooxygenase (CHMO), and an anaerobic system utilizing 2-haloacrylate reductase (CAA43). In all the reporting systems, replacing NAD(+)-dependent GapA activity with NADP(+)-dependent GapB activity increased the synthesis of NADPH-dependent compounds. The increase was more pronounced when NAD kinase was also overexpressed in the case of the one-step reaction catalyzed by CAA43 which approximately doubled the product yield. These results validate this novel approach to improve NADPH bioavailability in E. coli and suggest that the strategy can be applied in E. coli or other bacterium-based production of NADPH-dependent compounds.

  12. Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant

    PubMed Central

    Mohamed-Hussein, Zeti-Azura; Ng, Chyan Leong

    2016-01-01

    Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that’s highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity

  13. Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant.

    PubMed

    Seman-Kamarulzaman, Ahmad-Faris; Mohamed-Hussein, Zeti-Azura; Ng, Chyan Leong; Hassan, Maizom

    2016-01-01

    Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards

  14. Nicotinamide-induced mitophagy: event mediated by high NAD+/NADH ratio and SIRT1 protein activation.

    PubMed

    Jang, So-young; Kang, Hyun Tae; Hwang, Eun Seong

    2012-06-01

    Active autophagy coupled with rapid mitochondrial fusion and fission constitutes an important mitochondrial quality control mechanism and is critical to cellular health. In our previous studies, we found that exposure of cells to nicotinamide causes a decrease in mitochondrial content and an increase in mitochondrial membrane potential (MMP) by activating autophagy and inducing mitochondrial fragmentation. Here, we present evidence to show that the effect of nicotinamide is mediated through an increase of the [NAD(+)]/[NADH] ratio and the activation of SIRT1, an NAD(+)-dependent deacetylase that plays a role in autophagy flux. The [NAD(+)]/[NADH] ratio was inversely correlated with the mitochondrial content, and an increase in the ratio by the mobilization of the malate-aspartate shuttle resulted in autophagy activation and mitochondrial transformation from lengthy filaments to short dots. Furthermore, treatment of cells with SIRT1 activators, fisetin or SRT1720, induced similar changes in the mitochondrial content. Importantly, the activators induced mitochondrial fragmentation only when SIRT1 expression was intact. Meanwhile, MMP did not increase when the cells were treated with the activators, suggesting that the change in MMP is not induced by the mitochondrial turnover per se and that elevation of the [NAD(+)]/[NADH] ratio may activate additional mechanisms that cause MMP augmentation. Together, our results indicate that a metabolic state resulting in an elevated [NAD(+)]/[NADH] ratio can modulate mitochondrial quantity and quality via pathways that may include SIRT1-mediated mitochondrial autophagy.

  15. The structural requirements of organophosphorus insecticides (OPI) for reducing chicken embryo NAD(+) content in OPI-induced teratogenesis in chickens.

    PubMed

    Seifert, Josef

    2016-05-01

    The objective of this study was to determine the structural requirements of organophosphorus insecticides (OPI) for reducing chicken embryo nicotinamide adenine dinucleotide (NAD(+)) content in OPI-induced teratogenesis and compare them with those needed for OPI inhibition of yolk sac membrane kynurenine formamidase (KFase), the proposed primary target for OPI teratogens in chicken embryos. The comparative molecular field analysis (COMFA) of three-dimensional quantitative structure-activity relationship (3D QSAR) revealed the electrostatic and steric fields as good predictors of OPI structural requirements to reduce NAD(+) content in chicken embryos. The dominant electrostatic interactions were localized at nitrogen-1, nitrogen-3, nitrogen of 2-amino substituent of the pyrimidinyl of pyrimidinyl phosphorothioates, and at the oxygen of crotonamide carbonyl in crotonamide phosphates. Bulkiness of the substituents at carbon-6 of the pyrimidinyls and/or N-substituents of crotonamides was the steric structural component that contributed to superiority of those OPI for reducing embryonic NAD(+) levels. Both electrostatic and steric requirements are similar to those defined in our previous study for OPI inhibition of chicken embryo yolk sac membrane KFase. The findings of this study provide another piece of evidence for the cause-and-effect relationship between yolk sac membrane KFase inhibition and reduced embryo NAD(+) content in NAD-associated OPI-induced teratogenesis in chickens.

  16. Predicted Role of NAD Utilization in the Control of Circadian Rhythms during DNA Damage Response

    PubMed Central

    Luna, Augustin; McFadden, Geoffrey B.; Aladjem, Mirit I.; Kohn, Kurt W.

    2015-01-01

    The circadian clock is a set of regulatory steps that oscillate with a period of approximately 24 hours influencing many biological processes. These oscillations are robust to external stresses, and in the case of genotoxic stress (i.e. DNA damage), the circadian clock responds through phase shifting with primarily phase advancements. The effect of DNA damage on the circadian clock and the mechanism through which this effect operates remains to be thoroughly investigated. Here we build an in silico model to examine damage-induced circadian phase shifts by investigating a possible mechanism linking circadian rhythms to metabolism. The proposed model involves two DNA damage response proteins, SIRT1 and PARP1, that are each consumers of nicotinamide adenine dinucleotide (NAD), a metabolite involved in oxidation-reduction reactions and in ATP synthesis. This model builds on two key findings: 1) that SIRT1 (a protein deacetylase) is involved in both the positive (i.e. transcriptional activation) and negative (i.e. transcriptional repression) arms of the circadian regulation and 2) that PARP1 is a major consumer of NAD during the DNA damage response. In our simulations, we observe that increased PARP1 activity may be able to trigger SIRT1-induced circadian phase advancements by decreasing SIRT1 activity through competition for NAD supplies. We show how this competitive inhibition may operate through protein acetylation in conjunction with phosphorylation, consistent with reported observations. These findings suggest a possible mechanism through which multiple perturbations, each dominant during different points of the circadian cycle, may result in the phase advancement of the circadian clock seen during DNA damage. PMID:26020938

  17. The NAD(+) salvage pathway modulates cancer cell viability via p73.

    PubMed

    Sharif, T; Ahn, D-G; Liu, R-Z; Pringle, E; Martell, E; Dai, C; Nunokawa, A; Kwak, M; Clements, D; Murphy, J P; Dean, C; Marcato, P; McCormick, C; Godbout, R; Gujar, S A; Lee, P W K

    2016-04-01

    The involvement of the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD(+) salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD(+) salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.

  18. Nuclear resonance vibrational spectroscopy reveals the FeS cluster composition and active site vibrational properties of an O2-tolerant NAD+-reducing [NiFe] hydrogenase

    DOE PAGES

    Lauterbach, Lars; Wang, Hongxin; Horch, Marius; Gee, Leland B.; Yoda, Yoshitaka; Tanaka, Yoshihito; Zebger, Ingo; Lenz, Oliver; Cramer, Stephen P.

    2014-10-30

    Hydrogenases are complex metalloenzymes that catalyze the reversible splitting of molecular hydrogen into protons and electrons essentially without overpotential. The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha is capable of H2 conversion even in the presence of usually toxic dioxygen. The molecular details of the underlying reactions are largely unknown, mainly because of limited knowledge of the structure and function of the various metal cofactors present in the enzyme. Here, all iron-containing cofactors of the SH were investigated by 57Fe specific nuclear resonance vibrational spectroscopy (NRVS). Our data provide experimental evidence for one [2Fe2S] center and four [4Fe4S] clusters, whichmore » is consistent with the amino acid sequence composition. Only the [2Fe2S] cluster and one of the four [4Fe4S] clusters were reduced upon incubation of the SH with NADH. This finding explains the discrepancy between the large number of FeS clusters and the small amount of FeS cluster-related signals as detected by electron paramagnetic resonance spectroscopic analysis of several NAD+-reducing hydrogenases. For the first time, Fe–CO and Fe–CN modes derived from the [NiFe] active site could be distinguished by NRVS through selective 13C labeling of the CO ligand. This strategy also revealed the molecular coordinates that dominate the individual Fe–CO modes. The present approach explores the complex vibrational signature of the Fe–S clusters and the hydrogenase active site, thereby showing that NRVS represents a powerful tool for the elucidation of complex biocatalysts containing multiple cofactors.« less

  19. Transcriptional activation of NAD{sup +}-dependent protein deacetylase SIRT1 by nuclear receptor TLX

    SciTech Connect

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

    2009-09-04

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD{sup +}-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  20. p-Hydroxyphenylacetic Acid Metabolism in Pseudomonas putida F6

    PubMed Central

    O'Connor, Kevin E.; Witholt, Bernard; Duetz, Wouter

    2001-01-01

    Pseudomonas putida F6 was found to metabolize p-hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxybenzaldehyde. Cell extracts of P. putida F6 catalyze the NAD(P)H-independent hydroxylation of p-hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid. Oxidation and decarboxylation of the latter yields 3,4-dihydroxybenzaldehyde. A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds. 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage. PMID:11208791

  1. 23Na magic-angle spinning nuclear magnetic resonance of central and satellite transitions in the characterization of the anhydrous, dihydrate, and mixed phases of sodium molybdate and tungstate.

    PubMed

    Skibsted, J; Jakobsen, H J

    1994-02-01

    23Na Magic-angle spinning nuclear magnetic resonance (MAS NMR) spectra of pure phases for Na2MoO4, Na2MoO4 x 2H2O, Na2WO4, and Na2WO4 x 2H2O have led to the determination of accurate values for the quadrupole coupling parameters and isotropic chemical shifts for all Na sites. The analysis of the spectra involves a combination of simulations of the line shapes for the central transitions and the manifold of spinning sidebands for the satellite transitions. The spectral parameters for the pure phases represent a prerequisite for a correct assignment and quantitative evaluation of 23Na MAS spectra at different magnetic field strengths observed for mixtures of the anhydrous and dihydrate phases. Such phase mixtures are observed, for example, for some commercial samples of Na2MoO4 or may be generated by (i) exposure of the anhydrous phases to a humid atmosphere or (ii) gently heating the dihydrates. The quadrupole coupling parameters for the two Na sites in the dihydrates are tentatively assigned to the two crystallographically distinct Na atoms in the asymmetric unit by calculations of an approximate dependency of the electric field gradient tensor on the local geometry for the Na sites.

