Absence Of Nicotinamide Research Articles

Characterization of the electrochemical deposition of aluminum from an AlCl3:N-methylacetamide eutectic solvent modified with nicotinamide

Aluminum currently sees extensive use in a wide range of applications that include cars, the aviation industry, consumer appliances, container vessels, and electronics. Aluminum electroplating is typically carried out using conventional ionic liquids, but these are typically toxic, costly, and relatively sensitive to air. This research will investigate the electrodeposition of aluminum for the first time from a (1:1 M ratio of) AlCl3:N-methylacetamide-based eutectic solvent on different substrates at room temperature in the presence and absence of nicotinamide as an additive. The electrochemical behavior of the aluminum in N-methylacetamide electrolyte was studied via chronocoulometry and cyclic voltammetry. The redox peak current was found to decrease relative to the pure Al solution when nicotinamide is added. The surface morphologies and composition of any aluminum films produced have been investigated via SEM/EDX, while the crystal structures of the products and their roughnesses were examined using XRD and AFM, respectively. The roughness of the Al film produced from the bath containing 0.02 M nicotinamide decreased to approximately 312 nm. The corrosion resistance of the aluminum coatings obtained from the systems containing 0.1 M and 0.2 M nicotinamide were found to be improved.

Transcriptional and Metabolic Profiling of Nicotinamide-Enhanced Natural Killer (NAM-NK) Cells (GDA-201)

Adoptive transfer of NK cells is a promising immunotherapeutic modality, however limited NK cell persistence and proliferation in vivo have historically been barriers to clinical success. Nicotinamide (NAM), an allosteric inhibitor of NAD-dependent enzymes, has been shown to preserve cell function and prevent differentiation in ex vivo culture of NK (NAM-NK) and other cells. Clinical responses were observed in a Phase 1 trial of NAM-NK (GDA-201) in patients with refractory non-Hodgkin lymphoma (Bachanova, et. al., Blood 134:777, 2019). We now use transcriptional and metabolic profiling to characterize the mechanisms underlying the activity of NAM-NK.CD3 negative lymphocytes obtained from healthy donors were cultured for 14 days with IL-15 in the presence or absence of NAM (7 mM). Next generation sequencing (NGS), liquid chromatography-mass spectrometry (LC-MS)-based metabolite quantification, and glycolytic/mitochondrial respiration measurements were performed. Transcriptome and pathway enrichment analyses were performed with Ingenuity Pathway Analysis software. Extracted cellular and medium metabolites were analyzed on a Thermo Q-Exactive Plus mass spectrometer coupled with a Vanquish UHPLC system. Extracellular acidification (ECAR) and oxygen consumption rates (OCR) were quantified using a Seahorse Extracellular Flux Analyzer. Glycolysis/citric acid cycle (TCA) rates were measured using isotope-labelled glucose incorporation assays.Transcriptome analyses defined 1,204 differentially expressed (DE) genes in NAM-NK vs. control NK. Biological/functional enrichment and pathway analyses of DE-genes predicted upregulation of cell cycle, DNA replication (CDK4/CDKN2D, CyclinD/E, MAD2L), RNA transcription, translation (SMN1/2, ABCF1, EIF4B, RPL13, RPS6), protein synthesis (EIF2, PABPC1, SOS, 60S complex) mitochondrial energy metabolism (NDUFB8, ATP5G2/E, COX7B/C) migration, homing (CD62L, CD44, DNAM1), and cytokine/chemokine response (IL18R, CXCR3, CCR5, XCL1, SOCS3, LFA1) pathways, with concomitant downregulation of cell exhaustion, senescence (BATF1, FOXP1, STAT1, CD86, LGALS9, LAG3), apoptosis, necrosis (CASP1, MDM2, IKK3), stress response (CALR, HSP90, HSPH1), and lymphoid cellular maturation (IL-2Ra, CD40L, GATA3) pathways in NAM-NK. Metabolomic analyses showed a significant increase of intracellular NAD, NADH, NADP, NADPH, high-energy triphosphates (ATP, UTP, GTP) and overall energy charge ([ATP+0.5*ADP]/[ATP+ADP+AMP]) in NAM-NK. Cellular metabolic fitness analyses revealed increased basal and ATP-linked respiration, mitochondrial maximal respiratory capacity, and glycolytic capacity in NAM-NK compared to control NK. In addition, NAM increased the rate of glucose incorporation into TCA cycle intermediates (acetyl-CoA, succinyl-CoA), consistent with a more rapid glycolysis rate, increased TCA cycling, and improved glucose consumption efficiency.Taken together, results of transcriptome, metabolomic, mitochondrial respiration, and glycolytic rate analyses suggest that NAM pleiotropically modulates key cellular metabolic functions in ex vivo-expanded NK cells, resulting in increased response to cytokine stimulation and enhanced potency. NAM inhibits differentiation, cellular stress, and exhaustion pathways that are typically activated in culture. Moreover, NAM increases cellular metabolic fitness, energy charge, and efficiency of glucose consumption, potentially imparting a protective effect against oxidative stress in the tumor microenvironment. These data offer insight into the mechanism of improved persistence, proliferation, and cytotoxicity observed in in vivo and clinical studies of GDA-201. DisclosuresYackoubov: Gamida Cell: Current Employment. Pato: Gamida Cell: Current Employment. Rifman: Gamida Cell: Current Employment. Cohen: Gamida Cell: Current Employment. Hailu: Gamida Cell: Current Employment. Persi: Gamida Cell: Current Employment. Berhani-Zipori: Gamida Cell: Current Employment. Edri: Gamida Cell: Current Employment. Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding. Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Rabinowitz: Gamida Cell: Research Funding. Lodie: Gamida Cell: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Adams: Gamida Cell: Current Employment. Simantov: Gamida Cell: Current Employment. Geffen: Gamida Cell: Current Employment.

