NADH Balance Research Articles

Chloroquine Triggers Cell Death and Inhibits PARPs in Cell Models of Aggressive Hepatoblastoma.

Background: Hepatoblastoma (HB) is the most common pediatric liver malignancy. Despite advances in chemotherapeutic regimens and surgical techniques, the survival of patients with advanced HB remains poor, underscoring the need for new therapeutic approaches. Chloroquine (CQ), a drug used to treat malaria and rheumatologic diseases, has been shown to inhibit the growth and survival of various cancer types. We examined the antineoplastic activity of CQ in cell models of aggressive HB.Methods: Seven human HB cell models, all derived from chemoresistant tumors, were cultured as spheroids in the presence of relevant concentrations of CQ. Morphology, viability, and induction of apoptosis were assessed after 48 and 96 h of CQ treatment. Metabolomic analysis and RT-qPCR based Death Pathway Finder array were used to elucidate the molecular mechanisms underlying the CQ effect in a 2-dimensional cell culture format. Quantitative western blotting was performed to validate findings at the protein level.Results: CQ had a significant dose and time dependent effect on HB cell viability both in spheroids and in 2-dimensional cell cultures. Following CQ treatment HB spheroids exhibited increased caspase 3/7 activity indicating the induction of apoptotic cell death. Metabolomic profiling demonstrated significant decreases in the concentrations of NAD+ and aspartate in CQ treated cells. In further investigations, oxidation of NAD+ decreased as consequence of CQ treatment and NAD+/NADH balance shifted toward NADH. Aspartate supplementation rescued cells from CQ induced cell death. Additionally, downregulated expression of PARP1 and PARP2 was observed.Conclusions: CQ treatment inhibits cell survival in cell models of aggressive HB, presumably by perturbing NAD+ levels, impairing aspartate bioavailability, and inhibiting PARP expression. CQ thus holds potential as a new agent in the management of HB.

Using high-throughput data and dynamic flux balance modeling techniques to identify points of constraint in xylose utilization in Saccharomyces cerevisiae

Several enzymes and cofactors have been identified as contributing to the slow utilization of xylose by xylose-fermenting strains of Saccharomyces cerevisiae. However, there has been no consensus on which of these possible bottlenecks are the most important to address. A previous strain characterization study from our lab suggested that insufficient NAD+ limits fermentation and may be the most important bottleneck affecting utilization of xylose for the production of ethanol. The development and validation of a genome scale dynamic flux balance model would help to verify the existence and extent of this and other metabolic bottlenecks and suggest solutions to guide future strain development thereby minimizing bottleneck impact on process economics. A dynamic flux balance model was developed to identify bottlenecks in several strains of S. cerevisiae, both with wild-type pentose phosphate pathway expression and with the pathway over expressed. ZWF1 was found to be limiting in the oxidative portion of the pentose phosphate pathway under oxygen replete conditions. This pathway is used to regenerate NADPH. Under oxygen limiting conditions, respiration of xylose was limited by the lack of oxygen as a terminal electron acceptor. Ethanol production was also limited under these conditions due to the inability to balance NAD+/NADH. The model suggests the use of the anaplerotic glyoxylate pathway to improve NAD+/NADH balance, increasing ethanol production by 50% while producing succinate as a coproduct at upwards of 20 g/l. In the production of high value chemicals from biomass, the use of the respiratory metabolism is a waste of feedstock carbon. Bottlenecks previously identified in the oxidative pentose phosphate pathway are currently only relevant under oxygen-replete conditions and cannot impact the partitioning of carbon between the respiratory and fermentative pathways. Focusing future efforts on the non-respiratory balancing of NAD+/NADH, perhaps through the glyoxylate pathway, would improve the economics of ethanol production both directly and through coproduct formation.

NADH Ties One-Carbon Metabolism to Cellular Respiration

In this issue of Cell Metabolism, Yang etal., 2020 report that serine is a source of mitochondrial NADH derived from one-carbon metabolism. Serine becomes a major source of NADH when cellular respiration is inhibited, and the un-utilized, accumulated NADH inhibits the TCA cycle and slows proliferation.

Genetic Analysis of Peroxisomal Genes Required for Longevity in a Yeast Model of Citrin Deficiency.

Citrin is a liver-specific mitochondrial aspartate–glutamate carrier encoded by SLC25A13. Citrin deficiency caused by SLC25A13 mutation results in carbohydrate toxicity, citrullinemia type II, and fatty liver diseases, the mechanisms of some of which remain unknown. Citrin shows a functional homolog in yeast aspartate-glutamate carrier (Agc1p) and agc1Δ yeasts are used as a model organism of citrin deficiency. Here, we found that agc1Δ yeasts decreased fat utilization, impaired NADH balance in peroxisomes, and decreased chronological lifespan. The activation of GPD1-mediated NAD+ regeneration in peroxisomes by GPD1 over-expression or activation of the malate–oxaloacetate NADH peroxisomal shuttle, by increasing flux in this NADH shuttle and over-expression of MDH3, resulted in lifespan extension of agc1Δ yeasts. In addition, over-expression of PEX34 restored longevity of agc1Δ yeasts as well as wild-type cells. The effect of PEX34-mediated longevity required the presence of the GPD1-mediated NADH peroxisomal shuttle, which was independent of the presence of the peroxisomal malate–oxaloacetate NADH shuttle and PEX34-induced peroxisome proliferation. These data confirm that impaired NAD+ regeneration in peroxisomes is a key defect in the yeast model of citrin deficiency, and enhancing peroxisome function or inducing NAD+ regeneration in peroxisomes is suggested for further study in patients’ hepatocytes.

