Nicotinamide Adenine Dinucleotide Phosphate Content Research Articles

Evaluation of light irradiation on anthocyanins and energy metabolism of grape (Vitis vinifera L.) during storage

Grape is the world’s economic horticultural crop; it is perishable due to various pathogens and abiotic stress attributed to water loss-induced issues. To address these postharvest problems, this research investigates the effects of light irradiation on anthocyanins synthesis and energy metabolism in stored grapes to enhance their postharvest quality. The activities of chlorophyllase (1.17 U gk−1), Mg-dechelatase (351.69 U gk−1), chlorophyll-degraded peroxidase (3.49 U gk−1), and pheophytinase (0.85 U gk−1) were significantly higher in the control fruit than in the treated fruit at the end of storage. The red-light treatment showed higher levels of anthocyanins biosynthesis-related enzymes than green, blue-light, and control treatments. Additionally, light irradiation resulted in a decrease in adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, and energy charge. This was attributed to decreased activity of energy metabolism enzymes, reduced nicotinamide adenine dinucleotide phosphate content, and increased nicotinamide adenine dinucleotide content. These findings offer a theoretical foundation for optimizing grape coloration and energy metabolism during storage, thus prolonging the shelf-life of grapes by improving quality attributes. This research highlights the potential of light irradiation as a technique for enhancing the postharvest quality of agricultural products.

Insights into carbon utilization under mixotrophic conditions in Chlamydomonas

Mixotrophic microalgae cultivation with various carbon resources is considered as a strategy that could increase biomass. However, the mechanism of carbon utilization between inorganic carbon (IC) and organic carbon (OC) remains unknown. In this study, IC and OC consumption, chlorophyll fluorescence parameters, intracellular Nicotinamide adenine dinucleotide phosphate content and transcriptional changes in related genes were characterized. The results showed that IC was utilized preferentially, whereas 76% IC was consumed at 8 h. Subsequently, OC was the dominant carbon resource for fermentation. The cell density in the IC group was 100% higher than that in the group without IC at 24 h. Bicarbonate addition enhanced photosynthesis by dissipating less energy and generating more electrons and energy, which benefited OC assimilation. This finding was verified by qRT-PCR analysis. These results elucidate the carbon utilization mechanism under mixotrophic conditions, which provide clues for promoting microalgae growth by regulating carbon utilization.

Distinct redox state regulation in the seedling performance of Norway maple and sycamore

Norway maple and sycamore, two Acer genus species, have an important ecological value and different sensitivity to stressing factors being currently aggravated by climate change. Seedling growth is postulated to be the main barrier for successful plant establishment under the climate change scenarios. Therefore, the differences in redox regulation during the seedling performance of Norway maple and sycamore were investigated. Seeds of the two Acer species exhibited an identical high germination capacity, whereas seedling emergence was higher in sycamores. PCA analyses revealed that there is more diversification in the leaf characteristics than roots. Norway maple displayed a higher chlorophyll content index (CCI) with a similar leaf mass whereas sycamore seedlings exhibited a higher normalized difference vegetation index (NDVI), higher water content, higher root biomass and higher shoot height. Based on NDVI, sycamore seedlings appeared as very healthy plants, whereas Norway maple seedlings displayed a moderate healthy phenotype. Therefore, redox basis of seedling performance was investigated. The total pool of glutathione was four times higher in sycamore leaves than in Norway maple leaves and was reflected in highly reduced half-cell reduction potential of glutathione. Sycamore leaves contained more ascorbate because the content of its reduced form (AsA) was twice as high as in Norway maple. Therefore, the AsA/DHA ratio was balanced in sycamore leaves, reaching 1, and was halved in Norway maple leaves. Nicotinamide adenine dinucleotide phosphate content was twice as high in sycamore leaves than in Norway maples; however, its reduced form (NADPH) was predominant in Norway maple seedlings. Norway maple leaves exhibited the highest anabolic and catabolic redox charge. The higher reduction capacity and the activity of NADPH-dependent reductases in Norway maple leaves possibly resulted in higher CCI, whereas the larger root system contributed to higher NDVI in sycamore. The different methods of controlling redox parameters in Acer seedlings grown at controlled conditions provided here can be useful in understanding how tree species can cope with a changing environment in the future.