  2. Hydroperoxidic inhibitor of horse liver alcohol dehydrogenase activity, tightly bound to the enzyme-NAD+ complex, characteristically degrades the coenzyme.

    PubMed

    Skurský, L; Rezác, M; Khan, A N; Zídek, L; Rocek, J

    1992-01-01

    The strong inhibition of horse liver alcohol dehydrogenase (HLAD) by p-methylbenzyl hydroperoxide (XyHP) is only transient, XyHP behaves also as a pseudo-substrate of the enzyme and in the presence of NAD+, is degraded by HLAD to (as yet unidentified) non-inhibiting products while the NAD+ is converted to a derivative similar to the "NADX", originally observed in an analogous reaction of HLAD with hydrogen peroxide. The apparent KM for XyHP is approximately 10(4) times smaller than that for H2O2. The catalytic constant kcat for HLAD degradation of XyHP is two orders of magnitude less than that for ethanol dehydrogenation. XyHP inhibits both directions of the alcohol-aldehyde interconversion with equal potency. The first step of the inhibition mechanism is a tight binding of XyHP to the binary HLAD-NAD+ complex. PMID:1284958

  3. Seroprevalence of antibodies against fHbp and NadA, two potential vaccine antigens for Neisseria meningitidis.

    PubMed

    Jacobsson, Susanne; Mölling, Paula; Olcén, Per

    2009-09-25

    The IgG antibody levels directed against fHbp and NadA, two potential vaccine antigens for Neisseria meningitidis, were examined in order to investigate the extent of natural immunisation against these antigens in different age groups. As a comparison, the IgG antibody levels against Haemophilus influenzae type b were examined. In the two youngest age groups, below 10 years of age, relatively low levels of both anti-fHbp and anti-NadA were measured. A 15-fold higher geometric mean concentration of anti-fHbp was noted in the age group 20-29 years as compared to the age group 15-19 years. The peak concentration was found at 30-39 years, followed by decreased levels with age. Anti-NadA showed a certain increase up to 9 years followed by an even increase up to 40-49 years.

  4. Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38

    SciTech Connect

    Aksoy, Pinar; Escande, Carlos; White, Thomas A.; Thompson, Michael; Soares, Sandra; Benech, Juan Claudio; Chini, Eduardo N. . E-mail: chini.eduardo@mayo.edu

    2006-10-13

    The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes.

  5. Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

    PubMed Central

    Ma, Yan; Ludden, Paul W.

    2001-01-01

    Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria. Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of the nifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding. PMID:11114923

  6. Transgenic Perturbation of the Decarboxylation Phase of Crassulacean Acid Metabolism Alters Physiology and Metabolism But Has Only a Small Effect on Growth1[OPEN

    PubMed Central

    Dever, Louisa V.; Boxall, Susanna F.; Kneřová, Jana

    2015-01-01

    Mitochondrial NAD-malic enzyme (ME) and/or cytosolic/plastidic NADP-ME combined with the cytosolic/plastidic pyruvate orthophosphate dikinase (PPDK) catalyze two key steps during light-period malate decarboxylation that underpin secondary CO2 fixation in some Crassulacean acid metabolism (CAM) species. We report the generation and phenotypic characterization of transgenic RNA interference lines of the obligate CAM species Kalanchoë fedtschenkoi with reduced activities of NAD-ME or PPDK. Transgenic line rNAD-ME1 had 8%, and rPPDK1 had 5% of the wild-type level of activity, and showed dramatic changes in the light/dark cycle of CAM CO2 fixation. In well-watered conditions, these lines fixed all of their CO2 in the light; they thus performed C3 photosynthesis. The alternative malate decarboxylase, NADP-ME, did not appear to compensate for the reduction in NAD-ME, suggesting that NAD-ME was the key decarboxylase for CAM. The activity of other CAM enzymes was reduced as a consequence of knocking out either NAD-ME or PPDK activity, particularly phosphoenolpyruvate carboxylase (PPC) and PPDK in rNAD-ME1. Furthermore, the circadian clock-controlled phosphorylation of PPC in the dark was reduced in both lines, especially in rNAD-ME1. This had the consequence that circadian rhythms of PPC phosphorylation, PPC kinase transcript levels and activity, and the classic circadian rhythm of CAM CO2 fixation were lost, or dampened toward arrhythmia, under constant light and temperature conditions. Surprisingly, oscillations in the transcript abundance of core circadian clock genes also became arrhythmic in the rNAD-ME1 line, suggesting that perturbing CAM in K. fedtschenkoi feeds back to perturb the central circadian clock. PMID:25378692

  7. Treatment with NAD(+) inhibited experimental autoimmune encephalomyelitis by activating AMPK/SIRT1 signaling pathway and modulating Th1/Th17 immune responses in mice.

    PubMed

    Wang, Jueqiong; Zhao, Congying; Kong, Peng; Sun, Huanhuan; Sun, Zhe; Bian, Guanyun; Sun, Yafei; Guo, Li

    2016-10-01

    Nicotinamide adenine dinucleotide (NAD(+)) plays vital roles in mitochondrial functions, cellular energy metabolism and calcium homeostasis. In this study, we investigated the effect of NAD(+) administration for the treatment of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. EAE, a classical animal model of multiple sclerosis (MS), was induced by subcutaneous injection of myelin oligodendrocyteglycoprotein (MOG). The mice were treated with 250mg/kg (body weight) NAD(+) in PBS administered intraperitoneally once daily. We observed that NAD(+) treatment could lessen the severity of EAE. Additionally, NAD(+) treatment attenuated pathological injuries of EAE mice. We also found that the AMP-activated protein kinase (AMPK)/silent mating-type information regulation 2 homolog 1(SIRT1) pathway was activated in the NAD(+)-treated mice and NAD(+) treatment suppressed pro-inflammatory T cell responses. Our findings demonstrated that NAD(+) could be an effective and promising agent to treat multiple sclerosis and its effects on other autoimmune diseases should be explored. PMID:27500459

  8. Involvement of the nadA gene in formation of G-group aflatoxins in Aspergillus parasiticus.

    PubMed

    Cai, Jingjing; Zeng, Hongmei; Shima, Yoko; Hatabayashi, Hidemi; Nakagawa, Hiroyuki; Ito, Yasuhiro; Adachi, Yoshikazu; Nakajima, Hiromitsu; Yabe, Kimiko

    2008-07-01

    The nadA gene is present at the end of the aflatoxin gene cluster in the genome of Aspergillus parasiticus as well as in Aspergillus flavus. RT-PCR analyses showed that the nadA gene was expressed in an aflatoxin-inducible YES medium, but not in an aflatoxin-non-inducible YEP medium. The nadA gene was not expressed in the aflR gene-deletion mutant, irrespective of the culture medium used. To clarify the nadA gene's function, we disrupted the gene in aflatoxigenic A. parasiticus. The four nadA-deletion mutants that were isolated commonly accumulated a novel yellow-fluorescent pigment (named NADA) in mycelia as well as in culture medium. When the mutants and the wild-type strain were cultured for 3 days in YES medium, the mutants each produced about 50% of the amounts of G-group aflatoxins that the wild-type strain produced. In contrast, the amounts of B-group aflatoxins did not significantly differ between the mutants and the wild-type strain. The NADA pigment was so unstable that it could non-enzymatically change to aflatoxin G(1) (AFG(1)). LC-MS measurement showed that the molecular mass of NADA was 360, which is 32 higher than that of AFG(1). We previously reported that at least one cytosol enzyme, together with two other microsome enzymes, is necessary for the formation of AFG(1) from O-methylsterigmatocystin (OMST) in the cell-free system of A. parasiticus. The present study confirmed that the cytosol fraction of the wild-type A.parasiticus strain significantly enhanced the AFG(1) formation from OMST, whereas the cytosol fraction of the nadA-deletion mutant did not show the same activity. Furthermore, the cytosol fraction of the wild-type strain showed the enzyme activity catalyzing the reaction from NADA to AFG(1), which required NADPH or NADH, indicating that NADA is a precursor of AFG(1); in contrast, the cytosol fraction of the nadA-deletion mutant did not show the same enzyme activity. These results demonstrated that the NadA protein is the cytosol enzyme

  9. NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms.

    PubMed

    Love, Nick R; Pollak, Nadine; Dölle, Christian; Niere, Marc; Chen, Yaoyao; Oliveri, Paola; Amaya, Enrique; Patel, Sandip; Ziegler, Mathias

    2015-02-01

    Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD(+) kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca(2+)/calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms.

  10. NADPH regulates human NAD kinase, a NADP⁺-biosynthetic enzyme.

    PubMed

    Ohashi, Kazuto; Kawai, Shigeyuki; Koshimizu, Mari; Murata, Kousaku

    2011-09-01

    NAD kinase (NADK, EC 2.7.1.23) is the sole NADP(+)-biosynthetic enzyme that catalyzes phosphorylation of NAD(+) to yield NADP(+) using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69-74, 2001), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of NADP(+), on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by NADP(+).

