mM For NAD Research Articles

Silk Gland Factor 1 Plays a Pivotal Role in Larval Settlement of the Fouling Mussel Mytilopsis sallei.

Most fouling organisms have planktonic larval and benthic adult stages. Larval settlement, the planktonic-benthic transition, is the critical point when biofouling begins. However, our understanding of the molecular mechanisms of larval settlement is limited. In our previous studies, we identified that the AMP-activated protein kinase-silk gland factor 1 (AMPK-SGF1) pathway was involved in triggering the larval settlement in the fouling mussel M. sallei. In this study, to further confirm the pivotal role of SGF1, multiple targeted binding compounds of SGF1 were obtained using high-throughput virtual screening. It was found that the targeted binding compounds, such as NAD+ and atorvastatin, could significantly induce and inhibit the larval settlement, respectively. Furthermore, the qRT-PCR showed that the expression of the foot proteins' genes was significantly increased after the exposure to 10 μM NAD+, while the gene expression was significantly suppressed after the exposure to 10 μM atorvastatin. Additionally, the production of the byssus threads of the adults was significantly increased after the exposure to 10-20 μM of NAD+, while the production of the byssus threads was significantly decreased after the exposure to 10-50 μM of atorvastatin. This work will deepen our understanding of SGF1 in triggering the larval settlement in mussels and will provide insights into the potential targets for developing novel antifouling agents.

Non-invasive diagnosis of bacterial and non-bacterial inflammations using a dual-enzyme-responsive fluorescent indicator.

Bacterial infections, as the second leading cause of global death, are commonly treated with antibiotics. However, the improper use of antibiotics contributes to the development of bacterial resistance. Therefore, the accurate differentiation between bacterial and non-bacterial inflammations is of utmost importance in the judicious administration of clinical antibiotics and the prevention of bacterial resistance. However, as of now, no fluorescent probes have yet been designed for the relevant assessments. To this end, the present study reports the development of a novel fluorescence probe (CyQ) that exhibits dual-enzyme responsiveness. The designed probe demonstrated excellent sensitivity in detecting NTR and NAD(P)H, which served as critical indicators for bacterial and non-bacterial inflammations. The utilization of CyQ enabled the efficient detection of NTR and NAD(P)H in distinct channels, exhibiting impressive detection limits of 0.26 μg mL-1 for NTR and 5.54 μM for NAD(P)H, respectively. Experimental trials conducted on living cells demonstrated CyQ's ability to differentiate the variations in NTR and NAD(P)H levels between A. baumannii, S. aureus, E. faecium, and P. aeruginosa-infected as well as LPS-stimulated HUVEC cells. Furthermore, in vivo zebrafish experiments demonstrated the efficacy of CyQ in accurately discerning variations in NTR and NAD(P)H levels resulting from bacterial infection or LPS stimulation, thereby facilitating non-invasive detection of both bacterial and non-bacterial inflammations. The outstanding discriminatory ability of CyQ between bacterial and non-bacterial inflammation positions it as a promising clinical diagnostic tool for acute inflammations.

Involvement of SlSTOP1 regulated SlFDH expression in aluminum tolerance by reducing NAD+ to NADH in the tomato root apex.

Formate dehydrogenase (FDH; EC 1.2.1.2.) has been implicated in plant responses to a variety of stresses, including aluminum (Al) stress in acidic soils. However, the role of this enzyme in Al tolerance is not yet fully understood, and how FDH gene expression is regulated is unknown. Here, we report the identification and functional characterization of the tomato (Solanum lycopersicum) SlFDH gene. SlFDH encodes a mitochondria-localized FDH with Km values of 2.087mm formate and 29.1μm NAD+ . Al induced the expression of SlFDH in tomato root tips, but other metals did not, as determined by quantitative reverse transcriptase-polymerase chain reaction. CRISPR/Cas9-generated SlFDH knockout lines were more sensitive to Al stress and formate than wild-type plants. Formate failed to induce SlFDH expression in the tomato root apex, but NAD+ accumulated in response to Al stress. Co-expression network analysis and interaction analysis between genomic DNA and transcription factors (TFs) using PlantRegMap identified seven TFs that might regulate SlFDH expression. One of these TFs, SlSTOP1, positively regulated SlFDH expression by directly binding to its promoter, as demonstrated by a dual-luciferase reporter assay and electrophoretic mobility shift assay. The Al-induced expression of SlFDH was completely abolished in Slstop1 mutants, indicating that SlSTOP1 is a core regulator of SlFDH expression under Al stress. Taken together, our findings demonstrate that SlFDH plays a role in Al tolerance and reveal the transcriptional regulatory mechanism of SlFDH expression in response to Al stress in tomato.