Glutamic-oxaloacetic transaminase 1 regulates adipocyte differentiation by altering nicotinamide adenine dinucleotide phosphate content.

ObjectiveThis study was performed to examine whether the porcine glutamic-oxaloacetic transaminase 1 (GOT1) gene has important functions in regulating adipocyte differentiation.MethodsPorcine GOT1 knockout and overexpression vectors were constructed and transfected into the mouse adipogenic 3T3-L1 cells. Lipid droplets levels were measured after 8 days of differentiation. The mechanisms through which GOT1 participated in lipid deposition were examined by measuring the expression of malate dehydrogenase 1 (MDH1) and malic enzyme (ME1) and the cellular nicotinamide adenine dinucleotide phosphate (NADPH) content.ResultsGOT1 knockout significantly decreased lipid deposition in the 3T3-L1 cells (p< 0.01), whereas GOT1 overexpression significantly increased lipid accumulation (p<0.01). At the same time, GOT1 knockout significantly decreased the NADPH content and the expression of MDH1 and ME1 in the 3T3-L1 cells. Overexpression of GOT1 significantly increased the NADPH content and the expression of MDH1 and ME1, suggesting that GOT1 regulated adipocyte differentiation by altering the NADPH content.ConclusionThe results preliminarily revealed the effector mechanisms of GOT1 in regulating adipose differentiation. Thus, a theoretical basis is provided for improving the quality of pork and studies on diseases associated with lipid metabolism.

Inhibition of downy blight and enhancement of resistance in litchi fruit by postharvest application of melatonin

Litchis are tasty fruit with economic importance. However, the extreme susceptibility of harvested litchis to litchi downy blight caused by Peronophythora litchii leads to compromised quality. This study aimed to study the effects of melatonin on postharvest resistance to P. litchii in ‘Feizixiao’ litchis. Results showed that melatonin restricted lesion expansion in litchis after P. litchi inoculation. Melatonin enhanced the activities of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase and 4-hydroxycinnamate CoA ligase while promoting the accumulations of phenolics and flavonoids. Nicotinamide adenine dinucleotide phosphate content and glucose-6-phosphate dehydrogenase and 6-phosphogluconic acid dehydrogenase activities were higher in treated fruit than control fruit. Higher energy status along with elevated H+-ATPase, Ca2+-ATPase, succinate dehydrogenase and cytochrome C oxidase activities were observed in treated fruit. Ultrastructural observation showed reduced damage in mitochondria in treated fruit. The results suggest that melatonin induced resistance in litchis by modulating the phenylpropanoid and pentose phosphate pathways as well as energy metabolism..

Strengthening NADPH Regeneration for Improving Photo-biocatalytic Ketones Asymmetric Reduction Reaction by Synechocystis Through Overexpression of FNR

Microalgae are excellent biocatalyst candidates for photo-biocatalytic-asymmetric reduction of prochiral ketones to produce enantiomer alcohols. In the biocatalysis asymmetric reduction of carbonyl group process, the cofactor, NADPH, plays a key role. So the current study focused on the effect of NADPH availability on the reduction. Photosynthesis is a main pathway for NADPH regeneration in microalgae, and the ferredoxin-NADP+ oxidoreductase (FNR) is the key enzyme in this process. In this work, we constructed an engineered cyanobacterium Synechocystis sp. PCC6803::Ω-PpetE-petH to overexpress FNR gene petH to strengthen NADPH regeneration. The results show that the intracellular NADPH content was increased 80.8% compared to the wild type. Applying the Synechocystis sp. PCC6803::Ω-PpetE-petH to catalyze the model asymmetric reduction reaction (reduction of acetophenone to S-phenylethanol), the yield was improved 61.2% compared to the wild-type. This work makes the microalgal biocatalysis asymmetric reduction process become more preponderant via providing a new route to enhance the regeneration of cofactor NADPH. Putative metabolic process of nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and the photo-biocatalytic-asymmetric reduction reaction of prochiral ketones in microalgal cells.