  11. Subcellular localization of the NAD+-dependent alcohol dehydrogenase in Entamoeba histolytica trophozoites.

    PubMed

    Avila, Eva E; Martínez-Alcaraz, Edith R; Barbosa-Sabanero, Gloria; Rivera-Baron, Elda I; Arias-Negrete, Sergio; Zazueta-Sandoval, Roberto

    2002-04-01

    The protozoan parasite Entamoeba histolytica is an ancient eukaryotic cell that shows morphologically atypical organelles and differs metabolically from higher eukaryotic cells. The aim of this study was to determine the subcellular localization of ameba NAD+-dependent alcohol dehydrogenase (ADH2). The enzyme activity was present in soluble and mainly in particulate material whose density was 1.105 in a sucrose gradient. By differential centrifugation, most of the ADH activity sedimented at 160,000 g (160,000-g pellet), similar to the Escherichia coli polymeric ADHE. In the Coomassie staining of the 160,000-g pellet analyzed by electrophoresis, a 96-kDa protein was more prominent than in other fractions; this band was recognized by antibodies against Lactococcus lactis ADHE. By gold labeling, the antibodies recognized the granular material that mainly constitutes the 160,000-g pellet and a material that sedimented along with the internal membrane vesicles. By negative staining, the 160,000-g fraction showed helical rodlike structures with an average length of 103 nm; almost no membrane vesicles were observed in this pellet. In internal membrane fractions, no rodlike structures were found, but protomerlike round structures were observed. These results indicate that the main amebic NAD+-dependent ADH2 activity is naturally organized as rodlike helical particles, similar to bacterial ADHE. Detection of ADH2 in membrane fractions might be explained by cosedimentation of the multimeric ADH during membrane purification.

  12. A High Fat Diet and NAD+ Rescue Premature Aging in Cockayne Syndrome

    PubMed Central

    Scheibye-Knudsen, Morten; Mitchell, Sarah J.; Fang, Evandro F.; Iyama, Teruaki; Ward, Theresa; Wang, James; Dunn, Christopher A.; Singh, Nagendra; Veith, Sebastian; Hasan, M. Mahdi; Mangerich, Aswin; Wilson, Mark A.; Mattson, Mark P.; Bergersen, Linda H.; Cogger, Victoria C.; Warren, Alessandra; Le Couteur, David G.; Moaddel, Ruin; Wilson, David M.; Croteau, Deborah L.; de Cabo, Rafael; Bohr, Vilhelm A.

    2014-01-01

    Summary Cockayne syndrome (CS) is an accelerated aging disorder characterized by progressive neurodegeneration caused by mutations in the genes encoding the DNA repair proteins CSA or CSB. Csbm/m mice were given a high fat, caloric restricted or resveratrol supplemented diet. The high fat diet rescued the phenotype of Csbm/m mice at the metabolic, transcriptomic and behavioral levels. Additional analysis suggests that the premature aging seen in CS mice, nematodes and human cells results from aberrant PARP activation due to deficient DNA repair leading to decreased SIRT1 activity and mitochondrial dysfunction. Notably, β-hydroxybutyrate levels are increased by the high fat diet; and β-hydroxybutyrate, PARP inhibition, or NAD+ supplementation can activate SIRT1 and rescue CS-associated phenotypes. Mechanistically, CSB is able to displace activated PARP1 from damaged DNA to limit its activity. This study connects two emerging longevity metabolites, β-hydroxybutyrate and NAD+, through the deacetylase SIRT1 and suggests possible interventions for CS. PMID:25440059

  13. Atomic Resolution Crystal Structure of NAD+-Dependent Formate Dehydrogenase from Bacterium Moraxella sp. C-1

    PubMed Central

    Shabalin, I.G.; Polyakov, K.M.; Tishkov, V.I.

    2009-01-01

    The crystal structure of the ternary complex of NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Moraxella sp. C-1 with the cofactor (NAD+) and the inhibitor (azide ion) was established at 1.1 A resolution. The complex mimics the structure of the transition state of the enzymatic reaction. The structure was refined with anisotropic displacitalicents parameters for non-hydrogen atoms to a R factor of 13.4%. Most of the nitrogen, oxygen, and carbon atoms were distinguished based on the analysis of the titalicperature factors and electron density peaks, with the result that side-chain rotamers of histidine residues and most of asparagine and glutamine residues were unambiguously determined. A comparative analysis of the structure of the ternary complex determined at the atomic resolution and the structure of this complex at 1.95 A resolution was performed. In the atomic resolution structure, the covalent bonds in the nicotinamide group are somewhat changed in agreitalicent with the results of quantum mechanical calculations, providing evidence that the cofactor acquires a bipolar form in the transition state of the enzymatic reaction. PMID:22649619

  14. A simple plate-assay for the screening of L-malic acid producing microorganisms.

    PubMed

    Peleg, Y; Rokem, J S; Goldberg, I

    1990-02-01

    A simple plate-assay has been developed to screen microorganisms for L-malic acid production. Acid producing organisms were identified, after microbial colony growth on media containing glucose or fumaric acid as sole carbons sources, by formation of a dark halo of formazan. The halo was observed when the plate was covered with a soft agar overlay containing NAD(+)-malate dehydrogenase, NAD+, phenazine methosulfate (PMS) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay developed is simple, specific for L-malic acid and therefore can be used to identify L-malic acid producing filamentous fungi using glucose as carbon source (e.g. Aspergillus strains). The assay is also applicable for screening bacteria with high fumarase activity, able to convert fumaric acid to L-malic acid.

  15. Decoupling the effects of surface chemistry and humidity on solid-state hydrolysis of aspirin in the presence of dicalcium phosphate dihydrate.

    PubMed

    Cassidy, Andrew M; Gardner, Catherine E; Auffret, Tony; Aldous, Barry; Jones, William

    2012-04-01

    Atomic force microscopy (AFM) cantilevers were functionalized with particles of dicalcium phosphate dihydrate (DCP), and AFM, in force-displacement mode, was used to bring these probes into contact with aspirin (100) and (001) surfaces in order to investigate the effect of aspirin surface chemistry on the interaction between the two materials as a function of relative humidity (RH). The force of adhesion measurements showed a strong dependence on RH for the interactions between DCP and the aspirin (100) surface, with stronger interactions occurring at higher humudities. Relatively much weaker interactions were measured between DCP and the aspirin (001) surface under all RH conditions. Topographic imaging showed that contact between DCP and the aspirin (100) surface at high RH led to localised development of etch pits and, in some cases, growth normal to the surface. The methodology allows for the creation of a localised solid-solid interface between pharmaceutically relevant materials, providing a means of studying solid-state excipient-active ingredient decomposition reactions.

  16. Study on third order nonlinear optical properties of a metal organic complex-Monothiourea-cadmium Sulphate Dihydrate single crystals grown in silica gel

    NASA Astrophysics Data System (ADS)

    Sivanandan, T.; Kalainathan, S.

    2015-04-01

    The third order nonlinear optical properties of Monothiourea-cadmium Sulphate Dihydrate crystal were measured using a He-Ne laser (λ=632.8 nm) by a Z-scan technique. The magnitude of nonlinear refractive index (n2) and nonlinear absorption coefficient was found to be 4.4769×10-11 m2/W and 1.233×10-2 m/W respectively. The third order non-linear optical susceptibility χ(3) was found to be in the order of 3.6533×10-2 esu. The negative sign of non-linear refractive index shows the self-defocusing nature of the gel grown crystal. The second-order molecular hyperpolarizability γ of the grown crystal is 1.2822×10-33 esu. Laser damage threshold was measured by using an Nd: YAG laser (1064 nm). Photoconductivity studies of the gel grown crystal revealed that the crystal possesses positive photoconducting nature. The results obtained from Z-scan, laser damage threshold and photoconducting studies reveal that the crystal can be a possible candidate material for photonics device, optical switches, and optical power limiting application.

  17. Foraminal deposition of calcium pyrophosphate dihydrate crystals in the thoracic spine: possible relationship with disc herniation and implications for surgical planning. Report of two cases.

    PubMed

    Paolini, Sergio; Ciappetta, Pasquale; Guiducci, Antonio; Principi, Massimo; Missori, Paolo; Delfini, Roberto

    2005-01-01

    The authors report two cases of nodular calcium pyrophosphate dihydrate (CPPD) crystal deposition close to the thoracic neural foramen, which caused chronic radiculopathy. Preoperatively, the lesions were interpreted as calcified disc herniations. Both patients underwent surgery in which an extended transfacet pedicle-sparing approach was used. Incision of the posterior longitudinal ligament released soft degenerated material. In both cases, histological examination showed abundant degenerative debris along with CPPD crystals. Spinal CPPD deposition is a comparatively rare disease that almost invariably involves the posterior aspect of the spinal canal, typically the ligamentum flavum. The exceptional foraminal location of the lesions reported here, combined with the surgical findings, indicated that the CPPD crystals were deposited on a laterally herniated disc fragment. A distinctive feature in both cases was the soft consistency of the resected tissue. The consistency of the disc material and the location of the lesion in the axial plane (that is, median compared with lateral) are key factors in determining the optimal surgical approach to thoracic disc herniations. In describing consistency, terms such as "calcified" and "hard" have been used interchangeably in the literature. In the cases reported here, what appeared on computerized tomography and magnetic resonance imaging studies to be densely calcified lesions were shown intraoperatively to be soft herniations. The authors' experience underscores that not all densely calcified herniated discs are hard. Although detection of this discrepancy would have left surgical planning for the lateral disc herniations unchanged, it could have altered planning for centrally or centrolaterally located disc herniations.

  18. Analysis of DBC1 and its homologs suggests a potential mechanism for regulation of Sirtuin domain deacetylases by NAD metabolites

    PubMed Central

    Anantharaman, Vivek; Aravind, L.