Nicotinamide (NAM) Modulates Transcriptional Signature of Ex Vivo Cultured UCB CD34+ Cells (Omidubicel) and Preserves Their Stemness and Engraftment Potential

Historical efforts at expansion of umbilical cord blood (UCB) derived CD34+ hematopoietic stem cells (HSCs) ex vivo with cytokines yielded large numbers of progenitors for transplantation but impaired their long-term engraftment ability. We used nicotinamide (NAM), an allosteric inhibitor of NAD-enzymes, to create omidubicel, an investigational cell therapy designed to improve the expansion of CD34+ HSCs for bone marrow transplant. A Phase 1/2 clinical study of omidubicel in patients with high-risk hematologic malignancies showed rapid neutrophil engraftment and a more favorable immune reconstitution profile in patients compared to historical controls.1 We hypothesized that NAM treatment maintains the stemness and engraftment potential of omidubicel, which is associated with clinical benefit.2 We performed transcriptome, transcription factor (TF), and pathway analysis by next generation sequencing (NGS) to discern the mechanism of action of NAM and to elucidate the pathways leading to the preservation of engraftment after ex vivo expansion of omidubicel compared to CD34+ cells grown in the absence of NAM. Transcriptome analysis revealed that treatment of CD34+ cells with cytokines alone (stem cell factor [SCF], thrombopoietin [TPO], IL-6, and FLT3 ligand) led to an increase in pathways responsible for cell proliferation and differentiation, apoptotic stress, and production of reactive oxygen species (ROS), and matrix metalloproteinases (MMPs), all of which were attenuated by NAM. TF enrichment analysis of NAM-upregulated genes and downregulated genes demonstrated that NAM modulated several TFs critically involved in pathways of HSC cell self-renewal, differentiation, apoptosis and migration. Specifically, NF-kB, C-Jun, LXR/RXR and PPARα/RXRα, and AMPK-mTor signaling were all reduced in NAM-treated CD34+ cells compared to controls. Reduced expression of key genes involved in the production of ROS and reactive nitrogen species (RNS) including NADPH-oxidase-related genes (CYBB, NCF2 and NCF4) and iNOS, suggested that NAM-expanded CD34+ cells were less exposed to oxygen and nitrogen free radical stress than controls. NAM also downregulated the expression of several matrix metalloproteinases (MMP) genes including MMP7, MMP9, MMP12 and MMP19. NAM-induced downregulation of MMPs may explain the increase in engraftment in patients receiving omidubicel. Pathway analysis of differentially expressed (DE) genes was conducted using ingenuity (IPA) software. IPA analysis of DE genes showed significant downregulation of growth factor activating pathways including SCF, TPO, FLT, and GM-CSF and Endothelin-1 and P2Y Purigenic Receptor, which was confirmed by a reduction in cell cycling rates of labeled cells. IPA analysis also pointed to genes in 3 key cellular pathways that were downregulated by NAM: stress induction of apoptosis, production of ROS and RNS, and production of MMPs. NAM treatment also uniquely upregulated genes linked to cellular metabolism including the Sirtuin family genes, TCA cycle genes, and HIF1a. Interestingly, NAM upregulated genes responsible for telomerase expression further validating our hypothesis that NAM preserves cell stemness. In summary, NGS transcriptome analysis revealed that ex vivo expansion of UCB derived CD34+ cells in the presence of NAM attenuated TFs responsible for proliferation and differentiation of stem cells. In addition, NAM treatment downregulated genes regulating the production ROS, RNS, and MMPs and upregulated genes controlling metabolism and senescence, thus allowing for the expansion of CD34+ cells with preserved function and long-term engraftment ability. Our gene expression data leads to a better understanding of the mechanisms by which NAM modulates CD34+ cells in omidubicel to preserve their function. These data provide further scientific rationale for the favorable clinical engraftment and patient outcomes observed in the Phase 1/2 clinical study of omidubicel.1 An international, randomized, multi-center Phase 3 study of omidubicel in patients with high-risk hematologic malignancies is underway.2 [1]Horwitz M.E., et. al., J Clin Oncol. 2019 Feb 10;37(5):367-374. [2] ClinicalTrials.gov identifier NCT02730299. Disclosures Lodie: Gamida Cell: Employment, Equity Ownership. Adams:Gamida Cell: Employment, Equity Ownership. Yackoubov:GAMIDA CELL: Employment, Other: unexecuted shares of the company . Peled:Gamida Cell: Employment, Equity Ownership.