PP2A catalytic subunit silence by microRNA-429 activates AMPK and protects osteoblastic cells from dexamethasone

Activation of AMP-activated protein kinase (AMPK) could efficiently protect osteoblasts from dexamethasone (Dex). Here, we aim to induce AMPK activation through miRNA-mediated downregulating its phosphatase, protein phosphatase 2A (PP2A). We discovered that microRNA-429 (“miR-429”) targets the catalytic subunit of PP2A (PP2A-c). Significantly, expression of miR-429 downregulated PP2A-c and activated AMPK (p-AMPKα1 Thr172) in human osteoblastic cells (OB-6 and hFOB1.19 lines). Remarkably, miR-429 expression alleviated Dex-induced osteoblastic cell death and apoptosis. On the other hand, miR-429-induced AMPK activation and osteoblast cytoprotection were almost abolished when AMPKα1 was either silenced (by targeted shRNA) or mutated (T172A inactivation). Further studies showed that miR-429 expression in osteoblastic cells increased NADPH (nicotinamide adenine dinucleotide phosphate) content to significantly inhibit Dex-induced oxidative stress. Such effect by miR-429 was again abolished with AMPKα1 silence or mutation. Together, we propose that PP2A-c silence by miR-429 activates AMPK and protects osteoblastic cells from Dex.

Low-concentration of perifosine surprisingly protects cardiomyocytes from oxygen glucose deprivation

Here we found that low-concentration of perifosine, an Akt inhibitor, surprisingly protected cardiomyocytes from oxygen glucose deprivation (OGD)/re-oxygenation. In H9c2 cardiomyocytes, non-cytotoxic perifosine (0.1–0.5 μM) suppressed OGD/re-oxygenation-induced reactive oxygen species (ROS) production, p53 mitochondrial translocation and cyclophilin D complexation, as well as mitochondrial membrane potential (MMP) reduction. Molecularly, perifosine activated AMP-activated kinase (AMPK) signaling to increase intracellular NADPH (nicotinamide adenine dinucleotide phosphate) content in H9c2 cells. On the other hand, AMPK inhibition by AMPKα1 shRNA-knockdown in H9c2 cells significantly reduced perifosine-induced NADPH production, and alleviated perifosine-mediated anti-oxidant and cytoprotective activities against OGD/re-oxygenation. In primary murine cardiomyocytes, perifosine similarly activated AMPK signaling, and offered significant protection against OGD/re-oxygenation, which was largely attenuated with siRNA knockdown of AMPKα1. We demonstrate an unexpected function of perifosine (low-concentration) in protecting cardiomyocytes from OGD/re-oxygenation.

Glucose-6-phosphate dehydrogenase protects Escherichia coli from tellurite-mediated oxidative stress.

The tellurium oxyanion tellurite induces oxidative stress in most microorganisms. In Escherichia coli, tellurite exposure results in high levels of oxidized proteins and membrane lipid peroxides, inactivation of oxidation-sensitive enzymes and reduced glutathione content. In this work, we show that tellurite-exposed E. coli exhibits transcriptional activation of the zwf gene, encoding glucose 6-phosphate dehydrogenase (G6PDH), which in turn results in augmented synthesis of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Increased zwf transcription under tellurite stress results mainly from reactive oxygen species (ROS) generation and not from a depletion of cellular glutathione. In addition, the observed increase of G6PDH activity was paralleled by accumulation of glucose-6-phosphate (G6P), suggesting a metabolic flux shift toward the pentose phosphate shunt. Upon zwf overexpression, bacterial cells also show increased levels of antioxidant molecules (NADPH, GSH), better-protected oxidation-sensitive enzymes and decreased amounts of oxidized proteins and membrane lipids. These results suggest that by increasing NADPH content, G6PDH plays an important role in E. coli survival under tellurite stress.