    2008-01-01

    Deleted in Breast Cancer-1 (DBC1) and its paralog CARP-1 are large multi-domain proteins, with a nuclear or perinuclear localization, and a role in promoting apoptosis upon processing by caspases. Recent studies on human DBC1 show that it is a specific inhibitor of the sirtuin-type deacetylase, Sirt1, which deacetylates histones and p53. Using sensitive sequence profile searches and HMM-HMM comparisons we show that the central conserved globular domain present in the DBC1 and it homologs from diverse eukaryotes is a catalytically inactive version of the Nudix hydrolase (MutT) domain. Given that Nudix domains are known to bind nucleoside diphosphate sugars and NAD, we predict that this domain in DBC1 and its homologs binds NAD metabolites such as ADP-ribose. Hence, we propose that DBC1 and its homologs are likely to regulate the activity of SIRT1 or related deacetylases by sensing the soluble products or substrates of the NAD-dependent deacetylation reaction. The complex domain architectures of the members of the DBC1 family, which include fusions to the RNA-binding S1-like domain, the DNA-binding SAP domain and EF-hand domains, suggest that they are likely to function as integrators of distinct regulatory signals including chromatin protein modification, soluble compounds in NAD metabolism, apoptotic stimuli and RNA recognition. PMID:18418069

  19. The respiratory chain provides salt stress tolerance by maintaining a low NADH/NAD+ ratio in Zymomonas mobilis.

    PubMed

    Hayashi, Takeshi; Kato, Tsuyoshi; Watakabe, Satoshi; Song, Wonjoon; Aikawa, Shizuho; Furukawa, Kensuke

    2015-12-01

    The respiratory chain of ethanol-producing Zymomonas mobilis shows an unusual physiological property in that it is not involved in energy conservation, even though this organism has a complete electron transport system. We reported previously that respiratory-deficient mutants (RDMs) of Z. mobilis exhibit higher growth rates and enhanced ethanol productivity under aerobic and high-temperature conditions. Here, we demonstrated that the salt tolerance of RDM strains was drastically decreased compared with the wild-type strain. We found that the NADH/NAD+ ratio was maintained at low levels in both the wild-type and the RDM strains under non-stress conditions. However, the ratio substantially increased in the RDM strains in response to salt stress. Complementation of the deficient respiratory-chain genes in the RDM strains resulted in a decrease in the NADH/NAD+ ratio and an increase in the growth rate. In contrast, expression of malate dehydrogenase, activity of which increases the supply of NADH, in the RDM strains led to an increased NADH/NAD+ ratio and resulted in poor growth. Taken together, these results suggest that the respiratory chain of Z. mobilis functions to maintain a low NADH/NAD+ ratio when the cells are exposed to environmental stresses, such as salinity. PMID:26432557

  20. Cytosolic NADH-NAD+ Redox Visualized in Brain Slices by Two-Photon Fluorescence Lifetime Biosensor Imaging

    PubMed Central

    Mongeon, Rebecca; Venkatachalam, Veena

    2016-01-01

    Abstract Aim: Cytosolic NADH-NAD+ redox state is central to cellular metabolism and a valuable indicator of glucose and lactate metabolism in living cells. Here we sought to quantitatively determine NADH-NAD+ redox in live cells and brain tissue using a fluorescence lifetime imaging of the genetically-encoded single-fluorophore biosensor Peredox. Results: We show that Peredox exhibits a substantial change in its fluorescence lifetime over its sensing range of NADH-NAD+ ratio. This allows changes in cytosolic NADH redox to be visualized in living cells using a two-photon scanning microscope with fluorescence lifetime imaging capabilities (2p-FLIM), using time-correlated single photon counting. Innovation: Because the lifetime readout is absolutely calibrated (in nanoseconds) and is independent of sensor concentration, we demonstrate that quantitative assessment of NADH redox is possible using a single fluorophore biosensor. Conclusion: Imaging of the sensor in mouse hippocampal brain slices reveals that astrocytes are typically much more reduced (with higher NADH:NAD+ ratio) than neurons under basal conditions, consistent with the hypothesis that astrocytes are more glycolytic than neurons. Antioxid. Redox Signal. 25, 553–563. PMID:26857245

  1. Membrane permeability of redox active metal chelators: an important element in reducing hydroxyl radical induced NAD+ depletion in neuronal cells.

    PubMed

    Jayasena, T; Grant, R S; Keerthisinghe, N; Solaja, I; Smythe, G A

    2007-03-01

    There is substantial evidence implicating increased production of the hydroxyl radical and oxidative stress in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD). Significant amounts of hydroxyl radicals will be produced in the presence of hydrogen peroxide and redox active iron via Fenton chemistry. Increased iron levels within the cytoplasm of vulnerable neurons suggest that this may also be an important site of oxidative activity. We investigated the likelihood that intracellular, rather than extracellular chelation of ferrous or ferric iron may be more effective in reducing hydroxyl radical induced cell damage and preserving NAD(+) levels and cell viability. Using intracellular NAD(H) measurements as an indicator of cell viability we found that membrane permeable ferrous chelators were most efficient in preserving cellular NAD(+) levels. Hydrophilic, ferrous or ferric chelators and lipophilic ferric chelators were essentially ineffective in preventing cellular NAD(+) depletion when added at physiological concentrations. We propose that lipophilic ferrous chelators, due to their actions inside the cell, are effective agents for moderating neuronal damage in conditions such as AD where intracellular oxidative stress plays a significant role in disease pathology. PMID:17210195

  2. The Differences between NAD-ME and NADP-ME Subtypes of C4 Photosynthesis: More than Decarboxylating Enzymes

    PubMed Central

    Rao, Xiaolan; Dixon, Richard A.

    2016-01-01

    As an adaptation to changing climatic conditions that caused high rates of photorespiration, C4 plants have evolved to display higher photosynthetic efficiency than C3 plants under elevated temperature, high light intensities, and drought. The C4 plants independently evolved more than 60 times in 19 families of angiosperms to establish similar but not uniform C4 mechanisms to concentrate CO2 around the carboxylating enzyme Rubisco (ribulose bisphosphate carboxylase oxygenase). C4 photosynthesis is divided into at least two basic biochemical subtypes based on the primary decarboxylating enzymes, NAD-dependent malic enzyme (NAD-ME) and NADP-dependent malic enzyme (NADP-ME). The multiple polygenetic origins of these subtypes raise questions about the association of C4 variation between biochemical subtypes and diverse lineages. This review addresses the differences in evolutionary scenario, leaf anatomy, and especially C4 metabolic flow, C4 transporters, and cell-specific function deduced from recently reported cell-specific transcriptomic, proteomic, and metabolic analyses of NAD-ME and NADP-ME subtypes. Current omic analysis has revealed the extent to which component abundances differ between the two biochemical subtypes, leading to a better understanding of C4 photosynthetic mechanisms in NAD-ME and NADP-ME subtypes. PMID:27790235

  3. A modular system for regeneration of NAD cofactors using graphite particles modified with hydrogenase and diaphorase moieties.

    PubMed

    Reeve, Holly A; Lauterbach, Lars; Ash, Philip A; Lenz, Oliver; Vincent, Kylie A

    2012-02-01

    Pyrolytic graphite particles modified with hydrogenase and an NAD(+)/NADH cycling enzyme provide a modular heterogeneous catalyst system for regeneration of oxidised or reduced nicotinamide cofactors using H(2) and H(+) as electron source or sink. Particles can be tuned for cofactor supply under different conditions by appropriate choice of hydrogenase. PMID:21986817

  4. Streptococcus mutans NADH Oxidase Lies at the Intersection of Overlapping Regulons Controlled by Oxygen and NAD+ Levels

    PubMed Central

    Baker, J. L.; Derr, A. M.; Karuppaiah, K.; MacGilvray, M. E.; Kajfasz, J. K.; Faustoferri, R. C.; Rivera-Ramos, I.; Bitoun, J. P.; Lemos, J. A.; Wen, Z. T.

    2014-01-01

    NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD+. The critical nature of Nox is 2-fold: it serves to regenerate NAD+, a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD+ have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD+ affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress. PMID:24682329

  5. NAD(+)- dependent deacetylase SIRT3 regulates mitochondrial protein synthesis by deacetylation of the ribosomal protein MRPL10

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A member of the sirtuin family of NAD (+)-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated ...

  6. [Report on a measles epidemic in the Ústí nad Labem Region].

    PubMed

    Trmal, J; Limberková, R

    2015-09-01

    The Czech Republic is a measles free country where only isolated, mostly imported cases have been reported. A measles epidemic that occurred in the Ústí nad Labem Region is presented, with the first case diagnosed early in February 2014 and the last one reported in August 2014. The index case and source of infection to other susceptible patients was an adult male with a history of travel to India. The diagnosis of measles was difficult to make as the patient presented with feverish condition due to co-infection with dengue fever, chikungunya, and measles. The primary measles outbreak occurred in contacts and spread to health workers of the Masaryk Hospital in Ústí nad Labem. The infection further spread to the general population of adults. In total, 305 persons presented with suspected measles. One hundred and eighty-six and of them (61%) met the criteria for a confirmed case (positive clinical symptoms and laboratory test). Fifty (16.4%) patients developed typical clinical symptoms and were epidemiologically linked to confirmed cases, but turned out to be antibody negative. In 69 (22.6%) patients, measles were ruled out. Cases were confirmed by the detection of IgM and IgG antibodies against the measles virus or by RT--PCR. Nearly all cases were verified by the National Reference Laboratory for Rubella, Measles, Mumps, and Parvovirus B 19 of the National Institute of Public Health in Prague. In response to the epidemic, apart from common anti-epidemic measures, emergency vaccination was provided to health professionals of the Masaryk Hospital in Ústí nad Labem. Within two weeks after vaccination, a considerable decline in cases was seen in the vaccinated group. Measles most often occurred in persons born in 1970-1980, but were also observed in the smallest, non-vaccinated children (32 cases). Isolated cases also emerged in duly vaccinated children and adolescents. General recommendations are provided based on practical experience from the epidemic. PMID

  7. [Report on a measles epidemic in the Ústí nad Labem Region].