Combination of hydrotropic nicotinamide with nanoparticles for enhancing tacrolimus percutaneous delivery.

Tacrolimus (FK506), an effective immunosuppressant for treating inflammatory skin diseases, hardly penetrates into and through the skin owing to its high hydrophobicity and molecular weight. The aim of this study was to develop a hybrid system based on nicotinamide (NIC) and nanoparticles (NPs) encapsulating FK506, such as FK506–NPs–NIC, for facilitating percutaneous delivery, which exploited virtues of both NIC and NPs to obtain the synergetic effect. Solubility and percutaneous permeation studies were carried out. The results showed that NIC could increase the solubility and permeability of FK506 and that 20% (w/v) NIC presented higher FK506 permeability and was thus chosen as the hydrotropic solution to solubilize FK506 and prepare FK506–NPs–NIC. Hyaluronic acid (HA) was chemically conjugated with cholesterol (Chol) to obtain amphiphilic conjugate of HA–Chol, which self-assembled NPs in 20% NIC solution containing FK506. The particle size, zeta potential, and morphology of NPs were characterized. The encapsulation efficiency and in vitro percutaneous permeation of NPs were evaluated in the presence and absence of NIC. The results demonstrated that hydrotropic solubilizing FK506 was readily encapsulated into NPs with a higher encapsulation efficiency of 79.2%±4.2%, and the combination of NPs with NIC exhibited a significantly synergistic effect on FK506 deposition within the skin (2.39±0.53 μg/cm2) and penetration through the skin (13.38±2.26 μg/cm2). The effect of the combination of NPs with NIC on drug permeation was further visualized by confocal laser scanning microscope through in vivo permeation studies, and the results confirmed that NPs–NIC synergistically enhanced the permeation of the drug into the skin. The cellular uptake performed in HaCaT cells presented a promoting effect of NPs on cellular uptake. These overall results demonstrated that HA–Chol–NPs–NIC can synergistically improve the percutaneous delivery of FK506, and it is a novel potential strategy based on a nano-sized carrier for FK506 to treat skin diseases.

MP20-04 DNA DAMAGE REPAIR GENES REGULATE BLADDER INFLAMMATION

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Kidney & Bladder II1 Apr 2015MP20-04 DNA DAMAGE REPAIR GENES REGULATE BLADDER INFLAMMATION Christopher Dru, Subhash Haldar, and Neil Bhowmick Christopher DruChristopher Dru More articles by this author , Subhash HaldarSubhash Haldar More articles by this author , and Neil BhowmickNeil Bhowmick More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.976AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Chemical cystitis is a prevalent and bothersome inflammatory bladder condition characterized by dysuria and associated with cyclophosphamide and radiation therapy. It can progress to life-threatening gross hematuria and ulceration. The bladder mounts a multi-factorial immune response involving various pro- and anti- inflammatory pathways to mediate acute and chronic inflammation. The goal of this project was to determine the mechanisms underlying detrusor smooth muscle responsiveness caused by cyclophosphamide and identify the role of nicotinamide, a nutraceutical well-known for its anti-inflammatory effects, in inflammation attenuation. METHODS Ovariectomized C57Bl/6 mice received daily intraperitoneal injections of nicotinamide or placebo followed by administration of cyclophosphamide to induce chemical cystitis. Additionally, mouse bladder fibroblasts were cultured in the presence or absence of nicotinamide before induction of inflammation with acrolein. Tissues and cells were collected to determine the degree of biochemical and histological inflammation as well as changes in gene expression and cell death. RESULTS All mice and cultured cells pre-treated with nicotinamide before administration of cyclophosphamide or acrolein demonstrated a histological and biochemical decrease in inflammation. Nicotinamide increased DNA damage repair gene expression of Neil-1, Parp-1, OGG-1, and RAD-50 to reduce inflammation. FACS analysis using markers for cellular apoptosis demonstrated higher levels of cell survival (74% v 17%) when nicotinamide was added to cell-culture media before addition of acrolein. CONCLUSIONS Nicotinamide is an important regulator of inflammation and prevention of cell death in both mice treated with cyclophosphamide and cultured mouse bladder fibroblasts. This is evidenced by increased expression of DNA damage repair genes and alterations in cytokine regulation to protect the urothelium from reactive hyperplasia and ulceration. We propose that nicotinamide reduces inflammation through preservation of DNA integrity by maintaining physiologic levels of the DNA damage repair genes Neil-1, Parp-1, OGG-1, and RAD-50. These findings have future implications for the treatment of chemotherapy-induced cystitis as well as in the treatment of other types of inflammation. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e224 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christopher Dru More articles by this author Subhash Haldar More articles by this author Neil Bhowmick More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP8-12 NICOTINAMIDE REGULATES BLADDER INFLAMMATION THROUGH EPIGENETIC MECHANISMS