Impaired activation of glucose oxidation and NADPH supply in human endothelial cells exposed to H2O2 in high-glucose medium.

The effects of glucose concentration on D-glucose oxidation and reduced nicotinamide adenine dinucleotide phosphate (NADPH) supply were studied during exposure of cultured human umbilical vein endothelial cells to hydrogen peroxide (H2O2). The activation of glucose oxidation via the pentose phosphate pathway (PPP), induced by exposure of cells to 200 mumol/l H2O2 for 1 h, was reduced by 50% (P < 0.01) in cells cultured for 5-7 days in 33 mmol/l D-glucose (HG) versus those cultured in 5.5 mmol/l D-glucose without (NG) or with (HR) 27.5 mmol/l D-raffinose. The intracellular NADPH content in HG cells, but not in NG or HR cells, was decreased by 42% (P < 0.01) by exposing cells to 200 mumol/l H2O2. The decrease in NADPH was dependent on D-glucose concentration in the medium and was prevented in glutathione (GSH)-depleted cells. The latter observation suggests that the decrease in NADPH is associated with activation of the GSH redox cycle. In the presence of 200 mumol/l H2O2, lactate release into the medium, NADH/NAD ratio, and phosphofructokinase activity in HG cells were 56, 53, and 68% greater, respectively, than in the NG group, which indicates that inhibition of glycolysis by H2O2 is less marked in the HG group compared with NG group. These results indicate that activation of the PPP was impaired in endothelial cells cultured under conditions of high-glucose and oxidative stress, resulting in a decreased supply of NADPH to various NADPH-dependent pathways, including the GSH redox cycle.

Metabolic and functional effects of the nucleoside transport inhibitor R75231 in the ischaemic and blood reperfused rabbit heart

The ability of R75231, a nucleoside transport inhibitor, to influence adenine nucleotide metabolism and enhance postischaemic functional recovery was assessed in the blood perfused rabbit heart. Hearts (n = 8 per group) from donor animals were excised and perfused with blood at 37 degrees C from a support rabbit. After 20 min of aerobic perfusion hearts were arrested with St Thomas' Hospital cardioplegic solution (2 min at 37 degrees C) and rendered globally ischaemic for 60 min. This was followed by 60 min of reperfusion. R75231 (0.1 mg.kg-1, intravenously) was given to donor and support rabbits 1 h before the experiment, control rabbits receiving the same volume of vehicle. Treatment with R75231 resulted in a 45% reduction in coronary vascular resistance in aerobically perfused control hearts, an effect that was absent during postischaemic reperfusion. Thus, before ischaemia, coronary flow was greater in R75231 treated hearts [6.6(SEM 0.8) ml.min-1] than in controls [4.3(0.6) ml.min-1; p < 0.05] but during reperfusion no significant difference was observed [4.0(0.6) v 3.6(0.3) ml.min-1]. The mean time to onset and extent of contracture during ischaemia was similar in R75231 treated and control groups, at 42(4) v 41(4) min and 27(3) v 26(6) mm Hg, respectively. Left ventricular developed pressure recovered to approximately 50% of its preischaemic value during the first 40 min of reperfusion in both groups; however, after longer durations of reperfusion, it tended to deteriorate in the R75231 treated group whereas it was maintained at a constant level in the controls [37(10) v 53(6) mm Hg, respectively; NS]. At the end of reperfusion, tissue adenosine content was 13-fold greater in the R75231 treated group, at 0.40(0.09) v 0.03(0.01) mumol.g-1 dry wt in controls; p < 0.05; the nucleotide pool, nicotinamide adenine dinucleotide phosphate content, and the energy charge potential were similar in groups. R75231 decreased coronary vascular resistance and increased coronary flow during aerobic perfusion in control hearts, an effect that was lost after ischaemia and reperfusion. R75231 also increased greatly the tissue content of adenosine but, despite this, failed to improve either the recovery of cardiac contractile function or the replenishment of the adenine nucleotide pool.