    PubMed

    Trmal, J; Limberková, R

    2015-09-01

    The Czech Republic is a measles free country where only isolated, mostly imported cases have been reported. A measles epidemic that occurred in the Ústí nad Labem Region is presented, with the first case diagnosed early in February 2014 and the last one reported in August 2014. The index case and source of infection to other susceptible patients was an adult male with a history of travel to India. The diagnosis of measles was difficult to make as the patient presented with feverish condition due to co-infection with dengue fever, chikungunya, and measles. The primary measles outbreak occurred in contacts and spread to health workers of the Masaryk Hospital in Ústí nad Labem. The infection further spread to the general population of adults. In total, 305 persons presented with suspected measles. One hundred and eighty-six and of them (61%) met the criteria for a confirmed case (positive clinical symptoms and laboratory test). Fifty (16.4%) patients developed typical clinical symptoms and were epidemiologically linked to confirmed cases, but turned out to be antibody negative. In 69 (22.6%) patients, measles were ruled out. Cases were confirmed by the detection of IgM and IgG antibodies against the measles virus or by RT--PCR. Nearly all cases were verified by the National Reference Laboratory for Rubella, Measles, Mumps, and Parvovirus B 19 of the National Institute of Public Health in Prague. In response to the epidemic, apart from common anti-epidemic measures, emergency vaccination was provided to health professionals of the Masaryk Hospital in Ústí nad Labem. Within two weeks after vaccination, a considerable decline in cases was seen in the vaccinated group. Measles most often occurred in persons born in 1970-1980, but were also observed in the smallest, non-vaccinated children (32 cases). Isolated cases also emerged in duly vaccinated children and adolescents. General recommendations are provided based on practical experience from the epidemic.

  8. Crystallization and immersion freezing ability of oxalic and succinic acid in multicomponent aqueous organic aerosol particles

    NASA Astrophysics Data System (ADS)

    Wagner, Robert; Höhler, Kristina; Möhler, Ottmar; Saathoff, Harald; Schnaiter, Martin

    2015-04-01

    This study reports on heterogeneous ice nucleation efficiency of immersed oxalic and succinic acid crystals in the temperature range from 245 to 215 K, as investigated with expansion cooling experiments using suspended particles. In contrast to previous laboratory work with emulsified solution droplets where the precipitation of solid inclusions required a preceding freezing/evaporation cycle, we show that immersed solids readily form by homogeneous crystallization within aqueous solution droplets of multicomponent organic mixtures, which have noneutonic compositions with an excess of oxalic or succinic acid. Whereas succinic acid crystals did not act as heterogeneous ice nuclei, immersion freezing by oxalic acid dihydrate crystals led to a reduction of the ice saturation ratio at freezing onset by 0.066-0.072 compared to homogeneous freezing, which is by a factor of 2 higher than previously reported laboratory data. These observations emphasize the importance of oxalic acid in heterogeneous ice nucleation.

  9. Kinetic and structural basis for acyl-group selectivity and NAD+-dependence in Sirtuin-catalyzed deacylation

    PubMed Central

    Thelen, Julie N.; Ito, Akihiro; Yoshida, Minoru; Denu, John M.

    2015-01-01

    Acylation of lysine is an important protein modification regulating diverse biological processes. It was recently demonstrated that members of the human Sirtuin family are capable of catalyzing long-chain deacylation, in addition to the well-known NAD+-dependent deacetylation activity.1 Here we provide a detailed kinetic and structural analysis that describes the interdependence of NAD+ and acyl-group length for a diverse series of human Sirtuins, SIRT1, SIRT2, SIRT3 and SIRT6. Steady-state and rapid-quench kinetic analyses indicated that differences in NAD+ saturation and susceptibility to nicotinamide inhibition reflect unique kinetic behavior displayed by each Sirtuin and depend on acyl-substrate chain length. Though the rate of nucleophilic attack of the 2′-hydroxyl on the C1′-O-alkylimidate intermediate varies with acyl substrate chain length, this step remains rate-determining for SIRT2 and SIRT3; however for SIRT6, this step is no longer rate-limiting for long-chain substrates. Co-crystallization of SIRT2 with myristoylated peptide and NAD+ yielded a co-complex structure with reaction product 2′-O-myristoyl-ADP-ribose, revealing a latent hydrophobic cavity to accommodate the long chain acyl group, and suggesting a general mechanism for long chain deacylation. Comparing two separately solved co-complex structures containing either a myristoylated peptide or 2′-O-myristoyl-ADP-ribose indicate there are conformational changes at the myristoyl-ribose linkage with minimal structural differences in the enzyme active site. During the deacylation reaction, the fatty acyl group is held in a relatively fixed position. We describe a kinetic and structural model to explain how various Sirtuins display unique acyl-substrate preferences and how different reaction kinetics influence NAD+ dependence. The biological implications are discussed. PMID:25897714

  10. Crystal Structure of Human Dihydrolipoamide Dehydrogenase: NAD[superscript +]/NADH Binding and the Structural Basis of Disease-causing Mutations

    SciTech Connect

    Brautigam, Chad A.; Chuang, Jacinta L.; Tomchick, Diana R.; Machius, Mischa; Chuang, David T.

    2010-07-13

    Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial {alpha}-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: noncovalently bound FAD and a transiently bound substrate, NAD{sup +}. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD{sup +} or NADH have been determined at resolutions of 2.5 {angstrom} and 2.1 {angstrom}, respectively. Although the overall fold of the enzyme is similar to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD{sup +} bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD{sup +} or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD{sup +}-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.

  11. Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803.

    PubMed Central

    Valverde, F; Losada, M; Serrano, A

    1997-01-01

    The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD- to NADP-dependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis

  12. A NAD-dependent glutamate dehydrogenase coordinates metabolism with cell division in Caulobacter crescentus.

    PubMed

    Beaufay, François; Coppine, Jérôme; Mayard, Aurélie; Laloux, Géraldine; De Bolle, Xavier; Hallez, Régis

    2015-07-01

    Coupling cell cycle with nutrient availability is a crucial process for all living cells. But how bacteria control cell division according to metabolic supplies remains poorly understood. Here, we describe a molecular mechanism that coordinates central metabolism with cell division in the α-proteobacterium Caulobacter crescentus. This mechanism involves the NAD-dependent glutamate dehydrogenase GdhZ and the oxidoreductase-like KidO. While enzymatically active GdhZ directly interferes with FtsZ polymerization by stimulating its GTPase activity, KidO bound to NADH destabilizes lateral interactions between FtsZ protofilaments. Both GdhZ and KidO share the same regulatory network to concomitantly stimulate the rapid disassembly of the Z-ring, necessary for the subsequent release of progeny cells. Thus, this mechanism illustrates how proteins initially dedicated to metabolism coordinate cell cycle progression with nutrient availability.

  13. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2.

    PubMed

    Lavado-Roldán, Angela; Fernández-Chacón, Rafael

    2016-07-01

    One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation. PMID:27454736

  14. A NAD-dependent glutamate dehydrogenase coordinates metabolism with cell division in Caulobacter crescentus

    PubMed Central

    Beaufay, François; Coppine, Jérôme; Mayard, Aurélie; Laloux, Géraldine; De Bolle, Xavier; Hallez, Régis

    2015-01-01

    Coupling cell cycle with nutrient availability is a crucial process for all living cells. But how bacteria control cell division according to metabolic supplies remains poorly understood. Here, we describe a molecular mechanism that coordinates central metabolism with cell division in the α-proteobacterium Caulobacter crescentus. This mechanism involves the NAD-dependent glutamate dehydrogenase GdhZ and the oxidoreductase-like KidO. While enzymatically active GdhZ directly interferes with FtsZ polymerization by stimulating its GTPase activity, KidO bound to NADH destabilizes lateral interactions between FtsZ protofilaments. Both GdhZ and KidO share the same regulatory network to concomitantly stimulate the rapid disassembly of the Z-ring, necessary for the subsequent release of progeny cells. Thus, this mechanism illustrates how proteins initially dedicated to metabolism coordinate cell cycle progression with nutrient availability. PMID:25953831

  15. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2.

    PubMed

    Lavado-Roldán, Angela; Fernández-Chacón, Rafael

    2016-07-01

    One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation.

  16. Application of NAD-dependent polyol dehydrogenases for enzymatic mannitol/sorbitol production with coenzyme regeneration.

    PubMed

    Parmentier, S; Arnaut, F; Soetaert, W; Vandamme, E J

    2003-01-01

    D-Mannitol and D-sorbitol were produced enzymatically from D-fructose using NAD-dependent polyol dehydrogenases. For the production of D-mannitol the Leuconostoc mesenteroides mannitol dehydrogenase could be used. Gluconobacter oxydans cell extract contained however both mannitol and sorbitol dehydrogenase. When this cell extract was used, the reduction of D-fructose resulted in a mixture of D-sorbitol and D-mannitol. To determine the optimal bioconversion conditions the polyol dehydrogenases were characterized towards pH- and temperature-optimum and -stability. As a compromise between enzyme activity and stability, the bioconversion reactions were performed at pH 6.5 and 25 degrees C. Since the polyol dehydrogenases are NADH-dependent, an efficient coenzyme regeneration was needed. Regeneration of NADH was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2.