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Kidney & Bladder1 Apr 2014MP8-12 NICOTINAMIDE REGULATES BLADDER INFLAMMATION THROUGH EPIGENETIC MECHANISMS Christopher Dru, Subhash Haldar, and Neil Bhowmick Christopher DruChristopher Dru More articles by this author , Subhash HaldarSubhash Haldar More articles by this author , and Neil BhowmickNeil Bhowmick More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.355AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hemorrhagic cystitis (HC) is an inflammatory bladder condition characterized by hematuria, dysuria, and ulceration and is associated with anti-cancer and pelvic radiation therapy. The bladder mounts a multi-factorial immune response involving epigenetic mechanisms and various pro- and anti- inflammatory cytokines to mediate acute and chronic inflammation. The goal of this project was to determine the mechanism of detrusor smooth muscle responsiveness to bladder inflammation caused by cyclophosphamide. METHODS Ovariectomized C57Bl/6 mice received daily intraperitoneal injections of nicotinamide or placebo followed by administration of cyclophosphamide to induce HC. Additionally, mouse bladder fibroblasts were cultured in the presence or absence of nicotinamide before induction of inflammation with acrolein. Tissues and cells were collected to determine the degree of biochemical and histological inflammation as well as DNA promoter methylation. RESULTS All cells and tissues pre-treated with nicotinamide before administration of cyclophosphamide or acrolein had a decrease in inflammation. RT-PCR expression of Interleukin-1β, a chemo-attractant for macrophages, was reduced four-fold in bladder fibroblasts treated with acrolein and nicotinamide, compared to acrolein alone. Immunohistochemistry analysis of mouse bladders treated with cyclophosphamide and nicotinamide revealed decreased infiltration of macrophages on F4/80 staining and reduced detrusor edema on H&E preparation. DNA methylation studies showed a reduction in promoter methylation of glutathione-S-Transferase Pi-1 (GSTP-1) and 5-methylcytosine when mice and cultured fibroblasts were treated with nicotinamide before the inflammatory stimulus. FACS cell sorting for 7AAD, a marker of apoptosis, demonstrated quantitatively higher levels of cell survival (74% v 17%) when nicotinamide was added to cell-culture media before addition of acrolein. CONCLUSIONS Nicotinamide is an important regulator of inflammation, namely through DNA promoter methylation and cytokine gene regulation to protect the urothelium from reactive hyperplasia and ulceration. We propose that nicotinamide reduces inflammation through reduction of IL-1β to decrease macrophage recruitment and DNA promoter methylation of GSTP-1. The epigenetic silencing of GSTP-1 in the detrusor can result in greater accumulation of reactive oxygen to further bladder injury. These findings have future implications for the effective treatment of chemotherapy-induced cystitis as well as in the treatment of other types of inflammation. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e78-e79 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Christopher Dru More articles by this author Subhash Haldar More articles by this author Neil Bhowmick More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Nicotinamide downregulates gene expression of interleukin-6, interleukin-10, monocyte chemoattractant protein-1, and tumour necrosis factor-α gene expression in HaCaT keratinocytes after ultraviolet B irradiation

Ultraviolet (UV) radiation has profound effects on human skin, causing sunburn, inflammation, cellular-tissue injury, cell death, and skin cancer. Most of these effects are mediated by a number of cytokines produced by keratinocytes. In this study we investigated whether nicotinamide (NCT), the amide form of vitamin B3, might have a protective function in reducing the expression of interleukin (IL)-1β, IL-6, IL-8, IL-10, monocyte chemoattractant protein (MCP)-1 and tumour necrosis factor (TNF)-α in UV-irradiated keratinocytes. HaCaT cells were treated with UVB in the presence or absence of NCT, and cytokine mRNA levels were examined by quantitative real-time PCR. NCT significantly downregulated IL-6, IL-10, MCP-1 and TNF-α mRNA expression, whereas it did not exert any significant effect on IL-1β or IL-8 expression. Because of its ability to decrease these cytokine mediators after UV exposure, NCT is a possible therapy to improve or prevent conditions induced or aggravated by UV light.