ODP443 Targeting NAD+ availability modulates 11β- Hydroxysteroid dehydrogenase 1 activity in mouse and human cell lines and primary cells

Abstract Four nicotinamide adenine dinucleotide coenzymes – NAD+, NADH, NADP+, and NADPH – are the central catalysts of metabolism. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is NADPH-dependent reductase that catalyses the reduction of the inactive glucocorticoid cortisone) to active cortisol (11-dehydrocorticosterone to corticosterone in rodents). 11β-HSD1 is in the endoplasmic reticulum (ER) lumen, and the level of NADPH in the ER regulates enzyme activity. Little is known about the biochemical mechanisms controlling cytosolic/ER NAD(P)(H) crosstalk. Here we examined how perturbed cellular NAD+ availability modulates 11β-HSD1 enzyme activity in the ER across a range of mouse and human cell types. 11β-HSD1 activity was measured in a series of transformed and primary mouse and human liver (HepG2), muscle, and Human Dermal Fibroblast cells (HDFn) depleted for NAD+ using FK866 to inhibit nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. To replete NAD+ we supplemented cells with 0.5mM of NAD+ precursors nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN). FK866 (100nM, 48h) was effective at depleting NAD+ by approximately 90% in C2C12 myotubes and 50% in HepG2 liver cells. In each cell line, 11β-HSD1 reductase activity was decreased by FK866 compared to untreated controls (80% in C2C12 P<0. 0001, 55% in HepG2, P=0. 03 and 65% in HDFn P=0. 002). Restoring NAD+ level with NR (0.5mM, 6h) in C2C12 and (0.5mM, 24h) in HepG2 and HDFn rescued 11β-HSD1 enzyme activity compared with no precursor treatment (60% in C2C12 P=0. 0007, 52% in HepG2 P=0. 03 and 68% in HDFn P=0. 0006). Human dermal fibroblasts treated with TNFa (10nM, 24h) boosted 11β-HSD1 expression 4-fold, however this was not able to overcome the FK866 induced drop-in reductase activity. These data show that depletion of NAD+ and impaired 11β-HSD1 reductase activity in the ER is a generalised phenomenon across cell types. This suggests a crosstalk between cytosolic and ER NAD(P)(H) pools that can respond rapidly to changes in NAD+ precursor availability. This could provide new insight into the regulation of cellular glucocorticoid metabolism and ER NAD(P)(H) homeostasis. Presentation: No date and time listed

A system of co-immobilized dual-enzyme and coenzyme for in-situ coenzyme regeneration

Coenzymes are required in the reaction catalyzed by oxidoreductases. The research on coenzyme regeneration has always been a hot topic in the field of biological catalysis. In this work, we tried to construct a co-immobilization system of glucose dehydrogenase (GDH), (R)-1-phenylethanol dehydrogenase ((R)-PEDH) and coenzyme with functional magnetic nanoparticles as carriers, so as to realize chiral (R)-1-phenylethanol highly selective production and coenzyme effective recycling. The enzymatic properties of immobilized enzymes were explored. The results showed that the immobilized enzymes had improved stability and fine reusability. In addition, during the catalytic coupling reaction at a final concentration of 2.63 μM NAD+, the total turnover number (TTN) was as high as 924, and the volumetric productivity of (R)-1-phenylethanol was 37.06 mg·L−1·h−1. After reused for 10 cycles, the catalytic efficiency of immobilized coenzyme can still be maintained at more than 30%. From multiple perspectives, the immobilized dual-enzymes and coenzyme system seems to be a multifunctional system for (R)-1-phenylethanol production and coenzyme regeneration.