Increased Nicotinamide Adenine Dinucleotide Content and Synthesis in Plasmodium falciparum-Infected Human Erythrocytes

Plasmodium falciparum-infected red blood cells (RBCs) are characterized by increases in the activity of glycolytic enzymes. Because nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are cofactors in the reactions of glycolysis and pentose phosphate shunt, we have examined NAD and NADP content in P. falciparum-infected RBCs. Although NADP content was not significantly altered, NAD content was increased approximately 10-fold in infected RBCs (66% parasitemia) compared with uninfected control RBCs. To determine the mechanism for the increase in NAD content, we examined the activity of several NAD biosynthetic enzymes. It is known that normal human RBCs make NAD exclusively from nicotinic acid and lack the capacity to make NAD from nicotinamide. We demonstrate that infected RBCs have readily detectable nicotinamide phosphoribosyltransferase (NPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinamide, and abundant nicotinamide deamidase, the enzyme that converts nicotinamide to nicotinic acid, thereby indicating that infected RBCs can make NAD from nicotinamide. In addition, infected RBCs have a threefold increase in nicotinic acid phosphoribosyltransferase (NAPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinic acid. Thus, the increase in NAD content in P falciparum-infected RBCs appears to be mediated by increases in NAD synthesis from both nicotinic acid and nicotinamide.

Increased nicotinamide adenine dinucleotide content and synthesis in Plasmodium falciparum-infected human erythrocytes

Plasmodium falciparum-infected red blood cells (RBCs) are characterized by increases in the activity of glycolytic enzymes. Because nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are cofactors in the reactions of glycolysis and pentose phosphate shunt, we have examined NAD and NADP content in P. falciparum-infected RBCs. Although NADP content was not significantly altered, NAD content was increased approximately 10-fold in infected RBCs (66% parasitemia) compared with uninfected control RBCs. To determine the mechanism for the increase in NAD content, we examined the activity of several NAD biosynthetic enzymes. It is known that normal human RBCs make NAD exclusively from nicotinic acid and lack the capacity to make NAD from nicotinamide. We demonstrate that infected RBCs have readily detectable nicotinamide phosphoribosyltransferase (NPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinamide, and abundant nicotinamide deamidase, the enzyme that converts nicotinamide to nicotinic acid, thereby indicating that infected RBCs can make NAD from nicotinamide. In addition, infected RBCs have a threefold increase in nicotinic acid phosphoribosyltransferase (NAPRT), the first enzyme in the NAD biosynthetic pathway that uses nicotinic acid. Thus, the increase in NAD content in P falciparum-infected RBCs appears to be mediated by increases in NAD synthesis from both nicotinic acid and nicotinamide.

Decreased Erythrocyte Nicotinamide Adenine Dinucleotide Redox Potential and Abnormal Pyridine Nucleotide Content in Sickle Cell Disease

AbstractRBCs from individuals with sickle cell disease are more susceptible to oxidant damage. Because key antioxidant defense reactions are linked to the pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), we tested the hypothesis that the RBC redox potential as manifested by the NADH/[NAD+ + NADH] and NADPH/[NADP+ + NADPH] ratios is decreased in sickle erythrocytes. Our data demonstrate that sickle RBCs have a significant decrease in the NADH/[NAD+ + NADH] ratio compared with normal RBCs (P <. 00005). Interestingly, sickle RBCs also had a significant increase in total NAD content compared with normal RBCs (P < .00005). In contrast, although sickle RBCs had a significant increase in the total NADP content compared with normal RBCs (P < .00005), sickle RBCs had no significant alteration in the NADPH/[NADP+ + NADPH] ratio. High reticulocyte controls demonstrated that these changes were not related to cell age. Thus, sickle RBCs have a decrease in NAD redox potential that may be a reflection of their increased oxidant sensitivity. The changes in these pyridine nucleotides may have further metabolic consequences for the sickle erythrocyte.© 1988 by Grune & Stratton, Inc. 0006-4971/88/7102-0016$ 3.00/0

