Decrease In NADPH Research Articles

Abstract 1792: Synthetic lethal vulnerabilities stemming from inhibition of one carbon metabolism in anaplastic thyroid cancer

Abstract Reprogramming of metabolic processes by tumor cells is essential to cope with their increased proliferative activity, and with the challenges of the unique microenvironment they inhabit. These metabolic alterations are attractive targets to design novel therapeutic approaches.The most aggressive subtype of thyroid cancer, Anaplastic Thyroid Cancer (ATC), is often unresectable at presentation, highly resistant to therapy and usually lethal. We have recently discovered that ATCs overexpress several genes encoding enzymes involved in one-carbon metabolism (1C-Met), which utilizes serine and dietary folates to produce glycine and tetrahydrofolate-bound one-carbon units, required for nucleotide biosynthesis and for NADPH and glutathione (GSH) production. We have shown that i) the increased activity of the 1C-Met pathway supports the high purine demand of ATC; ii) inhibition of 1C-Met impairs tumor growth in vitro and in vivo, leading to growth arrest; iii) inhibition of 1C-Met renders thyroid cancer cells glycine-auxotroph.We now demonstrate that the profound addiction of ATC cells to 1C-Met creates a series of targetable vulnerabilities that can be harnessed to induce massive ATC cell death.Our new data demonstrate that:1- The purine depletion consequent impaired 1C-Met induces replicative stress and triggers the DNA Damage Response (DDR). Activation of DDR, coupled with the absence of functional TP53 in the vast majority of ATCs, leads to synthetic lethality between inhibition of 1C-Met and G2/M checkpoint kinases.2- Inhibition of 1C-Met in ATC cells generates an absolute dependence on extracellular glycine uptake. We have identified and validated the main glycine transporter(s) in ATC cells and show that inhibition of 1C-Met is synthetic lethal with inhibition of glycine uptake.3- Impaired 1C-Met leads to increased oxidative stress and decrease of NADPH and glutathione levels. These alterations create a synthetic lethality relationship with the glutathione-depleting activity of PRIMA-1 (APR246), a small molecule originally identified as a compound restoring mutant p53 conformation and function, but later shown to be converted within cells into the reactive electrophile methylene quinuclidinone, which acts in a p53-independent manner through a variety of mechanisms, including reduction of cellular GSH levels.Our data shed light on the molecular consequences of 1C-Met upregulation in ATC and support the efficacy of novel rationally designed therapeutic approaches with curative intent not only for ATC, but also for other aggressive, dedifferentiated solid tumors. Citation Format: Alexander Forrest, Anil Ahsan, Antonio Di Cristofano. Synthetic lethal vulnerabilities stemming from inhibition of one carbon metabolism in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1792.

Stability of Metabolomic Content during Sample Preparation: Blood and Brain Tissues.

Thermal and enzymatic reactions can significantly change the tissue metabolomic content during the sample preparation. In this work, we evaluated the stability of metabolites in human whole blood, serum, and rat brain, as well as in metabolomic extracts from these tissues. We measured the concentrations of 63 metabolites in brain and 52 metabolites in blood. We have shown that metabolites in the extracts from biological tissues are stable within 24 h at 4 °C. Serum and whole blood metabolomes are also rather stable, changes in metabolomic content of the whole blood homogenate become apparent only after 1–2 h of incubation at 4 °C, and become strong after 24 h. The most significant changes correspond to energy metabolites: the concentrations of ATP and ADP decrease fivefold, and the concentrations of NAD, NADH, and NADPH decrease below the detectable level. A statistically significant increase was observed for AMP, IMP, hypoxanthine, and nicotinamide. The brain tissue is much more metabolically active than human blood, and significant metabolomic changes occur already within the first several minutes during the brain harvest and sample homogenization. At a longer timescale (hours), noticeable changes were observed for all classes of compounds, including amino acids, organic acids, alcohols, amines, sugars, nitrogenous bases, nucleotides, and nucleosides.

Different Effects of RNAi-Mediated Downregulation or Chemical Inhibition of NAMPT in an Isogenic IDH Mutant and Wild-Type Glioma Cell Model.