Ex Vivo Lung Perfusion with β-Nicotinamide Adenine Dinucleotide (NAD+) Improves Ischemic Lung Function

Ischemia-reperfusion injury compromises short- and long-term outcomes after lung transplantation. The scarce existing data on NAD+ suggest effects on hypoxia-induced vasoconstriction, on reactive oxygen species and on tampering inflammation. We exposed rat lungs to 14 h of cold ischemic storage and perfused them in a rat ex vivo lung perfusion (EVLP) system for 4 h. A control group (n = 6) was compared to groups receiving 100 µM (n = 6) or 200 µM NAD+ (n = 6) in the preservation solution and groups receiving 200 µM (n = 4) or 2000 µM (n = 6) NAD+ every 30 min in the perfusate, starting at 1 h of EVLP. Compared to the control, significant effects were only achieved in the 2000 µM NAD+ group. During the 4 h of EVLP, we monitored higher vascular flow, lower mean pulmonary arterial pressure and increased oxygenation capacity. Tissue inflammation estimated with the myeloperoxidase assay was lower in the 2000 µM NAD+ group. We observed higher levels of anti-inflammatory IL-10, higher anti-inflammatory IL-6/IL-10 ratios and lower levels of pro-inflammatory IL-12 and IL-18 as well as a trend of more anti-inflammatory IFNy in the 2000 µM NAD+ perfusate. In the bronchoalveolar lavage, the pro-inflammatory levels of IL-1α and IL-1β were lower in the 2000 µM NAD+ group. NAD+ administered during EVLP is a promising agent with both anti-inflammatory properties and the ability to improve ischemic lung function.

Room-Temperature Persistent Luminescence in Metal Halide Perovskite Nanocrystals for Solar-Driven CO 2 Bioreduction

Room-Temperature Persistent Luminescence in Metal Halide Perovskite Nanocrystals for Solar-Driven CO ₂ Bioreduction

Inhibition of phosphate transport by NAD+/NADH brush border membrane vesicles.

Nicotinamide is an important regulator of Pi homeostasis after conversion into NAD+/NADH. In this work, we have studied the classical inhibition of Pi transport by these compounds in the brush border membrane vesicles (BBMV) of rat kidney and rat intestine, and we examined the effects in opossum kidney (OK) cells and in phosphate transporter-expressing Xenopus laevis oocytes. In BBMV, NAD+ required preincubation at either room temperature or on ice to inhibit Pi uptake in BBMV. However, no effects were observed in the known Slc34 or Slc20 Pi transporters expressed in Xenopus oocytes, in OK cells, or in isolated rat cortical nephron segments. In BBMV from jejunum or kidney cortex, the inhibition of Pi transport was specific, dose-related, and followed a competitive inhibition pattern, as shown by linear transformation and nonlinear regression analyses. A Ki value of 538 µM NAD+ in kidney BBMV was obtained. Ribosylation inhibitors and ribosylation assays revealed no evidence that this reaction was responsible for inhibiting Pi transport. An analysis of the persistence of NAD+/NADH revealed a half-life of just 2 min during preincubation. Out of several metabolites of NAD degradation, only ADP-ribose was able to inhibit Pi uptake. Pi concentration also increased during 30 min of preincubation, up to 0.67 mM, most likely as a metabolic end product. In conclusion, the classical inhibition of Pi transport by NAD+/NADH in BBMV seems to be caused by the degradation metabolites of these compounds during the preincubation time.