  17. Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2

    PubMed Central

    2016-01-01

    One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation. PMID:27454736

  18. Immunoreactive insulin in diabetes mellitus patient sera detected by ultrasensitive ELISA with thio-NAD cycling.

    PubMed

    Ito, Etsuro; Kaneda, Mugiho; Kodama, Hiromi; Morikawa, Mika; Tai, Momoko; Aoki, Kana; Watabe, Satoshi; Nakaishi, Kazunari; Hashida, Seiichi; Tada, Satoshi; Kuroda, Noriyuki; Imachi, Hitomi; Murao, Koji; Yamashita, Masakane; Yoshimura, Teruki; Miura, Toshiaki

    2015-12-01

    To minimize patient suffering, the smallest possible volume of blood should be collected for diagnosis and disease monitoring. When estimating insulin secretion capacity and resistance to insulin in diabetes mellitus (DM), increasing insulin assay immunosensitivity would reduce the blood sample volume required for testing. Here we present an ultrasensitive ELISA coupled with thio-NAD cycling to measure immunoreactive insulin in blood serum. Only 5 μL of serum was required for testing, with a limit of detection (LOD) for the assay of 10(-16) moles/assay. Additional recovery tests confirmed this method can detect insulin in sera. Comparisons between a commercially available immunoreactive insulin kit and our ultrasensitive ELISA using the same commercially available reference demonstrated good data correlation, providing further evidence of assay accuracy. Together, these results demonstrate our ultrasensitive ELISA could be a powerful tool in the diagnosis and treatment of not only DM but also many other diseases in the future.

  19. Exogenous NAD(+) decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy.

    PubMed

    Zhu, Ying; Zhao, Ke-Ke; Tong, Yao; Zhou, Ya-Li; Wang, Yi-Xiao; Zhao, Pei-Quan; Wang, Zhao-Yang

    2016-05-31

    Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD(+)) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD(+) administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD(+) administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD(+) administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD(+) against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD(+) administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD(+) administration might be potential value for the treatment of AMD.

  20. Exogenous NAD+ decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy

    PubMed Central

    Zhu, Ying; Zhao, Ke-ke; Tong, Yao; Zhou, Ya-li; Wang, Yi-xiao; Zhao, Pei-quan; Wang, Zhao-yang

    2016-01-01

    Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD+) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD+ administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD+ administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD+ administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD+ against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD+ administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD+ administration might be potential value for the treatment of AMD. PMID:27240523

  1. Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis

    SciTech Connect

    Xi, Wenjun

    2008-01-01

    A capillary electrophoresis (CE) method is developed to determine both NAD+ and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD+ and 1 amol NADH on a home-made CE-LIF setup. The method showed good reproducibility and specificity. After an intact cell was injected into the inlet of a capillary and lysed using a Tesla coil, intracellular NAD+ and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD+ levels of single cells of three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD+ and NADH levels with and without exposure to oxidative stress induced by H2O2, it was found that H9c2 cells respond to the stress by reducing both cellular NAD+ and NADH levels, while astrocytes respond by increasing cellular NADH/NAD+ ratio.

  2. Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes*

    PubMed Central

    Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.; Jensen, Pernille Rose; Lerche, Mathilde H.

    2014-01-01

    Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD+]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD+]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments. PMID:24302737

  3. MNADK, a Long-Awaited Human Mitochondrion-Localized NAD Kinase.

    PubMed

    Zhang, Ren

    2015-08-01

    Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, play essential roles in numerous cellular processes in all organisms. NADP maintains a pool of its reducing equivalent, NADPH, which regenerates cellular oxidative defense systems to counteract oxidative damages. Mitochondria represent a major source of oxidative stress, because the majority of superoxide, a reactive oxygen species, is generated from the mitochondrial respiratory chain. Therefore, as universal electron carriers in cellular electron transfer reactions, the pyridine nucleotides are required by mitochondria for both antioxidant protection and biosynthetic pathways. The NAD kinase (NADK) is the sole NADP biosynthetic enzyme. Because NADP is membrane-impermeable, eukaryotes need compartment-specific NADKs for different organelles. Consistently, in both yeast and plants, three compartment-specific NADKs have been identified. In contrast, even though the first human NADK, a cytosolic one, was identified in 2001, the identity of a hypothesized mitochondrial NADK remained elusive, until a recent discovery that the uncharacterized human gene C5ORF33 encodes a mitochondrion-localized NADK, referred to as MNADK. Three groups have characterized MNADK functions based on distinct systems involving yeast, mouse, and human studies, from aspects of both in vitro and in vivo evidence. MNADK is a mitochondrial NADK that is enriched and nutritionally-regulated in mouse liver, and a MNADK-deficient patient exhibits symptoms characteristic of mitochondrial disease. The identification of MNADK provides a key clue to the mechanism involved in mitochondrial NADPH production and the maintenance of redox balance in mammalian cells. The roles of MNADK in physiological and pathological processes have yet to be discovered.

  4. A continuous spectrophotometric assay for the activation of plant NAD kinase by calmodulin, calcium(II), and europium(III) ions.

    PubMed

    Amann, B T; Mulqueen, P; Horrocks, W D

    1992-12-01

    A continuous spectrophotometric assay has been developed to quantify the calmodulin, calcium(II) ion, and europium(III) ion dependence of the activation of NAD kinase from pea seedlings. Experimental enzyme activation data are compared with the theoretical curves for the binding of calcium(II) ions to the individual calcium binding sites of calmodulin. These results indicate that the binding of three calcium(II) ions is necessary for activation of plant NAD kinase. Further studies demonstrate that europium(III) ions can replace calcium(II) ions in calmodulin with retention of its ability to activate NAD kinase.

  5. The Unusual Acid-Accumulating Behavior during Ripening of Cherimoya (Annona cherimola Mill.) is Linked to Changes in Transcription and Enzyme Activity Related to Citric and Malic Acid Metabolism.

    PubMed

    González-Agüero, Mauricio; Tejerina Pardo, Luis; Zamudio, María Sofía; Contreras, Carolina; Undurraga, Pedro; Defilippi, Bruno G

    2016-04-25

    Cherimoya (Annona cherimola Mill.) is a subtropical fruit characterized by a significant increase in organic acid levels during ripening, making it an interesting model for studying the relationship between acidity and fruit flavor. In this work, we focused on understanding the balance between the concentration of organic acids and the gene expression and activity of enzymes involved in the synthesis and degradation of these metabolites during the development and ripening of cherimoya cv. "Concha Lisa". Our results showed an early accumulation of citric acid and other changes associated with the accumulation of transcripts encoding citrate catabolism enzymes. During ripening, a 2-fold increase in malic acid and a 6-fold increase in citric acid were detected. By comparing the contents of these compounds with gene expression and enzymatic activity levels, we determined that cytoplasmic NAD-dependent malate dehydrogenase (cyNAD-MDH) and mitochondrial citrate synthase (mCS) play important regulatory roles in the malic and citric acid biosynthetic pathways.

  6. Experimental synovitis induced by aluminium phosphate in rabbits. Comparison of the changes produced in synovial tissue and in articular cartilage by aluminium phosphate, carrageenin, calcium hydrogen phosphate dihydrate, and natural diamond powder.

    PubMed

    Delongeas, J L; Netter, P; Boz, P; Faure, G; Royer, R J; Gaucher, A

    1984-01-01

    The goal of this experimental study was to examine the effect on articular tissue of tribasic aluminium phosphate (crystalline and amorphous forms) after intraarticular injection in rabbit and to compare it with that of various phlogistic compounds such as carrageenin, calcium hydrogen phosphate dihydrate and diamond powder, as a control. Synovium and cartilage were studied with light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy dispersive micro-analysis (EDM). Crystalline and amorphous aluminium phosphate could induce a synovitis with articular effusion in rabbits. With TEM, lysosomal inclusions of phagocytosed material were observed. Through SEM coupled with EDM, aluminium associated with phosphate was found in cellular elements. PMID:6087947

  7. Experimental synovitis induced by aluminium phosphate in rabbits. Comparison of the changes produced in synovial tissue and in articular cartilage by aluminium phosphate, carrageenin, calcium hydrogen phosphate dihydrate, and natural diamond powder.

    PubMed

    Delongeas, J L; Netter, P; Boz, P; Faure, G; Royer, R J; Gaucher, A

    1984-01-01

    The goal of this experimental study was to examine the effect on articular tissue of tribasic aluminium phosphate (crystalline and amorphous forms) after intraarticular injection in rabbit and to compare it with that of various phlogistic compounds such as carrageenin, calcium hydrogen phosphate dihydrate and diamond powder, as a control. Synovium and cartilage were studied with light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy dispersive micro-analysis (EDM). Crystalline and amorphous aluminium phosphate could induce a synovitis with articular effusion in rabbits. With TEM, lysosomal inclusions of phagocytosed material were observed. Through SEM coupled with EDM, aluminium associated with phosphate was found in cellular elements.

  8. The prevalence of chondrocalcinosis (CC) of the acromioclavicular (AC) joint on chest radiographs and correlation with calcium pyrophosphate dihydrate (CPPD) crystal deposition disease

    PubMed Central

    Carrera, Guillermo; Baynes, Keith; Mautz, Alan; DuBois, Melissa; Cerniglia, Ross; Ryan, Lawrence M.