The IDH1R132H mutation in glioma results in the neoenzymatic function of IDH1, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG), alterations in energy metabolism and changes in the cellular redox household. Although shifts in the redox ratio NADPH/NADP+ were described, the consequences for the NAD+ synthesis pathways and potential therapeutic interventions were largely unexplored. Here, we describe the effects of heterozygous IDH1R132H on the redox system in a CRISPR/Cas edited glioblastoma model and compare them with IDH1 wild-type (IDH1wt) cells. Besides an increase in 2-HG and decrease in NADPH, we observed an increase in NAD+ in IDH1R132H glioblastoma cells. RT-qPCR analysis revealed the upregulation of the expression of the NAD+ synthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Knockdown of NAMPT resulted in significantly reduced viability in IDH1R132H glioblastoma cells. Given this dependence of IDH1R132H cells on NAMPT expression, we explored the effects of the NAMPT inhibitors FK866, GMX1778 and GNE-617. Surprisingly, these agents were equally cytotoxic to IDH1R132H and IDH1wt cells. Altogether, our results indicate that targeting the NAD+ synthesis pathway is a promising therapeutic strategy in IDH mutant gliomas; however, the agent should be carefully considered since three small-molecule inhibitors of NAMPT tested in this study were not suitable for this purpose.

Dual Functional Eudragit® S100/L30D-55 and PLGA Colon-Targeted Nanoparticles of Iridoid Glycoside for Improved Treatment of Induced Ulcerative Colitis.

AimIridoid glycosides (IG) as the major active fraction of Syringa oblata Lindl. has a proven anti-inflammatory effect for ulcerative colitis (UC). However, its current commercial formulations are hampered by low bioavailability and unable to reach inflamed colon. To overcome the limitation, dual functional IG-loaded nanoparticles (DFNPs) were prepared to increase the residence time of IG in colon. The protective mechanism of DFNPs on DSS-induced colonic injury was evaluated in rats.Materials and MethodsWe prepared DFNPs using the oil-in-water emulsion method. PLGA was selected as sustained-release polymer, and ES100 and EL30D-55 as pH-responsive polymers. The morphology and size distribution of NPs were measured by SEM and DLS technique. To evaluate colon targeting of DFNPs, DiR, was encapsulated as a fluorescent probe into NPs. Fluorescent distribution of NPs were investigated. The therapeutic potential and in vivo transportation of NPs in gastrointestinal tract were evaluated in a colitis model.ResultsSEM images and zeta data indicated the successful preparation of DFNPs. This formulation exhibited high loading capacity. Drug release results suggested DFNPs released less than 20% at the first 6 h in simulated gastric fluid (pH1.2) and simulated small intestine fluid (pH6.8). A high amount of 84.7% sustained release from NPs in simulated colonic fluid (pH7.4) was beyond 24 h. DiR-loaded NPs demonstrated a much higher colon accumulation, suggesting effective targeting due to functionalization with pH and time-dependent polymers. DFNPs could significantly ameliorate the colonic damage by reducing DAI, macroscopic score, histological damage and cell apoptosis. Our results also proved that the potent anti-inflammatory effect of DFNPs is contributed by decrease of NADPH, gene expression of COX-2 and MMP-9 and the production of TNF-α, IL-17, IL-23 and PGE2.ConclusionWe confirm that DFNPs exert protective effects through inhibiting the inflammatory response, which could be developed as a potential colon-targeted system.

Pharmacological Inhibition of Glucose‐6‐phosphate Dehydrogenase Produces Inhibition of L‐type Calcium Channel Current in Arterial Smooth Muscle Cells