Cloning, expression, and characterization of an arabitol dehydrogenase and coupled with NADH oxidase for effective production of L-xylulose

A novel arabitol dehydrogenase (ArDH) gene was cloned from a bacterium named Aspergillus nidulans and expressed heterologously in Escherichia coli. The purified ArDH exhibited the maximal activity in pH 9.5 Tris-HCl buffer at 40 °C, showed K m and V max of 1.2 mg/mL and 9.1 U/mg, respectively. The ArDH was used to produce the L-xylulose and coupled with the NADH oxidase (Nox) for the regeneration of NAD+. In further optimization, a high conversion of 84.6% in 8 hours was achieved under the optimal conditions: 20 mM of xylitol, 100 µM NAD+ in pH 9.0 Tris-HCl buffer at 30 °C. The results indicated the coupling system with cofactor regeneration provides a promising approach for L-xylulose production from xylitol.

Lactate metabolism in strictly anaerobic microorganisms with a soluble NAD+ -dependent l-lactate dehydrogenase.

Lactate is a universal metabolite and energy source, yet the mode of lactate metabolism in many strictly anaerobic microorganisms is still enigmatic. This sparked us to investigate the biochemistry and bioenergetics of lactate metabolism in the model acetogenic bacterium Moorella thermoacetica. Growth and metabolism were dependent on CO2 and the chemiosmotic gradient. We discovered a l-lactate:NAD+ oxidoreductase (LDH) in cell-free extracts, exhibiting an average specific activity of 362.8± 22.9mU mg-1 . The enzyme was reversible, most active at 65°C and pH9, with Km values of 23.1± 3.7 mM for l-lactate and 273.3± 39.1μM for NAD+ . In-gel activity assays and mass spectrometric proteomics revealed that the ldh gene encoded the characterized LDH. Transcriptomic and genomic analyses showed that ldh expression was induced by lactate and there was a single nucleotide polymorphism near the predicted NAD+ binding site. Genes encoding central redox and energy metabolism complexes, such as, the energetic coupling site Ech2, menaquinone, and the electron bifurcating EtfABCX and MTHFR were also upregulated in cells grown on lactate. These findings ultimately lead to a redox-balanced metabolic model that shows how growth on lactate can proceed in a microorganism that only has a conventional NAD+ -reducing LDH.

POS0394 NAD+ BOOSTERS REESTABLISH THE ALTERED NAD+ METABOLISM OF LEUKOCYTES FROM RHEUMATOID ARTHRITIS PATIENTS IMPROVING THEIR OXIDATIVE, APOPTOTIC AND INFLAMMATORY STATUS

Background:NAD+ is an important cofactor and second messenger for multiple cellular processes that exhibits antioxidant, anti-apoptotic and anti-inflammatory properties. Pre-clinical studies in animal models of Rheumatoid Arthritis (RA) have demonstrated the therapeutic potential of NAD+ boosters in the control of the disease activity. However, to date no studies have been set up to evaluate the NAD+ metabolism and the therapeutic effects of NAD+ boosters in RA patients.Objectives:1- To study the NAD+ metabolism in RA patients and its association with key clinical features. 2- To evaluate the effect of anti-TNF therapy in the NAD+ metabolism.3- To analyze the beneficial effects of NAD+ boosters in leukocytes from active RA patients.Methods:Plasma and PBMCs were purified from 100 RA patients and 50 healthy donors (HDs). Moreover, an additional cohort of 50 RA patients treated with Anti-TNF therapy was analyzed before and after 6 months of treatment. NAD+ levels were determined by using the NAD/NADH-Glo Assay. NAD+-consuming genes expression were analyzed by RT-PCR. In parallel, PBMCs from eight HDs and eight active RA patients were treated ex vivo with 1 mM of NAD+ boosters including nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN). After 24 hours, intracellular reactive oxygen species (ROS) levels (DFCHDA) and the percentage of apoptotic PBMCs (annnexin V/PI) were assessed by flow cytometry. Lastly, a panel of key pro-inflammatory genes were evaluated by RT-PCR.Results:NAD+ and NADH levels were significantly reduced in plasma and PBMCs of RA patients compared with HDs and directly related to disease activity (DAS28, CDAI, SDAI). Accordingly, the expression levels of genes involved in the consumption of NAD+ such as SIRT-1, CD38 and PARP-1 were found up-regulated in PBMCs from RA patients. Anti-TNF therapy for 6 months restored the altered NAD+ levels towards those showed by HDs. Furthermore, the clinical response promoted by Anti-TNF therapy (changes in DAS28) correlated with changes in NAD+ levels. The in vitro treatments of PBMCs isolated from active RA patients with NAD+ boosters significantly increased the NAD+ levels and promoted a deep reduction of intracellular ROS levels, the percentage of apoptotic cells and the expression levels of key inflammatory mediators, such as IL-6, IL-8, IL-1ß, TNF-α, CCL2, IL-23, and STAT-3.Conclusion:1. NAD+ metabolism is altered and associated with the disease activity of RA patients, involving both, reduced NAD+ levels and increased expression of NAD+-consuming genes. 2. Anti-TNF therapy restored NAD+ levels, which were directly linked to the clinical effectiveness. 3. NAD+ boosters reduced the oxidative, apoptotic and inflammatory profiles of RA leukocytes through the parallel increase of intracellular NAD+ levels. Thus, NAD+ boosters might be considered novel therapeutic tools for RA patients.Acknowledgements:Supported by PI18/00837, RIER RD16/0012/0015, RTI2018-100695-B-I00, P18-RT-4264 and CVI276, co-funded with FEDER.Disclosure of Interests:None declared