Survival and B-Cell Function of Mouse Pancreatic Islets Maintained in Culture after Concomitant Exposure to Streptozotocin and Nicotinamide

It has recently been suggested from experiments performed on isolated pancreatic islets in vitro, that streptozotocin (SZ) may exert a progressive damage to the islet B-cells. It may be that this damaging effect is not dependent on the acute activation of the enzyme poly(ADP-ribose) synthetase, and a subsequent depletion of the islet NAD content. In the present study we have exposed mouse pancreatic islets in vitro to 2.2 mM SZ for 30 min at 37 degrees C, in the presence or absence of 10 mM nicotinamide, an inhibitor of poly(ADP-ribose) synthetase and examined the islet function immediately (Day 0) or after six days of culture (Day 6). Nicotinamide protected the islets against an inhibition of the glucose-stimulated insulin release on day 0 and against a SZ-induced loss in islet number and islet insulin content on day 6. However, on day 6 the islets incubated with SZ in the presence of nicotinamide showed an inhibition of the insulin release comparable to that observed in islets treated with SZ in the absence of nicotinamide. Furthermore, on day 0 nicotinamide counteracted a SZ-induced impairment of islet glucose oxidation, whereas on day 6 islets incubated with SZ both in the absence or presence of nicotinamide showed a similar and more markedly impaired oxidation of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural Basis for Nicotinamide Inhibition and Base Exchange in Sir2 Enzymes

The Sir2 family of proteins consists of broadly conserved NAD(+)-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD(+) at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with related biochemical studies and structures, we identify a nicotinamide inhibition and base-exchange site that is distinct from the so-called "C pocket" binding site for the nicotinamide group of NAD(+). These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.

The Sirtuin Inhibitor Nicotinamide Enhances Neuronal Cell Survival During Acute Anoxic Injury Through AKT, BAD, PARP, and Mitochondrial Associated "Anti-Apoptotic" Pathways

Understanding the role of nicotinamide (NIC) in different cell systems represents a significant challenge in several respects. Recently, NIC has been reported to have diverse roles during cell biology. In the absence of NIC, sirtuin protein activity is enhanced and pyrazinamidase/nicotinamidase 1 (PNC1) expression, an enzyme that deaminates NIC to convert NIC into nicotinic acid, is increased to lead to lifespan extension during calorie restriction, at least in yeast. Yet, NIC may be critical for cell survival as well as the modulation of inflammatory injury during both experimental models as well as in clinical studies. We therefore investigated some of the underlying signal transduction pathways that could be critical for the determination of the neuroprotective properties of NIC. We examined neuronal injury by trypan blue exclusion, DNA fragmentation, phosphatidylserine (PS) exposure, Akt1 phosphorylation, Bad phosphorylation, mitochondrial membrane potential, caspase activity, cleavage of poly(ADP-ribose) polymerase (PARP), and mitogen-activated protein kinases (MAPKs) phosphorylation. Application of NIC (12.5 mM) significantly increased neuronal survival from 38 -/+ 3% of anoxia treated alone to 68 +/- 3%, decreased DNA fragmentation and membrane PS exposure from 67 -/+ 4% and 61 -/+ 5% of anoxia treated alone to 30 +/- 4% and 26 +/- 4% respectively. We further demonstrate that NIC functions through Akt1 activation, Bad phosphorylation, and the downstream modulation of mitochrondrial membrane potential, cytochrome c release, caspase 1, 3, and 8 - like activities, and PARP integrity to prevent genomic DNA degradation and PS externalization during anoxia. Yet, NIC does not alter the activity of either the MAPKs p38 or JNK, suggesting that protection by NIC during anoxia is independent of the p38 and JNK pathways. Additional investigations targeted to elucidate the cellular pathways responsible for the ability of NIC to modulate both lifespan extension and cytoprotection may offer critical insight for the development of new therapies for nervous system disorders.

Nicotinamide, a Potent SIRT2 Inhibitor, Delays Differentiation of Hematopoietic Progenitor Cells.

CD38, originally described as a differentiation marker, has emerged as an important multifunctional transmembrane protein. Its most intriguing and well-characterized function is its ability to catalyze the synthesis of cyclic ADP-ribose (cADPR) from NAD. Of particular interest is its presence on the inner membrane of the nucleus, suggesting that CD38/cADPR may play a direct role in mediating nuclear activation and gene expression. Our studies on ex vivo expansion of Hematopoietic Stem Cells (HSCs) have led us to test whether alteration of CD38 function carries the potential of affecting cell fate decisions of HSCs. Inhibition of CD38 enzymatic activity was achieved by treating CD34+ cell cultures with nicotinamide (NA), a well-known base-exchange inhibitor demonstrated to inhibit the synthesis of cADPR from NAD.We report here that exogenously added nicotinamide (5–10 mM) to CD34+ cell cultures supplemented with cytokines (SCF, TPO, IL-6, FLt3, +/− IL-3) resulted in significant enrichment of CD34+CD38− (79±9.3%, n=9) and CD34+CD38−Lin− (19±3%, n=8) cells, as compared with control cultures treated only with cytokines (6.3±1.8%, n=9, and 0.7±0.06%, n=8, respectively, p<0.01). The functionality of these early progenitor subsets was demonstrated using the extended LTC-CFC assay, performed in the absence of NA. These results raised the intriguing possibility that cADPR production may have a pivotal role in regulation of CD34+ cell fate. However, inhibition of cADPR downstream signal transduction pathways by its specific antagonist, 8-amino-cADPR did not yield any effect on CD34+ cell cultures, excluding the possibility that nicotinamide modulates CD34+ cell fate solely by inhibition of cADPR synthesis.Nicotinamide is also a well-known potent inhibitor of SIRT2, a unique NAD(+)-dependent type III histone deacetylase (HDAC) with mono-ADP-ribosyltransferase activity involved in gene silencing, metabolism, apoptosis and aging. NA blocks NAD(+) hydrolysis by binding to an adjacent conserved pocket, and is therefore suggested as the physiologically relevant regulator of SIRT2 enzymes. This additional function of nicotinamide raises the intriguing possibility that HSC enrichment achieved by nicotinamide treatment may be related to specific inhibition of SIRT2 deacetylase activity and modulation of chromatin architecture leading to re-activation of previously silenced genes.In line with this hypothesis, Milhem et all. recently reported that addition of trichostatin A, a specific HDAC (type I and II) inhibitor, along with a DNA hypomethylating agent, modulated HSC fate ex vivo resulting in the retention of stem cell phenotype, number, and function (Blood , 2004; 103; 4102).Ongoing work is aimed at elucidating whether inhibition of SIRT2 is specifically involved in NA mechanism of activity leading to modulation of hematopoietic stem cell fate in ex vivo conditions.