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme in pentose phosphate pathway (PPP). G6PD is involved in cellular redox regulation by reducing NADP+ to NADPH, a primary cellular reducing agent, and PPP produces ribose 5‐phosphate, a substrate necessary for nucleotide synthesis. We used two inhibitors of G6PD, 6‐aminonicotinamide (6‐AN) and dehydroepiandrosterone (DHEA), to clarify involvement of G6PD in the regulation of L‐type Ca2+ channel current (ICa,L) in arterial smooth muscle cells (ASMCs). 6‐AN is converted to 6‐ANADP in the cell to competitively inhibit NADP+ binding to G6PD. DHEA is an adrenal steroid hormone with the highest serum concentration among steroid hormones with its sulfate ester DHEAS. DHEA inhibits G6PD in a non‐competitive manner. The potency of DHEAS to inhibit G6PD is about 1/100 of DHEA. DHEA induced relaxation of high K+‐induced contraction in arterial strips excised from carotid artery and aorta of rat in a dose‐dependent manner. In contrast, DHEAS did not produce the relaxation. In previous meetings we reported that DHEA inhibits ICa,L in voltage‐dependent and ‐independent mechanisms in ASMCs. 6‐AN produced voltage‐independent inhibition of ICa,L recorded with 10 mM Ba2+ as the charge carrier in A7r5 ASMCs. When constant depolarization step was repetitively applied from hyperpolarized HP, 6‐AN (3 mM) gradually induced inhibition of ICa,L decreasing the peak amplitude of ICa,L (ICa,L, peak) by 25% in 5 min and 45% in 10 min. The inhibition was not reversible by washout of 6‐AN. This was in contrast to DHEA‐induced inhibition of ICa,L which exhibits rapid onset and rapid recovery by washout. 6‐AN little affected the time course of inactivation of ICa,L during depolarization steps, while DHEA markedly accelerated the time course. When I‐V relationship was obtained with 5 min interval before and during application of 6‐AN, ICa,L did not significantly change in control, but decreased in 5 min in the presence of 6‐AN and the inhibition was increased by another 5 min application of 6‐AN. In the presence of 6‐AN, 0.1 mM DHEA rapidly decreased ICa,L,peak and markedly accelerated the time course of inactivation in a reversible manner. Inhibition of G6PD by 6‐AN and DHEA could inhibit ICa,L,peak by thiol‐oxidation of CaV1.2 channel proteins via decrease of NADPH. DHEA additionally augmented voltage‐dependent inactivation of ICa,L in a mode of open channel block produced by Ca2+ channel blockers.Support or Funding InformationNational Heart, Lung, and Blood Institute Grant RO1‐HL‐085352 and ROI‐HL‐132574 to S.A.G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

200 - NADPH de novo Biosynthesis Regulates NOX4-Induced Cell Growth and Cellular Bioenergetics

Reactive oxygen species play an important role in cellular events controlling growth, differentiation and adaptation to stress. NADPH oxidase 4 (NOX4) is an inducible enzyme belonging to the family of NADPH oxidases (NOX), expressed in several cell types. As opposed to other members of NADPH oxidases family, NOX4 does not produce superoxide radical anion but releases hydrogen peroxide (H2O2). We postulated that NOX4 regulates cell resistance to injury by mechanisms dependent on de novo NADPH biosynthesis coupled to stimulated mitochondrial bioenergetics. NAD depletion by FK866 (10 nM, 24h) was followed by 44% decrease in NADPH in HEK-NOX4 cells while only a 20% decrease was detected in HEK cells. This result indicated a greater dependence of HEK-NOX4 cells activity and consumption than HEK on de novo NADPH synthesis. Extracellular flux analysis was used to monitor cellular oxygen consumption rates. HEK-NOX4 consistently showed a higher basal rate (72.7 vs 42.7 pmol/min/mg protein) than HEK cells, which was offset by pre-treatment with FK866. Since the extracellular flux assay media contained pyruvate, this inhibition was taken as indication of differences in mitochondrial mass. Consistently, western blot analysis revealed lower mitochondria proteins VDAC and COX4 in HEK than HEK-NOX4 cells. Depletion of NADPH content in FK866-treated HEK-NOX4 diminished H2O2 release as verified with coumarin boronate (CBA) assay. Supplementation of cells with nicotinamide mononucleotide, a precursor of both NAD and NADPH, 24 h prior assays increased both the release of H2O2 and mitochondrial respiration in HEK-NOX4 but not HEK cells. In combination, these results indicate that de novo NAD/NADPH by regulating NOX4 derived H2O2 improves cell growth and bioenergetics, which may be an important mechanism conferring cell resistance to injury.