Characterization of Sodium‐Dependent Pi Transport Inhibition by NAD

Hyperphosphatemia during end-stage renal disease causes many adverse outcomes. To prevent them, several Pi intestinal absorption inhibitors are necessary for targeting either the paracellular (diffusion) or the transcellular (transport) absorption pathway. Nicotinamide adenine dinucleotide (NADH/NAD+) is a classical inhibitor of intestinal and renal Pi transport and is a circadian modulator of phosphatemia. The mechanism of NAD transporter inhibition remains unclear, although a competitive inhibition mechanism has been suggested. In this work, we sought to determine if this mechanism has a direct effect on Pi transporters. The five known rat Na/Pi cotransporters (NaPi2a, NaPi2b, NaPi2c, PiT1, and PiT2) were expressed in Xenopus laevis oocytes. Uptake of 32P-Pi was performed for three days post-injection of 5 ng of the corresponding cRNAs per oocyte and after 30 minutes of preincubation with 0.5 mM NAD, NADH, or NAM. None of the compounds affected the Pi uptake of any of the expressed Pi transporters in oocytes. Pi transport in the brush border membrane vesicles (BBMV) of either rat kidney cortex or jejunum epithelium was also assayed. As expected, it was significantly inhibited by NADH and NAD when BBMV were preincubated for 30 min at room or ice-cold temperature. NAM had no effect, and the effect was specific for Na-dependent Pi uptake (D-glucose uptake was not affected). Dose-response analyses on jejunum or kidney BBMV revealed IC50 values in the micromolar range for 50 µM of Pi uptake. Several Michaelis-Menten kinetics were performed in the presence of different concentrations of NAD, and a Lineweaver-Burk plot suggested changes in affinity (i.e., competitive inhibition), which contrasts with the lack of an effect on the transporters expressed in oocytes. The competitive model was confirmed by non-linear regression of a global fit with shared parameters, providing a Ki value of 538 µM NAD. When the effect of NAD was assayed in Opossum Kidney cells, a proximal tubule cell line model expressing a regulated NaPi2a, no evidence of Pi transport inhibition was observed. To check whether NAD-mediated ribosylation was involved in Pi transport inhibition, a classical ribosylation assay was performed with 32P-NAD, using jejunum and renal BBMV. No evidence of ribosylation was found for NaPi2b or NaPi2a, even after one month of exposure to an X-ray film. This was confirmed using several ribosylation inhibitors (meta-iodobenzylguanidine, novobiocin, vitamin K1, vitamin K3, or 3-methoxybenzamide), which failed to prevent NAD inhibition of Pi transport in BBMV. In conclusion, despite the kinetic findings in BBMV, NAD inhibition of Na/Pi-cotransporters is, most likely, not mediated through a direct interaction with these transporters. Instead, inhibition seems to be mediated through an indirect mechanism acting on components that are absent in Xenopus oocytes and in OK cells, which ends in the modification of Pi transport affinity (Km) and occurs at ice-cold temperature. Nevertheless, the unlikely inhibition of a novel, unknown Pi transporter cannot be completely discarded.