Inhibition of Silencing and Accelerated Aging by Nicotinamide, a Putative Negative Regulator of Yeast Sir2 and Human SIRT1

The Saccharomyces cerevisiae Sir2 protein is an NAD(+)-dependent histone deacetylase that plays a critical role in transcriptional silencing, genome stability, and longevity. A human homologue of Sir2, SIRT1, regulates the activity of the p53 tumor suppressor and inhibits apoptosis. The Sir2 deacetylation reaction generates two products: O-acetyl-ADP-ribose and nicotinamide, a precursor of nicotinic acid and a form of niacin/vitamin B(3). We show here that nicotinamide strongly inhibits yeast silencing, increases rDNA recombination, and shortens replicative life span to that of a sir2 mutant. Nicotinamide abolishes silencing and leads to an eventual delocalization of Sir2 even in G(1)-arrested cells, demonstrating that silent heterochromatin requires continual Sir2 activity. We show that physiological concentrations of nicotinamide noncompetitively inhibit both Sir2 and SIRT1 in vitro. The degree of inhibition by nicotinamide (IC(50) < 50 microm) is equal to or better than the most effective known synthetic inhibitors of this class of proteins. We propose a model whereby nicotinamide inhibits deacetylation by binding to a conserved pocket adjacent to NAD(+), thereby blocking NAD(+) hydrolysis. We discuss the possibility that nicotinamide is a physiologically relevant regulator of Sir2 enzymes.

Nicotinamide reduces tumour interstitial fluid pressure in a dose- and time-dependent manner.

Nicotinamide radiosensitizes a number of experimental tumours, and increases blood flow and mean pO2 in some tumours. It has been suggested that nicotinamide reduces tumour interstitial fluid pressure (IFP), thereby reducing transient vessel non-perfusion and acute hypoxia, and radiosensitizing tumours. To test this hypothesis, tumour IFP, transient vessel non-perfusion, and radiosensitivity after nicotinamide administration were examined in the murine carcinoma NT. Nicotinamide at doses of 500 and 1000 mg kg-1 significantly reduced tumour IFP within 20 min of administration, with recovery to control values by 60-80 min; 100 mg kg-1 had no effect. The percentage of previously non-perfused vessels that became perfused 20 min after administering 1000 mg kg-1 of nicotinamide significantly exceeded the percentage that became perfused within 20 min in the absence of nicotinamide. By 90 min after nicotinamide administration, this differential effect was abolished. The correlation in the time courses of reduced IFP and increased vessel perfusion after nicotinamide administration suggest that decreased IFP may accompany vessel reperfusion. However, 1000 mg kg-1 of nicotinamide radiosensitized the NT carcinoma 80 min after administration, whilst no radiosensitization was seen within 10 min. Thus it is unlikely that increased vessel perfusion is the sole mechanism of nicotinamide-induced radiosensitization in this tumour.

Nicotinamide as a Repair Inhibitor In Vivo: Studies Using Single and Fractionated X-Ray Doses in Mouse Skin and Kidneys