Abstract 3790: Targeting the one-carbon metabolism protein MTHFD2 for cancer therapy: Exploiting the unique redox status of cancer cells

Abstract Two well-established hallmarks of cancer are the need for nucleotides to support continuous cell proliferation and the requirement for cancer cell redox balance. In that respect cancer cells are characterized by an increased rate of reactive oxygen species (ROS) production which can be toxic to cancer cells. In this sense, cancer cells often display an increased ROS scavenging capacity, through antioxidants and NADPH that prevents ROS levels from reaching cytotoxic levels. Mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is a critical component of one-carbon metabolism and promotes the reaction of 5, 10-methylene tetrahydrofolate to 10-formyl tetrahydrofolate which is coupled to purine synthesis and the production of NAD(P)H. In its role, MTHFD2 is required for maintaining nucleotide biosynthesis and cancer cell redox balance. Recent studies have shown that MTHFD2 expression is elevated in many cancers compared to normal tissue and expression is correlated with poor survival in breast cancer. In order to validate MTHFD2 as a potential cancer target we show that genetic knockdown of MTHFD2 led to impaired cancer cell survival and proliferation. In breast cancer cells MTHFD2 inhibition caused a decrease in NADPH, an increase in the levels of oxidized glutathione and also promoted the expression of DNA damage and death markers specifically in cancer cells with high MTHFD2 protein expression. Interestingly, ROS-scavenging antioxidants reversed these phenotypes in the presence of MTHFD2 knockdown. Normal cells and low MTHFD2 expressing breast cancer cells had a higher tolerance for the inhibition of the protein. Based on our data, MTHFD2 inhibition leads to the impairment of cancer cells’ antioxidant capacity and ROS-mediated cell death. Therefore, MTHFD2 inhibition may have clinical potential for the treatment of patients with breast cancer, and potentially various other cancers. Citation Format: Andrea Glasauer, Michael Steckel, Andrea Haegebarth, Marcus Bauser, Luisella Toschi. Targeting the one-carbon metabolism protein MTHFD2 for cancer therapy: Exploiting the unique redox status of cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3790.

Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones: Synthesis, enzymatic reduction and cytotoxic activity.

Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones (NPDOs), a new type of N-heterocycle-fused o-quinones, have been synthesized. They have been found to be efficient electron-accepting substrates of NADPH-dependent single-electron-transferring P-450R and two-electron transferring NQO1, generating reactive oxygen species (ROS) with a concomitant decrease in NADPH, which is consistent with redox-cycling. The reactivity of NPDOs toward P-450R (in terms of kcat/Km) varied in the range of 10(6)-10(7)M(-1)s(-1), while their reduction by NQO1 proceeded much faster, approaching the diffusion control limit (kcat/Km∼10(8)-10(9)M(-1)s(-1)). NPDOs exhibited relatively high cytotoxic activity against human lung carcinoma (A-549) and breast tumor (MCF-7) cell lines (LC50=0.1-8.3μM), while promyelocytic leukemia cells (HL-60) were less sensitive to NPDOs (LC50⩾10μM). 3-Nitro-substituted NPDO (11) revealed the highest potency against both A-549 and MCF-7 cell lines, with LC50 of 0.12±0.03μM and 0.28±0.08μM, respectively. Dicoumarol partly suppressed the activity of the compounds against A-594 and MCF-7 cell lines, suggesting that their cytotoxic action might be partially influenced by NQO1-mediated bioreductive activation.

The effects of catechin isolated from green tea GMB-4 on NADPH and nitric oxide levels in endothelial cells exposed to high glucose.

Aim:This study aimed to investigate whether a catechin isolated from GMB-4 green tea is able to increase the reducing equivalent system and nitric oxide (NO) level in endothelial cells exposed to high glucose (HG) level.Materials and Methods:Endothelial cells were obtained from human umbilical vascular tissues. At confluent, human endothelial cells were divided into five groups, which included control (untreated), endothelial cells exposed to HG (30 mM), endothelial cells exposed to HG in the presence of green tea catechin (HG + C) at the following three doses: 0.03; 0.3; and 3 mg/ml. Analysis of NADP+, NADPH, and NO levels were performed colorimetrically.Results:This decrease in NADPH was significantly (P < 0.05) attenuated by both the 0.3 and 3 mg/ml treatments of catechin. HG level significantly decreased NO compared with untreated cells. This increase in NO was significantly attenuated by the 0.3 mg/ml dose of the catechin.Conclusion:In conclusion, catechin isolated from GMB-4 green tea prohibits the decrease in NADPH and NO in endothelial cells induced by HG. Therefore this may provide a natural therapy for attenuating the endothelial dysfunction found in diabetes mellitus.