The investigation of the effect of NAD, H2O2 and weak magnetic field on the antibacterial mechanism of isoniazid (INH) that first line antibiotic against M. tuberculosis agent

Tuberculosis is an infectious disease, which is caused by the Mycobacterium tuberculosis complex. This disease leads to up to 1.3 million deaths out of more than eight million cases every year. A prodrug called isoniazid has been proven to be effective and widely used in the treatment of infections caused by tuberculosis. Despite its use for more than six decades clinically, the action mechanism of this prodrug is yet to be elucidated. INH action agains mycobacteria requires catalase−peroxidase (KatG) function, and IN-NAD adduct formation is catalyzed in vitro by M. tuberculosis KatG under a variety of conditions. Low-intensity EMF (Electromagnetic Field) has been used in the₂rapeutic practices in addition to its use in telecommunication systems and food protection. EMF is used in medicine and food industries especially for its bactericidal effects. In this study, we aimed to investigate the effects of weak magnetic field application and the addition of NAD and H₂O₂ on the action mechanism of isoniazid. We added H₂O₂ and NAD individually and together, to the different groups at varying concentrations. Also, one experimental group was exposed to a 5mT, 50Hz magnetic field for 4 to 5 hours per day (total of 45 hours in 10 days). The agar proportion method was used to evaluate the results. It was determined that the addition of 100 μM NAD and H₂O₂ together increased the effectiveness of isoniazid to some extent. However, the application of a weak magnetic field did not change the effectiveness of the drug.

Role of the malic enzyme in metabolism of the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z.

The bacteria utilizing methane as a growth substrate (methanotrophs) are important constituents of the biosphere. Methanotrophs mitigate the emission of anthropogenic and natural greenhouse gas methane to the environment and are the promising agents for future biotechnologies. Many aspects of CH4 bioconversion by methanotrophs require further clarification. This study was aimed at characterizing the biochemical properties of the malic enzyme (Mae) from the halotolerant obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z. The His6-tagged Mae was obtained by heterologous expression in Escherichia coli BL21 (DE3) and purified by affinity metal chelating chromatography. As determined by gel filtration and non-denaturating gradient gel electrophoresis, the molecular mass of the native enzyme is 260 kDa. The homotetrameric Mae (65x4 kDa) catalyzed an irreversible NAD+-dependent reaction of L-malate decarboxylation into pyruvate with a specific activity of 32 ± 2 units mg-1 and Km value of 5.5 ± 0.8 mM for malate and 57 ± 5 μM for NAD+. The disruption of the mae gene by insertion mutagenesis resulted in a 20-fold increase in intracellular malate level in the mutant compared to the wild type strain. Based on both enzyme and mutant properties, we conclude that the malic enzyme is involved in the control of intracellular L-malate level in Mtm. alcaliphilum 20Z. Genomic analysis has revealed that Maes present in methanotrophs fall into two different clades in the amino acid-based phylogenetic tree, but no correlation of the division with taxonomic affiliations of the host bacteria was observed.