Inhibitors of adenosine diphosphoribosyl transferase, like nicotinamide, 3-aminobenzamide and other analogues, can inhibit repair of radiation-induced sublethal and/or potentially lethal damage in some in vitro systems. Therefore, we have tested the effect of nicotinamide on repair parameters in vivo in two rodent normal tissues. In skin, the sensitivity to dose fractionation (1, 2, 5 or 10 X-ray fractions in 5 days) was monitored by defining the alpha/beta ratio in the presence or absence of nicotinamide (0.5 mg g-1) in air or carbogen. Pre- and postirradiation sensitization were investigated using an X-ray schedule of 5 fractions/5 days in carbogen alone or combined with nicotinamide given 1 h before, immediately after or 8 h after irradiation. Also, changes in the steepness of the underlying X-ray survival curve for the target skin clonogens, reflected by a change in the alpha/beta ratio, were investigated using the neutron top-up design. Underlying survival curves for oxygen +/- nicotinamide were obtained over the X-ray dose range 2.5 to 25 Gy, by administering single X-ray doses and following these with single top-up doses of d(4)-Be neutrons. Finally, in mouse kidney, recovery half-times (t1/2) were obtained by determining the time-dependent disappearance of X-ray damage using a split-dose design of two 6-Gy fractions separated by an interval which varied from 0 to 48 h and followed by two top-up doses from a neutron beam. No increase in alpha/beta for epidermal damage was seen with nicotinamide alone and, although sensitization was observed when the drug was given 1 h before irradiation, no postirradiation sensitization was detected. In kidney, there was no significant difference in the proportion of total repairable damage or in the half-life of recovery between treatments given with or without nicotinamide. Therefore, no decrease in normal tissue tolerance should be observed with the use of nicotinamide in clinical radiotherapy resulting either from reduced sparing with dose fractionation or from an increase in residual damage when shortening the interfraction interval. Finally, unless repair of radiation damage in normal tissues in vivo differs markedly from that of tumors, it is unlikely that the large sensitization seen in rodent tumors at 1.5 to 2 Gy per fraction, with carbogen and nicotinamide, can be attributed to nicotinamide acting as a repair inhibitor.

Polyamine requirements in nicotinamide-stimulated beta-cell differentiation in fetal porcine islet-like cell clusters.

The impact of nicotinamide on regulation of cell replication and hormone production by fetal porcine islet-like cell clusters (ICCs) containing a low fraction of beta-cells was investigated. For this purpose, ICCs were produced in tissue culture for 4 days in the presence or absence of nicotinamide (10 mM). ICCs formed in the presence of nicotinamide showed increased rates of (pro)insulin biosynthesis, contained more insulin, and displayed elevated contents of polyamines when compared with untreated ICCs. Immunocytochemical analyses of autoradiographed ICCs disclosed that nicotinamide expanded the relative size of the beta-cell population, and that the vitamin increased DNA synthesis of non-beta-cells only. The possibility that the effects of nicotinamide were conveyed by the increased polyamine content was explored by giving nicotinamide together with inhibitors of rate-limiting enzymes of polyamine biosynthesis, a maneuver that precluded the increases in polyamines of whole ICCs. Although this treatment prevented the increases in beta-cell population size, insulin production, and insulin content evoked by nicotinamide, the elevated cell replication nonetheless persisted. We conclude that the stimulatory effects of nicotinamide on insulin production and content by fetal porcine ICCs result from neoformation of beta-cells through differentiation. Since these effects were prevented by blocking the increased polyamine content, it is suggested that a stimulated synthesis of polyamines may be an important event in mediating the differentiating action of nicotinamide.

Nicotinamide partially reverses the interleukin-1β inhibition of glucose-induced insulin release in pancreatic islets

Interleukin-1β (IL-1β) is known to inhibit glucose-induced insulin release by pancreatic islets. We studied the effect of nicotinamide, an inhibitor of poly[adenosine diphosphate (ADP)-ribose] synthetase and a free-radical scavenger, on this IL-1β-induced inhibition using rat pancreatic islets. In static experiments, groups of five islets were incubated for 24 hours in culture medium CMRL-1066, with or without 50 U/mL IL-1β, in the presence or absence of nicotinamide (dose range, 0 to 50 mmol/L), and then exposed for 1 hour to either 1.4 or 19.4 mmol/L glucose, 10 mmol/L arginine, or 10 μmol/L glyburide. Basal insulin secretion was 183 ± 32 pg/islet/h (mean ± SE, n = 7) and 176 ± 39 (n = 7) in control islets and in islets exposed to 50 U/mL IL-1β, respectively. Glucose-stimulated insulin secretion was significantly reduced (185 ± 41) in IL-1β-exposed islets in comparison to control islets (2,037 ± 363). In parallel, arginine-stimulated insulin release was inhibited by IL-1β exposure (166 ± 31 pg/islet/h, mean ± SE, n = 3) in comparison to control islets (1,679 ± 307). In contrast, IL-1β exposure did not significantly reduced glyburide-induced insulin secretion (1,516 ± 231 and 1,236 ± 214 in control and IL-1β-exposed islets, respectively; mean ± SE, n = 3). When islets were simultaneously exposed to IL-1β and increasing concentrations of nicotinamide, a dose-dependent recovery of glucose-induced insulin secretion was observed, with the maximum effect at 25 mmol/L nicotinamide (1,007 ± 123, P < .001). Arginine-stimulated insulin release was also partially restored by the addition of 25 mmol/L nicotinamide. To study the phases of insulin release, groups of 100 islets exposed to IL-1β in the presence or absence of nicotinamide (as in static experiments) were perifused for 20 minutes at 37°C in phosphate buffer with glucose 1.4 mmol/L (basal secretion) and then stimulated with glucose 19.4 mmol/L for 35 minutes. In control islets, glucose-stimulated insulin secretion followed the characteristic biphasic pattern: the first peak was 54.9 ± 9.7 and the second one was 70.8 ± 12.5 pg/islet/min (mean ± SE, n = 7). In islets exposed to IL-1β, both phases were greatly reduced (4.9 ± 1.1 and 7.8 ± 2.2, respectively; P < .001). In the presence of 25 mmol/L nicotinamide, glucose-stimulated insulin secretion was partially restored (19.3 ± 3.9, P < .01 in the first phase, and 28.1 ± 6.7, P < .01 in the second phase). Nicotinamide, therefore, is able to partially prevent the effect of IL-1β on blunting both glucose- and arginine-induced insulin secretion.