Heme oxygenase 1 attenuates interleukin‐1β–induced cytosolic phospholipase A2 expression via a decrease in NADPH oxidase/reactive oxygen species/activator protein 1 activation in rheumatoid arthritis synovial fibroblasts

Reactive oxygen species (ROS) produced by cytokines induce the expression of inflammatory mediators in rheumatoid arthritis (RA). Heme oxygenase 1 (HO-1) exerts an antiinflammatory effect. The aim of this study was to examine the mechanisms underlying interleukin-1β (IL-1β)-induced cytosolic phospholipase A2 (cPLA2) expression through ROS generation as modulated by HO-1 in RA synovial fibroblasts (RASFs). IL-1β-induced ROS generation was determined by flow cytometry. The involvement of MAPKs and NADPH oxidase (NOX)/ROS in IL-1β-induced cPLA2 expression was investigated using pharmacologic inhibitors and transfection with small interfering RNAs (siRNAs) and was analyzed by Western blotting and promoter assay. Overexpression of HO-1 was performed by transfection of RASFs with a recombinant adenovirus containing human HO-1 plasmid. SCID mice with inflammation caused by IL-1β were infected with adenovirus containing HO-1. Histologic characterization of joint inflammation and local expression of cPLA2 were evaluated after treatment. IL-1β-induced cPLA2 expression was mediated through NOX activation/ROS production, which was attenuated by N-acetylcysteine (NAC; a scavenger of ROS), the inhibitors of NOX (diphenyleneiodonium chloride and apocynin), MEK-1/2 (U0126), and JNK-1/2 (SP600125), transfection with the respective siRNAs, and the overexpression of HO-1 in RASFs. IL-1β-induced cPLA2 expression was mediated through recruitment of activator protein 1 (AP-1) to the cPLA2 promoter region, which was attenuated by NAC and overexpression of HO-1. Furthermore, HO-1 overexpression inhibited IL-1β-mediated cPLA2 expression in SCID mice. In RASFs, IL-1β induced cPLA2 expression via activation of p42/p44 MAPK and JNK-1/2, leading to p47phox phosphorylation, ROS production, and AP-1 activation. The induction of HO-1 exerted protective effects on the pathogenesis of RA.

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT) Activity by Small Molecule GMX1778 Regulates Reactive Oxygen Species (ROS)-mediated Cytotoxicity in a p53- and Nicotinic Acid Phosphoribosyltransferase1 (NAPRT1)-dependent Manner

Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD(+) from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD(+) level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.

Characterization of a Cr(VI)-sensitive Pseudomonas corrugata 28 mutant impaired in a pyridine nucleotide transhydrogenase gene

Bacteria are known to adopt complex metabolic strategies in an effort to counteract the impact of numerous toxic compounds. In this study, a Cr(VI)-sensitive mutant of the Cr(VI)-hyperresistant bacterium Pseudomonas corrugata 28, obtained by insertional mutagenesis using the EZ-Tn5™ <R6Kγori/KAN-2>Tnp, was employed to gain a greater understanding of Cr(VI) resistance in bacteria. The insertion of the transposon, which occurred 16 bp upstream from the start codon of an ORF encoding a soluble pyridine nucleotide transhydrogenase (STH), negatively affected expression of the sth gene. The compromised expression of the sth gene in the mutant had two main effects on the pyridine nucleotide pools: (i) a decrease in NADPH and NADH fractions with a consequent shift in the redox state toward oxidation; and (ii) a decrease in the total concentration of the pyridine nucleotides. In the absence of a suitable pool of NADPH, the mutant failed to sustain an effective defense against the oxidative stress induced by Cr(VI).

S-RNase disrupts tip-localized reactive oxygen species and induces nuclear DNA degradation in incompatible pollen tubes of Pyrus pyrifolia

Pear (Pyrus pyrifolia L.) has an S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. However, RNA degradation might be only the beginning of the SI response, not the end. Recent in vitro studies suggest that S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia, and it seems that a relationship exists between self S-RNase, actin depolymerization and DNA degradation. To further uncover the SI response in pear, the relationship between self S-RNase and tip-localized reactive oxygen species (ROS) was evaluated. Our results show that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest of ROS formation in mitochondria and cell walls. The mitochondrial ROS disruption was related to mitochondrial alteration, whereas cell wall ROS disruption was related to a decrease in NADPH. Tip-localized ROS disruption not only decreased the Ca(2+) current and depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. These results indicate that tip-localized ROS disruption occurs in Pyrus pyrifolia SI. Importantly, we demonstrated nuclear DNA degradation in the incompatible pollen tube after pollination in vivo. This result validates our in vitro system in vivo.