Purification and characterization of malate dehydrogenase from sheep liver (Ovis aries): Application in AST assay diagnostic kit

Malate dehydrogenase enzyme is a major component in aspartate aminotransferase (AST) diagnostic kit that used in diagnosis and monitoring of liver diseases. This study aimed at purification and characterization of MDH enzyme from sheep liver for direct application in preparation of AST kit. Two malate dehydrogenase isoenzymes were resolved from sheep liver by chromatography on DEAE-cellulose column, one major sheep liver MDH (SLMDH) and another minor one. SLMDH was extracted and purified by ammonium sulfate fractionation and chromatographical separation on anion exchanger and gel filtration resins. The purified sheep liver MDH specific activity is 6.7 units / mg protein representing 11.6 purification folds and 27.7 % yield. The molecular mass of SLMDH intact protein is 68 ± 1.6 kDa. SLMDH turned out to be homogeneous and consists of two identical subunits of 34 ± 1.2 kDa each. The SLMDH isoelectric point (pI) value is estimated at pH 6.2 and displayed its optimum activity at pH 9.6 and has Km value of 1.4 mM for NAD and 11 mM for malate. FeCl2, NiCl2 and ZnCl2 inhibited the SLMDH activity. The purified SLMDH is applied in the preparation of AST diagnostic kit that found sensitive and comparable with a commercially available one.

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Isotype-specific agglutination-PCR (ISAP): A sensitive and multiplex method for measuring allergen-specific IgE

Abstract 4404: Novel approach to circumvent cisplatin resistant lung cancer by targeting metabolism

Abstract For the past three decades, there are no drugs which could reverse cisplatin resistance or selectively kill these resistant cells. We have discovered that cisplatin resistant (CR) lung cancer shares one common factor which is increase in reactive oxygen species (ROS). Decreased intracellular thioredoxin-1(TRX1) due to excessive secretion was found in vitro and in vivo, as well as in patients’ serum which could be a primary contributory factor to higher mitochondria-ROS levels. Furthermore, CR cells possess significantly increased number of mitochondria and consume higher rates of oxygen. Key glycolytic enzymes (HKII and LDHA) and lactate production were decreased in CR cells which suggests rewiring of tumor metabolic pathways. NAD+, crucial co-factor of all redox systems, is significantly reduced in all CR cells. Importantly, treatment with riluzole (the FDA approved drug that interferes with cystine/glutamate pump and results in reduced intracellular glutathione (GSH) levels) led to further decrease in NAD+ and LDHA, as well as heightened oxidative stress in CR cells. ROS levels were increased (2-3X) more in resistant cells after riluzole treatment while no significant change occurred in parental cells. The ID50 dosage of parental cells were 3-4 fold more than theirs CR cell counterparts and no cytotoxicity were found in normal lung fibroblast (n=8; p<0.05). Addition of 200 µM NAD+ re-stabilized LDHA, reduced ROS, and reversed riluzole induced cell death. To further determine whether LDHA correlates with ROS levels and sensitivity to riluzole, we inhibited LDHA expressions in parental cell lines S and H460 using siRNA. Lactate productions were decreased in knockdown cells and further attenuated upon riluzole treatment when compared with scrambled control. Higher basal levels of ROS were obtained from S+siLDHA and H460+siLDHA and further increased in ROS levels were observed when treated with riluzole. We then evaluated the viability of these cells under riluzole treatment. Both siLDHA clones were sensitive to riluzole particularly in S+siLDHA which showed 4 fold more sensitivity. Importantly, treatment of riluzole also led to further decrease in LDHA expressions at both protein and mRNA levels. Using riluzole in 2 mouse xenograft models (H460CR (KRAS mutant) and SC (wt)) resulted in completely disappeared in wild type mice and were significant reduced in KRAS mutant mice (n=5 per treatment group; p<0.002). Repurpose of riluzole should be considered for future treatment of CR lung cancer patients. Supported by Department of Veterans Affairs, CDA2 award (1K2BX001289) and Woman Cancer Association Fund. Citation Format: Medhi Wangpaichitr, Chunjing Wu, Ying Ying Li, Lynn G. Feun, Macus T. Kuo, Niramol Savaraj. Novel approach to circumvent cisplatin resistant lung cancer by targeting metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4404. doi:10.1158/1538-7445.AM2017-4404

Effects of exogenous nicotinamide adenine dinucleotide (NAD+) in the rat heart are mediated by P2 purine receptors.

