The Rate of NAD Breakdown Is Maintained Constant against Deletion or Overexpression of NAD-Degrading Enzymes in Mammalian Cells.

Cellular NAD is continuously degraded and synthesized under resting conditions. In mammals, NAD synthesis is primarily initiated from nicotinamide (Nam) by Nam phosphoribosyltransferase, whereas poly(ADP-ribose) polymerase 1 (PARP1) and 2 (PARP2), sirtuin1 (SIRT1), CD38, and sterile alpha and TIR motif containing 1 (SARM1) are involved in NAD breakdown. Using flux analysis with H-labeled Nam, we found that when mammalian cells were cultured in the absence of Nam, cellular NAD levels were maintained and NAD breakdown was completely suppressed.

Decrease in Glycosaminoglycan with Aging in Normal Rat Articular Cartilage Is Greater in Females than in Males.

Objective: Osteoarthritis (OA) is more prevalent in females. We hypothesized that changes in articular cartilage (AC) constituents with aging may cause differences. Herein, we aimed to compare the changes in AC constituents with aging in male and female normal rats.

Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry.

NAD+ is mainly synthesized from nicotinamide (Nam) by the rate-limiting enzyme Nam phosphoribosyltransferase (Nampt) and degraded to Nam by NAD+-degrading enzymes in mammals. Numerous studies report that tissue NAD+ levels decrease during aging and age-related diseases and suggest that NAD+ replenishment promotes healthy aging. Although increased expression of Nampt might be a promising intervention for healthy aging, forced expression of Nampt gene, inducing more than 10-fold increases in the enzyme protein level, has been reported to elevate NAD+ levels only 40-60% in mammalian cells.

Complete solubilization of cartilage using the heat-stable protease thermolysin for comprehensive GAG analysis.

Articular cartilage comprises collagens, proteoglycans, and glycosaminoglycans (GAGs) together with water, in hyaline matrixes. Articular cartilage is resistant to proteolytic solubilization for comprehensive GAG analyses partly because of assemblies of collagen fibers with thermolabile hydrogen bonds. In this study, we used the heat-stable protease thermolysin to digest collagen in solid articular cartilage at 70 °C and compared the efficiencies of collagen digestion and GAG extraction to those with collagenase digestion at 50 °C.

Quantitative analysis of glycosaminoglycans, chondroitin/dermatan sulfate, hyaluronic acid, heparan sulfate, and keratan sulfate by liquid chromatography-electrospray ionization-tandem mass spectrometry.

We developed a method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min.

Formation of [nicotinamide-²H₃]NAD⁺ from [²H₄]nicotinamide and [²H₄]nicotinic acid in human HepG2N cells and involvement of ²H/¹H exchange at the redox site of NAD⁺/NADH.

To determine the rates of cellular NAD⁺ synthesis and breakdown, incorporation of stable isotope-labeled precursors into NAD⁺ should be quantified. Although with ²H (D)-labeled precursors [2,4,5,6-D₄]nicotinamide ([D₄]Nam) and [2,4,5,6-D₄]nicotinic acid ([D₄]NA), [D₃]NAD⁺ is formed in human cells, why only three of four D atoms from [D₄]Nam and [D₄]NA are present in NAD⁺ remains unknown. Using a liquid chromatography-tandem mass spectrometry, we tested the involvement of D/¹H (H) exchange at the redox site of NAD⁺/NADH (C-4 carbon of the pyridine ring) by oxidoreductases exhibiting opposite stereospecificity for the coenzymes in the 1-Da mass decrease in the cellular NAD⁺ formation.

Nicotinamide phosphoribosyltransferase/visfatin does not catalyze nicotinamide mononucleotide formation in blood plasma.

Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN.

Interferon-gamma elevates nicotinamide N-methyltransferase activity and nicotinamide level in human glioma cells.

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide. NNMT is strongly expressed in tumor cells and an increase in NNMT activity may reduce cellular nicotinamide level and thereby promote cell survival in the cells. However, there has been no report of a relationship between NNMT activity and nicotinamide level in tumor cells.

Precursor ion scanning and sequencing of arginine-ADP-ribosylated peptide by mass spectrometry.

Arginine (Arg)-specific ADP-ribosylation is one of the posttranslational modifications of proteins and is thought to play an important role in reversibly regulating functions of the target proteins in eukaryotes. However, the physiological target protein has not been established. We examined the fragmentation pattern of both ADP-ribosyl-Arg (ADP-R-Arg) and Arg-ADP-ribosylated peptides by quadrupole tandem mass spectrometry and found a specific cleavage of ADP-R-Arg into N-(ADP-ribosyl)-carbodiimide (ADP-R-carbodiimide) and ornithine.

Indoleamine 2,3-dioxygenase as a new target for malignant glioma therapy. Laboratory investigation.

Object: Indoleamine 2,3-dioxygenase (IDO), a kynurenine pathway (KP) enzyme catalyzing oxidation of the essential amino acid tryptophan (Trp), is thought to be involved in the immune resistance of malignant tumors through T-cell inactivation caused by Trp depletion and metabolite accumulation. Human malignant gliomas may use this strategy to escape immune attack. The object of this study was to investigate the possibility of IDO-dependent Trp depletion by malignant gliomas and the practicability of using an IDO inhibitor together with anticancer drugs to reserve Trp without decreasing the cytotoxicity of the drugs.

Simultaneous measurement of tryptophan and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry.

We have expanded a liquid chromatographic-tandem mass spectrometric method that measures 3-hydroxykynurenine and 3-hydroxyanthranilic acid in addition to tryptophan and kynurenine both intra- and extracellularly. After reversed phase HPLC separation, the compounds were detected in the MS positive multiple reaction monitoring mode. We found a good linear response for each tryptophan metabolite.

