Abstract P378: Metabolic Dysregulation Of Endothelial Sk Channels Via Protein Kinase C

Introduction: Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease, which predisposes to ischemic cardiovascular events. Small conductance calcium-activated-potassium (SK) channels are largely responsible for coronary arteriolar relaxation mediated by endothelium-dependent hyperpolarizing factors. Diabetic inactivation/inhibition of endothelial SK channels contributes to endothelial dysfunction. Endothelial dysfunction during diabetes (DM) is also associated with increases in metabolites NADH, and PKC. The pyridine nucleotide NADH has been recently found to inactivate endothelial SK channels. The overexpressed and activated PKC has been shown to play an important role in diabetes-induced endothelial dysfunction. However, it is undefined if PKC is involved in the metabolite NADH dysregulation of endothelial SK channels. Hypothesis: We hypothesized that PKC in a signaling cascade whereby NADH dysregulates endothelial SK channels. Methods: SK channels currents of human coronary artery endothelial cells were measured by whole cell patch clamp method in the presence or absence of NADH, and/or PKC activator PMA, PKC inhibitors or endothelial PKC α /PKCβ knock-down by using short interfering RNA. Results: NADH (30-300μM, n=7-9) or PKC activator PMA (30-300μM, n=6-12) reduced endothelial SK current density (p<0.05 vs. control (n=15), Fig. A-D), whereas the selective PKC α inhibitor LY333531 (50nM, n=12) significantly reversed the NADH-induced SK channel inhibition (p>0.05 vs. control (n=15), Fig. E). PKC α knock-down failed to affect NADH (n=13) and PMA (n=10) inhibition of endothelial SK currents (Fig. F). In contrast, PKCβ knock-down significantly prevented NADH (n=12) and PMA (n=6)-induced SK inhibition (p>0.05, vs. control, Fig. G). Conclusions: The metabolite NADH dysregulation of endothelial SK channels was via PKCβ activation.

Ferulic acid production by metabolically engineered Escherichia coli

Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics. Production of FA by eco‐friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p-coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as caffeic acid O-methytransferase. Fermentation result revealed JM109(DE3) as a more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 or p15A), and the combination p15a-T5 works best. To further improve FA production, E. coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also overexpressed. Based on the flask fermentation data which show that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E. coli K-12 strains reported to the best of our knowledge.

Identification of Oxygen-Independent Pathways for Pyridine Nucleotide and Coenzyme A Synthesis in Anaerobic Fungi by Expression of Candidate Genes in Yeast

NAD (NAD + ) and coenzyme A (CoA) are central metabolic cofactors whose canonical biosynthesis pathways in fungi require oxygen. Anaerobic gut fungi of the Neocallimastigomycota phylum are unique eukaryotic organisms that adapted to anoxic environments.

Ferulic Acid production by metabolically engineered Escherichia coli

Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics areas. Production of FA by eco-friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding Tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p - coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as Caffeic acid O-methytransferase. Fermentation results revealed JM109(DE3) as more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 ori or p15a ori), and the combination p15a-T5 works best. To further improve FA production, E.coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also over expressed. Based on the flask fermentation data which shows that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E.coli K-12 strains reported to the best of our knowledge.

Abstract 17038: Influence of Cardiac Workload on Vascular Pyridine Nucleotide Redox State

Introduction: Myocardial perfusion is enhanced during periods of elevated cardiomyocyte oxygen consumption. The coupling between blood flow and myocardial oxygen demand is mediated by redox-dependent signals, yet the influence of cardiac workload on arterial myocyte pyridine nucleotide redox status is unknown. Hypothesis: Increased cardiac workload acutely alters intramyocardial arterial myocyte [NADH] i :[NAD + ] i . Methods and Results: The genetically-encoded fluorescent biosensor Peredox-mCherry was used to monitor real-time changes in intracellular [NADH] i :[NAD] i in isolated arterial myocytes. Treatment of cells expressing Peredox-mCherry with a range of [lactate] o :[pyruvate] o resulted in significant changes in green (Ex,Em: 400, 510 nm): red (Ex,Em: 585,615 nm) fluorescence ratio (1.903 ± 0.010, 10 mM lactate, 1.072 ± 0.073, 10 mM pyruvate), affording accurate quantification of arterial myocyte [NADH] i :[NAD] i . Increased arterial myocyte [NADH] i :[NAD] i was observed in response to reductions in oxygen tension from 5% (0.0021 ± 0.0001) to 1% (0.0050 ± 0.0001; P<0.05). [NADH] i :[NAD] i was not altered by direct stimulation of arterial myocytes with the beta-adrenergic agonist isoproterenol (1 μM; P = .0630). Furthermore, biosensor-expressing arterial myocytes in co-culture with human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) recapitulated the effects of elevated [lactate] o :[pyruvate] o and reduced oxygen tension, exhibiting frequency-dependent increases in [NADH] i :[NAD] I following electrical pacing (e.g., ~3.3 fold increase in 3 Hz vs. unpaced; P<0.05). Consistent with these results, intramyocardial coronary arteries in mice acutely treated with dobutamine (10 mg/kg, i.p.) to drive high cardiac work exhibited significantly greater lactate:pyruvate ratio, used as a surrogate for [NADH] i :[NAD] i , compared with those in hearts treated with vehicle (3.17 ± 0.23 vs. 2.21 ± 0.11; P<0.05). Conclusions: Our results indicate that acute increases in cardiac workload promote the elevation of [NADH] i :[NAD] i in intramyocardial arterial myocytes.

Linkage between Carbon Metabolism, Redox Status and Cellular Physiology in the Yeast Saccharomyces cerevisiae Devoid of SOD1 or SOD2 Gene

Saccharomyces cerevisiae yeast cells may generate energy both by fermentation and aerobic respiration, which are dependent on the type and availability of carbon sources. Cells adapt to changes in nutrient availability, which entails the specific costs and benefits of different types of metabolism but also may cause alteration in redox homeostasis, both by changes in reactive oxygen species (ROS) and in cellular reductant molecules contents. In this study, yeast cells devoid of the SOD1 or SOD2 gene and fermentative or respiratory conditions were used to unravel the connection between the type of metabolism and redox status of cells and also how this affects selected parameters of cellular physiology. The performed analysis provides an argument that the source of ROS depends on the type of metabolism and non-mitochondrial sources are an important pool of ROS in yeast cells, especially under fermentative metabolism. There is a strict interconnection between carbon metabolism and redox status, which in turn has an influence on the physiological efficiency of the cells. Furthermore, pyridine nucleotide cofactors play an important role in these relationships.