BackgroundRecently, NAD+ has been considered as an essential factor, participating in nerve control of physiological functions and intercellular communication. NAD+ also has been supposed as endogenous activator of P1 and P2 purinoreceptors. Effects of extracellular NAD+ remain poorly investigated in cardiac tissue. This study aims to investigate the effects of extracellular NAD+ in different types of supraventricular and ventricular working myocardium from rat and their potential mechanisms.MethodsThe standard technique of sharp microelectrode action potential recording in cardiac multicellular preparations was used to study the effects of NAD+.ResultsExtracellular NAD+ induced significant changes in bioelectrical activity of left auricle (LA), right auricle (RA), pulmonary veins (PV) and right ventricular wall (RV) myocardial preparations. 10–100 μM NAD+ produced two opposite effects in LA and RA – quickly developing and transient prolongation of action potentials (AP) and delayed sustained AP shortening, which follows the initial positive effect. In PV and RV only AP shortening was observed in response to NAD+ application. In PV preparations AP shortening induced by NAD+ may be considered as a potential proarrhythmic effect. Revealed cardiotropic effects of NAD+ are likely to be mediated by P2 purine receptors, since P1 blocker DPCPX failed to affect them and P2 antagonist suramin abolished NAD + −induced alterations of electrical activity. P2X receptors may be responsible for NAD + −induced short-lasting AP prolongation, while P2Y receptors mediate persistent AP shortening. The latter effect is partially removed by PLC inhibitor U73122 showing the potential involvement of phosphoinositide signaling pathway in mediation of NAD+ cardiotropic effects.ConclusionsExtracellular NAD+ is supposed to be a novel regulator of cardiac electrical activity. P2 receptors represent the main target of NAD+ at least in the rat heart.

Enzyme-Modified Particles for Selective Biocatalytic Hydrogenation by Hydrogen-Driven NADH Recycling.

We describe a new approach to selective H2-driven hydrogenation that exploits a sequence of enzymes immobilised on carbon particles. We used a catalyst system that comprised alcohol dehydrogenase, hydrogenase and an NAD+ reductase on carbon black to demonstrate a greater than 98 % conversion of acetophenone to phenylethanol. Oxidation of H2 by the hydrogenase provides electrons through the carbon for NAD+ reduction to recycle the NADH cofactor required by the alcohol dehydrogenase. This biocatalytic system operates over the pH range 6–8 or in un-buffered water, and can function at low concentrations of the cofactor (10 μm NAD+) and at H2 partial pressures below 1 bar. Total turnover numbers >130 000 during acetophenone reduction indicate high enzyme stability, and the immobilised enzymes can be recovered by a simple centrifugation step and re-used several times. This offers a route to convenient, atom-efficient operation of NADH-dependent oxidoreductases for selective hydrogenation catalysis.

Effects of Cofactors on Conformation Transition of Random Peptides Consisting of a Reduced Amino Acid Alphabet.

This study aims to explore the structure characteristic of random polypeptides constructed by origin early amino acid alphabet, as well as the effects of cofactors on conformation transition of random peptides. DNA library R8-4 encoding VNM random peptides were constructed by small cassette strategy. Subsequently, a random polypeptide library was constructed using in vitro translation. Expression and purification of VNM random peptides were also performed by a conventional method of recombinant. CD spectrum analysis indicated that VNM random polypeptides have a secondary structure characteristic of protein, such as the content of α-helix is greater than 60%, random coil is about 20% β sheet, and β turn is less than 10%. CD spectrum changed with the addition of 10-40 µM ATP and NADP, but slightly changed by NAD; no influence was observed with MgSO4. Bis-ANS binding assay indicated that fluorescent intensity of bis-ANS was strengthened slightly by 10 VNM random peptides. Fluorescent intensity was strengthened fourfold by adding 10-40 µM ATP, NAD, and NADH, whereas the inducing effect of NADPH and MgSO4 were negligible. VNM random peptides have a classic secondary structure and hydrophobic domain in water solution. Moreover, conformation transition and hydrophobic domain could be induced by cofactor, indicating the preliminary evidence for the hypothesis that "the origin of primitive protein was induced by small molecule."