A new detection method for arginine-specific ADP-ribosylation of protein -- a combinational use of anti-ADP-ribosylarginine antibody and ADP-ribosylarginine hydrolase.

Arginine-specific ADP-ribosylation is one of the posttranslational modifications of proteins by transferring one ADP-ribose moiety of NAD to arginine residues of target proteins. This modification, catalyzed by ADP-ribosyltransferase (Art), is reversed by ADP-ribosylarginine hydrolase (AAH). In this study, we describe a new method combining an anti-ADP-ribosylarginine antibody (alphaADP-R-Arg Ab) and AAH for detection of the target protein of ADP-ribosylation.

Elevation of cellular NAD levels by nicotinic acid and involvement of nicotinic acid phosphoribosyltransferase in human cells.

NAD plays critical roles in various biological processes through the function of SIRT1. Although classical studies in mammals showed that nicotinic acid (NA) is a better precursor than nicotinamide (Nam) in elevating tissue NAD levels, molecular details of NAD synthesis from NA remain largely unknown. We here identified NA phosphoribosyltransferase (NAPRT) in humans and provided direct evidence of tight link between NAPRT and the increase in cellular NAD levels.

FR-167653, a selective p38 MAPK inhibitor, exerts salutary effect on liver cirrhosis through downregulation of Runx2.

Liver cirrhosis remains a difficult-to-treat disease with a substantial morbidity and mortality rate. There is an emerging body of data purporting a pivotal role of the activated p38 mitogen-activated protein kinase (MAPK) in the process of cirrhosis. Several anticirrhotic agents have been developed over the past few years, and most of them exert their effects by indirectly inhibiting the p38 pathway.

Enhancement of morphine analgesic effect with induction of mu-opioid receptor endocytosis in rats.

Background: Morphine can desensitize mu-opioid receptor (MOR), but it does not cause internalization of the receptor after binding. Acute desensitization of MOR impairs the efficiency of signaling, whereas the receptor internalization restores the cell responsiveness to the agonists. Thereby, the property of morphine may limit the analgesic effects of this opiate drug.

The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry.

We have developed a liquid chromatographic-tandem mass spectrometric method that is sensitive and specific and that simultaneously measures cellular NAD(+) and related compounds. Using this method, NAD(+), NAAD, NMN, NAMN, NAM, NA, ADPR, and 5'AMP were first separated over a reverse-phase high-performance liquid chromatography resin in a mobile ammonium formate-methanol linear gradient. Then each compound was ionized at an electrospray source and detected in the positive multiple reaction monitoring mode of a triple-quadrupole tandem mass spectrometer.

Purification, characterization and molecular cloning of glycosylphosphatidylinositol-anchored arginine-specific ADP-ribosyltransferases from chicken.

Mono-ADP-ribosylation is a post-translational modification that regulates the functions of target proteins or peptides by attaching an ADP-ribose moiety. Here we report the purification, molecular cloning, characterization and tissue-specific distribution of novel arginine-specific Arts (ADP-ribosyltransferases) from chicken. Arts were detected in various chicken tissues as GPI (glycosylphosphatidylinositol)-anchored forms, and purified from the lung membrane fraction.

Synergistic augmentation of antimicrotubule agent-induced cytotoxicity by a phosphoinositide 3-kinase inhibitor in human malignant glioma cells.

Because the aberrantly activated phosphoinositide 3-kinase (PI3K)/Akt pathway renders tumor cells resistant to cytotoxic insults, including those related to anticancer drugs, inhibition of the pathway may possibly restore or augment the effectiveness of chemotherapy. Using the human malignant glioma cell lines U87, A172, LN18, and LN229, we examined effects of the PI3K inhibitor LY294002 on both apoptosis and cytotoxicity induced by chemotherapeutic agents, including antimicrotubule agents vincristine and paclitaxel, an alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea, a topoisomerase II inhibitor etoposide, and a DNA cross-linking agent cisplatin (cis-diamminedichloroplatinum), and we compared the LY294002-induced enhancement of effects of those agents. Ten to 20 micro M LY294002 augmented both apoptosis and caspase 3-like activity caused by antimicrotubule agents to a larger extent than induced by 1,3-bis(2-chloroethyl)-1-nitrosourea, etoposide, and cisplatin in all four malignant glioma cell lines examined.

Molecular identification of human glutamine- and ammonia-dependent NAD synthetases. Carbon-nitrogen hydrolase domain confers glutamine dependency.

NAD synthetase catalyzes the final step in the biosynthesis of NAD. In the present study, we obtained cDNAs for two types of human NAD synthetase (referred as NADsyn1 and NADsyn2). Structural analysis revealed in both NADsyn1 and NADsyn2 a domain required for NAD synthesis from ammonia and in only NADsyn1 an additional carbon-nitrogen hydrolase domain shared with enzymes of the nitrilase family that cleave nitriles as well as amides to produce the corresponding acids and ammonia.

Growth inhibition of human malignant glioma cells induced by the PI3-K-specific inhibitor.

Object: The phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a tumor suppressor by negatively regulating the growth/survival signals of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The PI3-K/Akt pathway in PTEN-deficient tumors may be one of the key targets for anticancer therapy. The authors examined the effects of the PI3-K inhibitor 2-(4-morpholinyl)-8-phenylchromone (LY294002) on human malignant glioma cells, and compared these effects on PTEN-deficient cells with those on PTEN-wild-type (PTEN-wt) cells.