Substrate availability for both endothelial nitric oxide synthase (eNOS) and glucose‐6‐phosphate dehydrogenase (G6PD) result in enzyme dysfunction as a result of ischemia/reperfusion injury: the role of NADPH in endothelial dysfunction.

Ischemia/Reperfusion (IR) injury causes dysfunction in the coronary vascular endothelium. Endothelial nitric oxide synthase is a critical enzyme involved in endothelial function. Previously, we showed endothelial dysfunction occurs without direct damage to eNOS, but rather via depletion of the essential eNOS cofactor, tetrahydrobiopterin (BH4). Repletion of BH4 lost during IR partially restored endothelial function. Recently, we have found that 30 min of IR injury also results in the depletion of the eNOS substrate NADPH, to ~47% of pre‐injury levels. In isolated hearts repletion of NADPH restored endothelial function (as measured by coronary flow) to near pre‐ischemic levels, highlighting the significance the IR‐induced decrease in NADPH. Our current work shows levels of NADPH are altered throughout IR injury, rising ~20% above control values at 10 min ischemia (increasing from 49.2±2.2 to 61.2±3.5 nmoles/gram tissue), and then decreasing to below baseline (31.8±7.8 nmoles/gram tissue) 20 min post‐ischemia. Glucose‐6‐phosphate dehydrogenase (G6PD) is the rate limiting enzyme in the production of NADPH. We observed that while total G6PD activity is not changed during IR injury, it is limited by substrate availability. In conclusion, the modulation of NADPH throughout IR injury, partially due to a change in substrate availability of G6PD, gives new insights to the intracellular response to IR injury.

Novel Thioredoxin-related Transmembrane Protein TMX4 Has Reductase Activity

In the endoplasmic reticulum (ER), a number of thioredoxin (Trx) superfamily proteins are present to enable correct disulfide bond formation of secretory and membrane proteins via Trx-like domains. Here, we identified a novel transmembrane Trx-like protein 4 (TMX4), in the ER of mammalian cells. TMX4, a type I transmembrane protein, was localized to the ER and possessed a Trx-like domain that faced the ER lumen. A maleimide alkylation assay showed that a catalytic CXXC motif in the TMX4 Trx-like domain underwent changes in its redox state depending on cellular redox conditions, and, in the normal state, most of the endogenous TMX4 existed in the oxidized form. Using a purified recombinant protein containing the Trx-like domain of TMX4 (TMX4-Trx), we confirmed that this domain had reductase activity in vitro. The redox potential of this domain (-171.5 mV; 30 degrees C at pH 7.0) indicated that TMX4 could work as a reductase in the environment of the ER. TMX4 had no effect on the acceleration of ER-associated degradation. Because TMX4 interacted with calnexin and ERp57 by co-immunoprecipitation assay, the role of TMX4 may be to enable protein folding in cooperation with these proteins consisting of folding complex in the ER.

Hypoxia, coronary dilation, and the pentose phosphate pathway

hypoxic vasorelaxation of resistance arteries is an important feedback control mechanism to maintain adequate oxygenation of metabolically active tissues and is essential in tissues where oxygen extraction is nearly maximized, as it is in the heart. Although the physiological importance of this

Old yellow enzyme interferes with Bax-induced NADPH loss and lipid peroxidation in yeast

The yeast transcriptional response to murine Bax expression was compared with the changes induced by H(2)O(2) treatment via microarray technology. Although most of the Bax-responsive genes were also triggered by H(2)O(2) treatment, OYE3, ICY2, MLS1 and BTN2 were validated to have a Bax-specific transcriptional response not shared with the oxidative stress trigger. In knockout experiments, only deletion of OYE3, coding for yeast Old yellow enzyme, attenuated the rate of Bax-induced growth arrest, cell death and NADPH decrease. Lipid peroxidation was completely absent in DeltaOYE3 expressing Bax. However, the absence of OYE3 sensitized yeast cells to H(2)O(2)-induced cell death, and increased the rate of NADPH decrease and lipid peroxidation. Our results clearly indicate that OYE3 interferes with Bax- and H(2)O(2)-induced lipid peroxidation and cell death in Saccharomyces cerevisiae.