Decreased erythrocyte nicotinamide adenine dinucleotide redox potential and abnormal pyridine nucleotide content in sickle cell disease

RBCs from individuals with sickle cell disease are more susceptible to oxidant damage. Because key antioxidant defense reactions are linked to the pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), we tested the hypothesis that the RBC redox potential as manifested by the NADH/[NAD+ + NADH] and NADPH/[NADP+ + NADPH] ratios is decreased in sickle erythrocytes. Our data demonstrate that sickle RBCs have a significant decrease in the NADH/[NAD+ + NADH] ratio compared with normal RBCs (P less than .00005). Interestingly, sickle RBCs also had a significant increase in total NAD content compared with normal RBCs (P less than .00005). In contrast, although sickle RBCs had a significant increase in the total NADP content compared with normal RBCs (P less than .00005), sickle RBCs had no significant alteration in the NADPH/[NADP+ + NADPH] ratio. High reticulocyte controls demonstrated that these changes were not related to cell age. Thus, sickle RBCs have a decrease in NAD redox potential that may be a reflection of their increased oxidant sensitivity. The changes in these pyridine nucleotides may have further metabolic consequences for the sickle erythrocyte.

A new in vitro model for the study of pancreatic A and B cells.

A method is developed for the preparation of single, pure, and viable rat pancreatic A and B cells in numbers sufficient for in vitro analysis. Islet isolation and dissociation techniques have been modified to increase the yield in islet cells per pancreas and per experiment. Islet cells are separated on the basis of their light scatter activity and flavin adenine dinucleotide autofluorescence into single non-B cells, single B cells, and structurally coupled B cells. Islet non-B cells are further purified into single A cells by autofluorescence-activated sorting according to the cellular nicotinamide adenine dinucleotide phosphate content at 20 mM glucose. Apart from offering the advantage of separating cells according to their functional characteristics, this procedure succeeds in the simultaneous isolation of 95-100% pure A and B cells. More than 50% of the cells in the initial islet preparation are recovered as single purified cells which can be maintained in culture. The isolated pancreatic A and B cells have been defined in terms of their cell volume, DNA and hormone content, and ultrastructural characteristics. The availability of pure pancreatic A and B cells is expected to contribute to our understanding of the regulation of glucagon and insulin release.

Change in electrophoretic mobility of glucose-6-phosphate dehydrogenase with aging of erythrocytes.

Extract: Electrophoretic properties of glucose-6-phosphate dehydrogenase (G6PD) were studied in mother-infant pairs and in young and old erythrocytes of adults. G6PD isoenzymes of erythrocytes of newborn infants migrated at rates 3–4% faster than the corresponding isoenzymes of maternal erythrocytes. Analysis of hemolysates of young and old erythrocytes of adults revealed that G6PD of young cells also moved about 3% faster than that of old cells. In contrast, active enzyme sedimentation analysis revealed that the G6PD enzyme proteins of old and young erythrocytes of adults, of newborns, and of their mothers sedimented with essentially identical rates (S20,w = 6.8). Migration in gels of different acrylamide concentrations and velocity sedimentation analysis indicated that the G6PD of old and of young erythrocytes was in the same state of aggregation. Extensive equilibration of the hemolysates with nicotinamide-adenine dinucleotide phosphate (NADP) by filtration through Sephadex G-25 induced a clear increase in the migration rate of the G6PD of old cells of adults and of mothers at term to the rate characteristic of the enzyme of neonates and of young cells of adults. Experiments in which NADP was labeled with 14C indicated that, in the process of gel filtration, the NADP content of the aged G6PD was restored. Thus the rate of migration of G6PD in acrylamide gel seems to be a function of the amount of NADP bound. Speculation: Rates of electrophoretic migration of G6PD of the erythrocytes of infants and of young cells of adults were faster than the rates observed in adult cells; this, at first, was thought to be genetically determined, as with fetal hemoglobin. However, thorough testing proved that the difference was one of charge mediated by NADP, the concentration of which is a function of the age of the erythrocytes. There are numerous examples of aging of enzymes in erythrocytes. It is possible that loss of bound cofactor is a general mechanism for the aging of enzymes.