    2016-01-01

    Digital imaging combined with picture archiving and communication system (PACS) access allows detailed image retrieval and magnification. Calcium pyrophosphate dihydrate (CPPD) crystals preferentially deposit in fibrocartilages, the cartilage of the acromioclavicular (AC) joint being one such structure. We sought to determine if examination of the AC joints on magnified PACS imaging of chest films would be useful in identifying chondrocalcinosis (CC). Retrospective radiographic readings and chart reviews involving 1,920 patients aged 50 or more who had routine outpatient chest radiographs over a 4-month period were performed. Knee radiographs were available for comparison in 489 patients. Medical records were reviewed to abstract demographics, chest film reports, and diagnoses. AC joint CC was identified in 1.1 % (21/1,920) of consecutive chest films. Patients with AC joint CC were 75 years of age versus 65.4 in those without CC (p<0.0002). Four hundred eighty-nine patients had knee films. Six of these patients had AC joint CC, and of these, five also had knee CC (83 %). Of the 483 without AC joint CC, 62 (12 %) had knee CC (p=0.002). Patients with AC joint CC were more likely to have a recorded history of CPPD crystal deposition disease than those without AC joint CC (14 versus 1 %, p=0.0017). The prevalence of AC joint CC increases with age and is associated with knee CC. A finding of AC joint CC should heighten suspicion of pseudogout or secondary osteoarthritis in appropriate clinical settings and, in a young patient, should alert the clinician to the possibility of an associated metabolic condition. PMID:23609408

  9. The prevalence of chondrocalcinosis (CC) of the acromioclavicular (AC) joint on chest radiographs and correlation with calcium pyrophosphate dihydrate (CPPD) crystal deposition disease.

    PubMed

    Parperis, Konstantinos; Carrera, Guillermo; Baynes, Keith; Mautz, Alan; Dubois, Melissa; Cerniglia, Ross; Ryan, Lawrence M

    2013-09-01

    Digital imaging combined with picture archiving and communication system (PACS) access allows detailed image retrieval and magnification. Calcium pyrophosphate dihydrate (CPPD) crystals preferentially deposit in fibrocartilages, the cartilage of the acromioclavicular (AC) joint being one such structure. We sought to determine if examination of the AC joints on magnified PACS imaging of chest films would be useful in identifying chondrocalcinosis (CC). Retrospective radiographic readings and chart reviews involving 1,920 patients aged 50 or more who had routine outpatient chest radiographs over a 4-month period were performed. Knee radiographs were available for comparison in 489 patients. Medical records were reviewed to abstract demographics, chest film reports, and diagnoses. AC joint CC was identified in 1.1 % (21/1,920) of consecutive chest films. Patients with AC joint CC were 75 years of age versus 65.4 in those without CC (p < 0.0002). Four hundred eighty-nine patients had knee films. Six of these patients had AC joint CC, and of these, five also had knee CC (83 %). Of the 483 without AC joint CC, 62 (12 %) had knee CC (p = 0.002). Patients with AC joint CC were more likely to have a recorded history of CPPD crystal deposition disease than those without AC joint CC (14 versus 1 %, p = 0.0017). The prevalence of AC joint CC increases with age and is associated with knee CC. A finding of AC joint CC should heighten suspicion of pseudogout or secondary osteoarthritis in appropriate clinical settings and, in a young patient, should alert the clinician to the possibility of an associated metabolic condition.

  10. Immunotoxic effects of sodium tungstate dihydrate on female B6C3F1/N mice when administered in drinking water.

    PubMed

    Frawley, Rachel P; Smith, Matthew J; White, Kimber L; Elmore, Susan A; Herbert, Ron; Moore, Rebecca; Staska, Lauren M; Behl, Mamta; Hooth, Michelle J; Kissling, Grace E; Germolec, Dori R

    2016-09-01

    Tungsten is a naturally occurring, high-tensile strength element that has been used in a number of consumer products. Tungsten has been detected in soil, waterways, groundwater, and human tissue and body fluids. Elevated levels of tungsten in urine were reported for populations exposed to tungstate in drinking water in areas where natural tungsten formations were prevalent. Published reports indicated that sodium tungstate may modulate hematopoiesis, immune cell populations, and immune responses in rodent models. The objective of this study was to assess potential immunotoxicity of sodium tungstate dihydrate (STD), a drinking water contaminant. Female B6C3F1/N mice received 0-2000 mg STD/L in their drinking water for 28 d, and were evaluated for effects on immune cell populations in spleen and bone marrow, and humoral-mediated, cell-mediated, and innate immunity. Three different parameters of cell-mediated immunity were similarly affected at 1000 mg STD/L. T-cell proliferative responses against allogeneic leukocytes and anti-CD3 were decreased 32%, and 21%, respectively. Cytotoxic T-lymphocyte activity was decreased at all effector:target cell ratios examined. At 2000 mg STD/L, the absolute numbers of CD3(+) T-cell progenitor cells in bone marrow were increased 86%, but the alterations in B-lymphocyte and other progenitor cells were not significant. There were no effects on bone marrow DNA synthesis or colony forming capabilities. STD-induced effects on humoral-mediated immunity, innate immunity, and splenocyte sub-populations were limited. Enhanced histopathology did not detect treatment-related lesions in any of the immune tissues. These data suggest exposure to STD in drinking water may adversely affect cell-mediated immunity. PMID:27223060

  11. Reinjury risk of nano-calcium oxalate monohydrate and calcium oxalate dihydrate crystals on injured renal epithelial cells: aggravation of crystal adhesion and aggregation

    PubMed Central

    Gan, Qiong-Zhi; Sun, Xin-Yuan; Bhadja, Poonam; Yao, Xiu-Qiong; Ouyang, Jian-Ming

    2016-01-01

    Background Renal epithelial cell injury facilitates crystal adhesion to cell surface and serves as a key step in renal stone formation. However, the effects of cell injury on the adhesion of nano-calcium oxalate crystals and the nano-crystal-induced reinjury risk of injured cells remain unclear. Methods African green monkey renal epithelial (Vero) cells were injured with H2O2 to establish a cell injury model. Cell viability, superoxide dismutase (SOD) activity, malonaldehyde (MDA) content, propidium iodide staining, hematoxylin–eosin staining, reactive oxygen species production, and mitochondrial membrane potential (Δψm) were determined to examine cell injury during adhesion. Changes in the surface structure of H2O2-injured cells were assessed through atomic force microscopy. The altered expression of hyaluronan during adhesion was examined through laser scanning confocal microscopy. The adhesion of nano-calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals to Vero cells was observed through scanning electron microscopy. Nano-COM and COD binding was quantitatively determined through inductively coupled plasma emission spectrometry. Results The expression of hyaluronan on the cell surface was increased during wound healing because of Vero cell injury. The structure and function of the cell membrane were also altered by cell injury; thus, nano-crystal adhesion occurred. The ability of nano-COM to adhere to the injured Vero cells was higher than that of nano-COD crystals. The cell viability, SOD activity, and Δψm decreased when nano-crystals attached to the cell surface. By contrast, the MDA content, reactive oxygen species production, and cell death rate increased. Conclusion Cell injury contributes to crystal adhesion to Vero cell surface. The attached nano-COM and COD crystals can aggravate Vero cell injury. As a consequence, crystal adhesion and aggregation are enhanced. These findings provide further insights into kidney stone

  12. Immunotoxic effects of sodium tungstate dihydrate on female B6C3F1/N mice when administered in drinking water.

    PubMed

    Frawley, Rachel P; Smith, Matthew J; White, Kimber L; Elmore, Susan A; Herbert, Ron; Moore, Rebecca; Staska, Lauren M; Behl, Mamta; Hooth, Michelle J; Kissling, Grace E; Germolec, Dori R

    2016-09-01

    Tungsten is a naturally occurring, high-tensile strength element that has been used in a number of consumer products. Tungsten has been detected in soil, waterways, groundwater, and human tissue and body fluids. Elevated levels of tungsten in urine were reported for populations exposed to tungstate in drinking water in areas where natural tungsten formations were prevalent. Published reports indicated that sodium tungstate may modulate hematopoiesis, immune cell populations, and immune responses in rodent models. The objective of this study was to assess potential immunotoxicity of sodium tungstate dihydrate (STD), a drinking water contaminant. Female B6C3F1/N mice received 0-2000 mg STD/L in their drinking water for 28 d, and were evaluated for effects on immune cell populations in spleen and bone marrow, and humoral-mediated, cell-mediated, and innate immunity. Three different parameters of cell-mediated immunity were similarly affected at 1000 mg STD/L. T-cell proliferative responses against allogeneic leukocytes and anti-CD3 were decreased 32%, and 21%, respectively. Cytotoxic T-lymphocyte activity was decreased at all effector:target cell ratios examined. At 2000 mg STD/L, the absolute numbers of CD3(+) T-cell progenitor cells in bone marrow were increased 86%, but the alterations in B-lymphocyte and other progenitor cells were not significant. There were no effects on bone marrow DNA synthesis or colony forming capabilities. STD-induced effects on humoral-mediated immunity, innate immunity, and splenocyte sub-populations were limited. Enhanced histopathology did not detect treatment-related lesions in any of the immune tissues. These data suggest exposure to STD in drinking water may adversely affect cell-mediated immunity.

  13. Fatty acid biosynthesis during the life cycle of Debaryomyces etchellsii.

    PubMed

    Arous, Fatma; Mechichi, Tahar; Nasri, Moncef; Aggelis, George

    2016-07-01

    Fatty acid biosynthesis during the life cycle of the ascomycetous yeast Debaryomyces etchellsii cultivated on a non-fermentable substrate, i.e. glycerol, in nitrogen rich media (NRM) and nitrogen limited media (NLM) has been studied. Although considerable activities of key lipogenic enzymes, such as ATP citrate lyase (ACL) and malic enzyme (ME), were detected in vegetative cells during asexual proliferation (which occurred in the first growth stages in both NRM and NLM), lipid accumulation was restricted due to the high activities of NAD+-isocitrate dehydrogenase (NAD+-ICDH). A similar enzymatic profile has been found in ascii and free ascospores produced in NRM; thus lipid accumulation was low. On the contrary, very high activities of both ACL and ME and low activities of NAD+-ICDH were detected in ascii and free ascospores produced in NLM resulting in lipid accumulation. Neutral lipids (NL) were the predominant fraction of cellular lipids produced in vegetative cells and ascospores in both NRM and NLM. On the other hand, phospholipids (P) were the major polar lipids while glycolipids (G) were synthesized in low proportions. During transition from asexual to sexual phase, the percentage of NL increased with a significant decrease of P and, to a lesser extent, of G. High quantities of linoleic acid were found esterified in polar lipids, especially in P, during the vegetative stage of growth, while, with a few exceptions, during transition from asexual to sexual stage, linoleic acid concentration decreased markedly, mainly in P, while oleic acid concentration increased. PMID:27129978

  14. Identification of Isn1 and Sdt1 as glucose- and vitamin-regulated nicotinamide mononucleotide and nicotinic acid mononucleotide [corrected] 5'-nucleotidases responsible for production of nicotinamide riboside and nicotinic acid riboside.