Alpha-Tocopherol and NADPH in the Erythrocytes and Plasma of Multiple Sclerosis Patients

Objectives: To investigate the influence of interferon-β-1b (INF-β-1b) therapy on blood antioxidants (α-tocopherol and NADPH) in multiple sclerosis (MS). Patients and Methods: Patients with relapsing-remitting MS (n = 14) have been studied during 6 months of INF-β-1b therapy. α-Tocopherol was determined by HPLC and UV or electrochemical detection; NADPH was quantified spectrophotometrically. Results: The erythrocyte α-tocopherol level was reduced (p < 0.001) before treatment, but had regained the control level by 6 months of therapy. The plasma α-tocopherol/lipid ratios were constant during therapy. Plasma triglyceride levels were transiently increased (p = 0.0270) after 1 month of treatment. INF-β-1b had also induced a transient decrease in NADPH after 1 month, but thereafter the level returned to approximately the initial value (p = 0.0248). Conclusion: INF-β-1b seems to exert a sparing effect toward the erythrocyte α-tocopherol content. The fall in NADPH in parallel to the rise in plasma triglycerides suggests stimulation of fatty acid synthesis by INF-β-1b.

Altered etioplast development in phytochrome chromophore-deficient mutants.

Inhibition of chromophore synthesis in the phytochrome-deficient aurea (au) and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum L.) results in a severe reduction of protochlorophyllide (Pchlide) accumulation in dark-grown hypocotyls. Experiments with apophytochrome-deficient mutants indicate that the inhibition of Pchlide accumulation results from two separate effects: one dependent on the activity of phytochromes A and B1 and one phytochrome-independent effect that is attributed to a feedback inhibition of the tetrapyrrole biosynthesis pathway. Cotyledons only show phytochrome-independent inhibition of Pchlide synthesis. Analysis of NADPH:protochlorophyllide oxidoreductase levels by western blotting showed that the reduction in Pchlide in au and yg-2 is accompanied by a correlative, but less substantial, decrease in NADPH:protochlorophyllide oxidoreductase. Consistent with this result, in vivo fluorescence spectra demonstrate that both mutants are primarily deficient in non-phototransformable Pchlide. Analysis of etioplast structure indicates that plastid development in au and yg-2 is retarded in hypocotyls and partially impaired in cotyledons, again correlating with the reduction in Pchlide. Since Pchlide synthesis is also reduced in chromophore-deficient mutants of pea (Pisum sativum L.) and Arabidopsis thaliana (L.) Heynh. (Landsberg erecta) these results may be significant for explaining aspects of the phenotype of this mutant class that are independent of the loss of phytochrome.

Determination of Nitrate in Biological Fluids Using Nitrate Reductase in a Flow System.

The concentration of nitrate in biological fluids was determined using nitrate reductase (NR) in a flow system. A merging zone method was applied, in which a zone of NR and that of nitrate in separate streams were merged, and allowed to react. The decrease in NADPH caused by the reaction between NR and nitrate was measured at 340 nm. The conditions were as follows: length of the reaction coil used for the enzymatic reaction was 250 cm; 0.1 M PIPES buffer (pH 7.5) was used as the first carrier (C1); 0.1 M PIPES buffer (pH 7.5) containing 0.1 mM NADPH was used as the second carrier (C2); the reaction coil was immersed in a water bath maintained at 32°C. Under these conditions, a linear calibration curve (r = 0.994) was plotted for the concentration of nitrate between 1 and 100 μM with a detection limit (S/N = 3) of 0.2 μM. The present method was applied to determine the amount of nitrate in serum, plasma and urine using samples that had not been deproteinized. The concentration of nitrate within each sample was calculated from differences observed in the peak areas obtained in the absence or presence of nitrate reductase. The recovery test of the nitrate added to biological fluids indicated the applicability of the present method to the determination of nitrate in them. The nitrate concentrations determined using this technique correspond well with those of other methods.