Change in Electrophoretic Mobility of Glucose-6-Phosphate Dehydrogenase with Aging of Erythrocytes

Extract: Electrophoretic properties of glucose-6-phosphate dehydrogenase (G6PD) were studied in mother-infant pairs and in young and old erythrocytes of adults. G6PD isoenzymes of erythrocytes of newborn infants migrated at rates 3–4% faster than the corresponding isoenzymes of maternal erythrocytes. Analysis of hemolysates of young and old erythrocytes of adults revealed that G6PD of young cells also moved about 3% faster than that of old cells. In contrast, active enzyme sedimentation analysis revealed that the G6PD enzyme proteins of old and young erythrocytes of adults, of newborns, and of their mothers sedimented with essentially identical rates (S20,w = 6.8). Migration in gels of different acrylamide concentrations and velocity sedimentation analysis indicated that the G6PD of old and of young erythrocytes was in the same state of aggregation. Extensive equilibration of the hemolysates with nicotinamide-adenine dinucleotide phosphate (NADP) by filtration through Sephadex G-25 induced a clear increase in the migration rate of the G6PD of old cells of adults and of mothers at term to the rate characteristic of the enzyme of neonates and of young cells of adults. Experiments in which NADP was labeled with 14C indicated that, in the process of gel nitration, the NADP content of the aged G6PD was restored. Thus the rate of migration of G6PD in acrylamide gel seems to be a function of the amount of NADP bound.

Inhibition of Nicotinate Phosphoribosyl Transferase by Nonsteroidal Anti-Inflammatory Drugs: A Possible Mechanism of Action

When incubated with 14C-nicotinic acid and phosphorylribose-1-pyrophosphate, human platelet lysate incorporates radioactivity into nicotinic acid mononucleotide. Under these conditions, the apparent Km of nicotinic acid for nicotinate phosphoribosyl transferase is 2.4 × 10−5M. Along with 2-hydroxynicotinic acid (Ki = 2.3 × 10−4M), the following nonsteroidal anti-inflammatory compounds competed reversibly with nicotinic acid for the enzyme: flufenamic acid (Ki = 4.6 × 10−5M), mefenamic acid (Ki = 7.6 × 10−5M), salicylic acid (Ki = 1.6 × 10−4M), phenylbutazone (Ki = 1.6 × 10−4M), and indomethacin (Ki = 4.2 × 10−4M). Such inhibition may explain the decreases in nicotinamide adenine dinucleotide phosphate content of rat liver after administration of salicylate. Furthermore, suppression of nicotinamide adenine dinucleotide biosynthesis by anti-inflammatory drugs may restrain mucopolysaccharide biosynthesis and, thereby, reduce inflammatory responsiveness. Hence, this antagonism of niacin may be involved in the pharmacologic activity and, particularly, the toxicity of anti-inflammatory drugs.

Altered fatty acid distribution in mutants of Neurospora crassa.

Morphological mutants of Neurospora with decreased levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduced nicotinamide ad enine dinucleotide (NADH) contained only 20% as much of a polyunsaturated fatty acid (linolenic acid) as the wild type in both the phospholipid and neutral lipid fractions. There was an excellent correlation between linolenic acid levels and morphological appearance as a function of total NADPH content, but no correlation with NADH content. The linolenic acid deficiency was balanced by a relative increase in the amounts of the less unsaturated fatty acids (oleic and linoleic acids), but the level of three other fatty acids did not appear to be changed. This accumulation of these two precursors suggests that the NADPH deficiency preferentially affected the final desaturation step, i.e., the conversion of linoleic to linolenic acid. The NADPH needed for this reaction in vivo was probably generated by the pentose phosphate shunt, since mutations affecting the shunt lead to the decreased levels of linolenic acid. It is not clear whether the changes in fatty acid distribution affect the morphogenesis of Neurospora, or if these changes are just part of the NADPH-deficiency syndrome.