    PubMed

    Bogan, Katrina L; Evans, Charles; Belenky, Peter; Song, Peng; Burant, Charles F; Kennedy, Robert; Brenner, Charles

    2009-12-11

    Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD(+), which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD(+) metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5'-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5'-nucleotidase, and Sdt1, initially classified as a pyrimidine 5'-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD(+). Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD(+) metabolism. PMID:19846558

  15. First experimental charge density study using a Bruker CMOS-type PHOTON 100 detector: the case of ammonium tetraoxalate dihydrate.

    PubMed

    Jarzembska, Katarzyna N; Kamiński, Radosław; Dobrzycki, Lukasz; Cyrański, Michał K

    2014-10-01

    The aim of this study was to test the applicability of a Bruker AXS CMOS-type PHOTON 100 detector for the purpose of a fine charge density quality data collection. A complex crystal containing oxalic acid, ammonium oxalate and two water molecules was chosen as a test case. The data was collected up to a resolution of 1.31 Å(-1) with high completeness (89.1%; Rmrg = 0.0274). The multipolar refinement and subsequent quantum theory of atoms in molecules (QTAIM) analysis resulted in a comprehensive description of the charge density distribution in the crystal studied. The residual density maps are flat and almost featureless. It was possible to derive reliable information on intermolecular interactions to model the anharmonic motion of a water molecule, and also to observe the fine details of the charge density distribution, such as polarization on O and H atoms involved in the strongest hydrogen bonds. When compared with our previous statistical study on oxalic acid data collected with the aid of CCD cameras, the complementary metal-oxide semiconductor (CMOS) detector can certainly be classified as a promising alternative in advanced X-ray diffraction studies.

  16. Plant NAD(H)-Glutamate Dehydrogenase Consists of Two Subunit Polypeptides and Their Participation in the Seven Isoenzymes Occurs in an Ordered Ratio

    PubMed Central

    Loulakakis, Konstantinos A.; Roubelakis-Angelakis, Kalliopi A.

    1991-01-01

    The structure and function of NAD(H)-glutamate dehydrogenase in plants was studied by using grapevine (Vitis vinifera L. cv Sultanina) callus grown under different nitrogen sources. The enzyme consists of two subunit-polypeptides, α and β, with similar antigenic properties but with different molecular mass and charge. The two polypeptides have molecular masses of 43.0 and 42.5 kilodaltons, respectively. The holoenzyme is hexameric and is resolved into seven isoenzymes by native gel electrophoresis. Two-dimensional native/SDS-PAGE revealed that the 1 and 7 isoenzymes are homohexamers and the isoenzymes 2 through 6 are hybrids of the two polypeptides following an ordered ratio. The total quantity of α- and β-polypeptides and the isoenzymic pattern was altered by the exogenous nitrogen source. The sample derived from callus grown on nitrate or glutamic acid contained a slightly greater amount of β-polypeptide and of the more cathodal isoenzymes, whereas α-polypeptide and the more anodal isoenzymes predominated in callus grown in the presence of either ammonium or glutamine. The anabolic reaction was correlated with the α- and the catabolic reaction with the β-polypeptide; this could suggest that each isoenzyme exhibits anabolic and catabolic function of different magnitude. The isoenzymic patterns did not obey the expected binomial distribution proportions. ImagesFigure 1Figure 2Figure 3Figure 4 PMID:16668355

  17. Molecular clone and expression of a NAD+-dependent glycerol-3-phosphate dehydrogenase isozyme gene from the halotolerant alga Dunaliella salina.

    PubMed

    Cai, Ma; He, Li-Hong; Yu, Tu-Yuan

    2013-01-01

    Glycerol is an important osmotically compatible solute in Dunaliella. Glycerol-3-phosphate dehydrogenase (G3PDH) is a key enzyme in the pathway of glycerol synthesis, which converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. Generally, the glycerol-DHAP cycle pathway, which is driven by G3PDH, is considered as the rate-limiting enzyme to regulate the glycerol level under osmotic shocks. Considering the peculiarity in osmoregulation, the cDNA of a NAD(+)-dependent G3PDH was isolated from D. salina using RACE and RT-PCR approaches in this study. Results indicated that the length of the cDNA sequence of G3PDH was 2,100 bp encoding a 699 amino acid deduced polypeptide whose computational molecular weight was 76.6 kDa. Conserved domain analysis revealed that the G3PDH protein has two independent functional domains, SerB and G3PDH domains. It was predicted that the G3PDH was a nonsecretory protein and may be located in the chloroplast of D. salina. Phylogenetic analysis demonstrated that the D. salina G3PDH had a closer relationship with the G3PDHs from the Dunaliella genus than with those from other species. In addition, the cDNA was subsequently subcloned in the pET-32a(+) vector and was transformed into E. coli strain BL21 (DE3), a expression protein with 100 kDa was identified, which was consistent with the theoretical value.

  18. 4-Pyridone-3-carboxamide-1-β-D-ribonucleoside triphosphate (4PyTP), a novel NAD metabolite accumulating in erythrocytes of uremic children: a biomarker for a toxic NAD analogue in other tissues?

    PubMed

    Synesiou, Elena; Fairbanks, Lynnette D; Simmonds, H Anne; Slominska, Ewa M; Smolenski, Ryszard T; Carrey, Elizabeth A

    2011-06-01

    We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP), which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD(+) metabolites (nicotinamide, N(1)-methylnicotinamide and 4Py-riboside) and the major nicotinamide metabolite N(1)-methyl-2-pyridone-5-carboxamide (2PY), with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD(+) from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD(+) analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD(+) analogue that inhibits IMP dehydrogenase in other cells. PMID:22069723

  19. Mutagenesis of Key Residues in the Binding Center of l-Aspartate-b-Semialdehyde Dehydrogenase from Escherichia coli Enhances Utilization of the Cofactor NAD(H).

    PubMed

    Xu, Xiaoshu; Chen, Jun; Wang, Qingzhuo; Duan, Chunlan; Li, Yan; Wang, Renxiao; Yang, Sheng

    2016-01-01

    L-Aspartate-β-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate pathway. In bacteria, ASADH is highly specific for the cofactor NADP(+) rather than NAD(+). Limited information on cofactor utilization is available, and neither the wild-type protein nor the available mutants could utilize NAD(+) efficiently. In this study, we identified several residues crucial for cofactor utilization by Escherichia coli ASADH (ecASADH) by mutating residues within the cofactor binding center. Among the investigated mutants, ecASADH-Q350N and ecASADH-Q350N/H171A, which exhibited markedly improved NAD(+) utilization, were further investigated by various biochemical approaches and molecular modeling. Relative to the wild type, the two mutants showed approximately 44-fold and 66-fold increases, respectively, in the constant kcat /Km of NAD(+). As desired, they could also utilize NADH efficiently to synthesize l-homoserine in cascade reactions in vitro.

  20. Occurrence of ferredoxin:NAD+ oxidoreductase activity and its ion specificity in several Gram-positive and Gram-negative bacteria

    PubMed Central

    Hess, Verena; Gallegos, Rene; Jones, J Andrew; Barquera, Blanca; Malamy, Michael H

    2016-01-01

    A ferredoxin:NAD+ oxidoreductase was recently discovered as a redox-driven ion pump in the anaerobic, acetogenic bacterium Acetobacterium woodii. The enzyme is assumed to be encoded by the rnf genes. Since these genes are present in the genomes of many bacteria, we tested for ferredoxin:NAD+ oxidoreductase activity in cytoplasmic membranes from several different Gram-positive and Gram-negative bacteria that have annotated rnf genes. We found this activity in Clostridium tetanomorphum, Clostridium ljungdahlii, Bacteroides fragilis, and Vibrio cholerae but not in Escherichia coli and Rhodobacter capsulatus. As in A. woodii, the activity was Na+-dependent in C. tetanomorphum and B. fragilis but Na+-independent in C. ljungdahlii and V. cholerae. We deleted the rnf genes from B. fragilis and demonstrated that the mutant has greatly reduced ferredoxin:NAD+ oxidoreductase activity. This is the first genetic proof that the rnf genes indeed encode the reduced ferredoxin:NAD+ oxidoreductase activity. PMID:26793417

  1. [Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

    PubMed

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage.

  2. [Activity of liver mitochondrial NAD+-dependent dehydrogenases of the krebs cycle in rats with acetaminophen-induced hepatitis developed under conditions of alimentary protein deficiency].

    PubMed

    Voloshchuk, O N; Kopylchuk, G P

    2016-01-01

    Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD(+)/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deprivation. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD(+)/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein caused a more pronounced decrease in the activity of NAD(+)-dependent dehydrogenases studied and a 2.2-fold increase of the mitochondrial NAD(+)/NADН ratio. This suggests that alimentary protein deprivation potentiated drug-induced liver damage. PMID:27143375

  3. The Soluble Recombinant Neisseria meningitidis Adhesin NadAΔ351–405 Stimulates Human Monocytes by Binding to Extracellular Hsp90

    PubMed Central

    Cecchini, Paola; Tavano, Regina; Polverino de Laureto, Patrizia; Franzoso, Susanna; Mazzon, Cristin