Reactivation of NAD(H) biosynthetic pathway by exogenous NAD+ in Nil cells severely depleted of NAD(H).

The culture of Nil hamster fibroblasts in MEM lacking nicotinamide (NAm-MEM) leads to: (1) the rapid loss of intracellular total nicotinamide adenine dinucleotide (NAD(H)) content in these cells from a level of 150-200 pmoles/10(5) cells to less than 20 pmoles/10(5) cells; (2) the cessation of cell division and inhibition of DNA synthesis; and (3) a reduction of glucose consumption and lactic acid production. In most situations, following nicotinamide starvation, the restoration of intracellular NAD(H) follows rapidly the readdition of NAD+ (oxidized), nicotinamide mononucleotide (NMN), nicotinamide, or nicotinic acid. Resumption of cell division occurs after only a lag of about 24 hours. Nil cells subcultured for three consecutive times in the absence of nicotinamide (3(0) NAm- cells) exhibit different behavior. These severely starved cells are incapable of quickly restoring their intracellular NAD(H) content to normal levels when provided with any pyridine ring compound except NAD+. One-hour exposure of such cells to NAD+ allows utilization of nicotinamide to rapidly restore intracellular NAD(H). This short incubation with NAD+ does not result in any significant restoration of intracellular NAD(H) or lead to the accumulation of an intracellular pool of some precursor. This function of NAD+ as a stimulatory signal to the NAD(H)-biosynthetic pathway in severely starved Nil cells is a previously unreported role of NAD+, and does not require protein synthesis.

Biosynthesis of vitamin B12. Some properties of the 5,6-dimethylbenzimidazole-forming system of Propionibacterium freudenreichii and Propionibacterium shermanii.

1. Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6-dimethylbenzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6-dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. 2. The stimulation of the 5,6-dimethylbenzimidazole-forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. 3. The 5,6-dimethylbenzimidazole-forming homogenate system is only active at a high buffer concentration (0.3--0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. 4. Flavin mononucleotide and flavin-adenine dinucleotide are better substrates for the 5,6-dimethylbenzimidazole-forming homogenate system than riboflavin. But with [1'-14C]riboflavin as substrate the specific radioactivity of 5,6-dimethylbenzimidazole is higher than the specific radioactivity of flavin--adenine dinucleotide and lower than the specific radioactivie substrate for the formation of 5,6-dimethylbenzimidazole. 5. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6-dimethylbenzimidazole is discussed.

The development of sporulation competence in Physarum polycephalum

Plasmodial cells of the slime mold Physarum polycephalum become “competent” for sporulation following a prolonged period of dark starvation in the presence of nicotinamide. Sporulation can then be induced by illumination. Plasmodia are found to release into the medium during starvation one or more cellular products that promote sporulation. These products exert their effect specifically during the dark starvation period, rather than during the final phase of fruiting body construction. The sporulation control factor(s) (SCF) is nondialyzable and can stimulate the development of sporulation competence in the absence of nicotinamide.

Protein binding of nicotinamide adenine dinucleotide and regulation of nicotinamide adenine dinucleotide glycohydrolase activity in homogenates of rabbit skeletal muscle

Gel-permeation chromatography and ultrafiltration have been used to study the free and bound forms of NAD in crude extracts prepared from rabbit muscle. Both techniques indicate that over 80% of the endogenous NAD is free. Nicotinamide inhibits the destruction of NAD in muscle homogenates (50% inhibition at 1.6 m m nicotinamide). In the absence of nicotinamide, there is a rapid destruction of free NAD, but a more gradual destruction of bound NAD. The latter result confirms earlier findings that bound NAD is protected from the hydrolytic action of NADase. However, this protection is unlikely to constitute an important mechanism for controlling NADase activity in muscle homogenates because such a small proportion of the endogenous NAD is bound. In the absence of nicotinamide, NAD also disappears rapidly from minced muscle. Interestingly, the NAD/NADH ratio remains constant (NAD/NADH = 18.1–18.5) during the disappearance of NAD in minced muscle. Upon homogenization of the mince, the NAD/NADH ratio abruptly decreases, then slowly increases during subsequent incubation. The latter rise in NAD/NADH ratio appears to be independent of absolute changes in NAD concentration brought about by the action of NADase or the addition of exogenous NAD.