Growth of carbon nanotubes on graphene as 3D biocathode for NAD+/NADH balance model and high-rate production in microbial electrochemical synthesis from CO2

Design and synthesis of three-dimensional (3D) structured carbon nanomaterials for achieving high-performance electrode for microbial electrochemical synthesis (MES).

Structural Analysis of Redox-sensing Transcriptional Repressor Rex from Thermotoga maritima

The cellular redox state of organisms continues to fluctuate during the metabolism. All organisms have various sensors that help detect and adapt to changes in the redox state. Nicotinamide adenine dinucleotides (NAD+/NADH), which are involved in various cellular metabolic activities as cofactors, have been revealed as the key molecules sensing the intra-cellular redox state. The Rex family members are well conserved transcriptional repressors that regulate the expression of respiratory genes by sensing the redox state according to the intra-cellular NAD+/NADH balance. Herein, we reported crystal structures of apo and NAD+- and NADH-bound forms of Rex from Thermotoga maritima to analyse the structural basis of transcriptional regulation depending on either NAD+ or NADH binding. The different orientation of the reduced nicotinamide group to helix α9 caused the rearrangement of N-terminal DNA binding domain, thus resulting in closed form of Rex to dissociate from cognate DNA. The structural data of Rex from T. maritima also support the previous redox-sensing mechanism models of Rex homologues.

SRNA-mediated activation of gene expression by inhibition of 5'-3' exonucleolytic mRNA degradation.

Post-transcriptional control by small regulatory RNA (sRNA) is critical for rapid adaptive processes. sRNAs can directly modulate mRNA degradation in Proteobacteria without interfering with translation. However, Firmicutes have a fundamentally different set of ribonucleases for mRNA degradation and whether sRNAs can regulate the activity of these enzymes is an open question. We show that Bacillus subtilis RoxS, a major trans-acting sRNA shared with Staphylococus aureus, prevents degradation of the yflS mRNA, encoding a malate transporter. In the presence of malate, RoxS transiently escapes from repression by the NADH-sensitive transcription factor Rex and binds to the extreme 5'-end of yflS mRNA. This impairs the 5'-3' exoribonuclease activity of RNase J1, increasing the half-life of the primary transcript and concomitantly enhancing ribosome binding to increase expression of the transporter. Globally, the different targets regulated by RoxS suggest that it helps readjust the cellular NAD+/NADH balance when perturbed by different stimuli.

Self-regulated 1-butanol production in Escherichia coli based on the endogenous fermentative control.

BackgroundAs a natural fermentation product secreted by Clostridium species, bio-based 1-butanol has attracted great attention for its potential as alternative fuel and chemical feedstock. Feasibility of microbial 1-butanol production has also been demonstrated in various recombinant hosts.ResultsIn this work, we constructed a self-regulated 1-butanol production system in Escherichia coli by borrowing its endogenous fermentation regulatory elements (FRE) to automatically drive the 1-butanol biosynthetic genes in response to its natural fermentation need. Four different cassette of 5′ upstream transcription and translation regulatory regions controlling the expression of the major fermentative genes ldhA, frdABCD, adhE, and ackA were cloned individually to drive the 1-butanol pathway genes distributed among three plasmids, resulting in 64 combinations that were tested for 1-butanol production efficiency. Fermentation of 1-butanol was triggered by anaerobicity in all cases. In the growth-decoupled production screening, only combinations with formate dehydrogenase (Fdh) overexpressed under FREadhE demonstrated higher titer of 1-butanol anaerobically. In vitro assay revealed that 1-butanol productivity was directly correlated with Fdh activity under such condition. Switching cells to oxygen-limiting condition prior to significant accumulation of biomass appeared to be crucial for the induction of enzyme synthesis and the efficiency of 1-butanol fermentation. With the selection pressure of anaerobic NADH balance, the engineered strain demonstrated stable production of 1-butanol anaerobically without the addition of inducer or antibiotics, reaching a titer of 10 g/L in 24 h and a yield of 0.25 g/g glucose under high-density fermentation.ConclusionsHere, we successfully engineered a self-regulated 1-butanol fermentation system in E. coli based on the natural regulation of fermentation reactions. This work also demonstrated the effectiveness of selection pressure based on redox balance anaerobically. Results obtained from this study may help enhance the industrial relevance of 1-butanol synthesis using E. coli and solidifies the possibility of strain improvement by directed evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0680-1) contains supplementary material, which is available to authorized users.

Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans.

Thermotolerant Bacillus coagulans is considered to be a more promising producer for bio-chemicals, due to its capacity to withstand harsh conditions. Two L-lactate dehydrogenase (LDH) encoding genes (ldhL1 and ldhL2) and one D-LDH encoding gene (ldhD) were annotated from the B. coagulans DSM1 genome. Transcriptional analysis revealed that the expression of ldhL2 was undetectable while the ldhL1 transcription level was much higher than that of ldhD at all growth phases. Deletion of the ldhL2 gene revealed no difference in fermentation profile compared to the wild-type strain, while ldhL1 single deletion or ldhL1ldhL2 double deletion completely blocked L-lactic acid production. Complementation of ldhL1 in the above knockout strains restored fermentation profiles to those observed in the wild-type strain. This study demonstrates ldhL1 is crucial for L-lactic acid production and NADH balance in B. coagulans DSM1 and lays the fundamental for engineering the thermotolerant B. coagulans strain as a platform chemicals producer.

NADH dehydrogenase of Trypanosoma brucei is important for efficient acetate production in bloodstream forms

In the slender bloodstream form, Trypanosoma brucei mitochondria are repressed for many functions. Multiple components of mitochondrial complex I, NADH:ubiquinone oxidoreductase, are expressed in this stage, but electron transfer through complex I is not essential. Here we investigate the role of the parasite’s second NADH:ubiquinone oxidoreductase, NDH2, which is composed of a single subunit that also localizes to the mitochondrion. While inducible knockdown of NDH2 had a modest growth effect in bloodstream forms, NDH2 null mutants, as well as inducible knockdowns in a complex I deficient background, showed a greater reduction in growth. Altering the NAD+/NADH balance would affect numerous processes directly and indirectly, including acetate production. Indeed, loss of NDH2 led to reduced levels of acetate, which is required for several essential pathways in bloodstream form T. brucei and which may have contributed to the observed growth defect. In conclusion our study shows that NDH2 is important, but not essential, in proliferating bloodstream forms of T. brucei, arguing that the mitochondrial NAD+/NADH balance is important in this stage, even though the mitochondrion itself is not actively engaged in the generation of ATP.

Succinic acid production by Actinobacillus succinogenes from batch fermentation of mixed sugars.

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7g/gh for glucose, but below 0.2g/gh for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8-26.8g/L and a total yield of 0.59-0.68g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14-0.20g/g and formate 0.08-0.13g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60g/g. In addition, it was found that CO2 sparging could replace carbonate supply in the form of MgCO3 without affecting the succinate yield.

Down-Regulation of NDUFB9 Promotes Breast Cancer Cell Proliferation, Metastasis by Mediating Mitochondrial Metabolism.

Despite advances in basic and clinical research, metastasis remains the leading cause of death in breast cancer patients. Genetic abnormalities in mitochondria, including mutations affecting complex I and oxidative phosphorylation, are found in breast cancers and might facilitate metastasis. Genes encoding complex I components have significant breast cancer prognostic value. In this study, we used quantitative proteomic analyses to compare a highly metastatic cancer cell line and a parental breast cancer cell line; and observed that NDUFB9, an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I), was down-regulated in highly metastatic breast cancer cells. Furthermore, we demonstrated that loss of NDUFB9 promotes MDA-MB-231 cells proliferation, migration, and invasion because of elevated levels of mtROS, disturbance of the NAD+/NADH balance, and depletion of mtDNA. We also showed that, the Akt/mTOR/p70S6K signaling pathway and EMT might be involved in this mechanism. Thus, our findings contribute novel data to support the hypothesis that misregulation of mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, suggesting that complex I deficiency is a potential and important biomarker for further basic research or clinical application.

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes.

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in Trypanosoma cruzi.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA.

A ROLE OF POPULATION HETEROGENEITY AND pH FACTOR IN NATURAL α-ACETOLACTATE SUPERSYNTHESIS IN A DIACETYL PRODUCER, Lactococcus lactis ssp. lactis bv. diacetylactis B2103/74

The effect of the population heterogeneity and medium pH on the found out by us supersynthesis of а-acetolactate (AL) by the L. lactis bv. diacetyllactis B2103/74 culture has been investigated. The nature of an enzyme system that is responsible for this effect due to lactate dehydrogenase essentially blocking by intercepting up to 80% NADH from it and was indicated as hemin-independent electron-transferring system (HIETS) is unknown. It was shown that the effect was disguised by the culture heterogeneity which was the most pronounced at neighboring to neutral pH values 6.1—6.5. It was established that at least three types of dissociants with different HIETS and, consequently, AL levels occurred in the population: active dissociant (AL accumulation up to 30 mM); inactive dissociant that lost HIETS activity (AL content of no more than 3 mM); and dissociant with moderate activity (AL content 15—17 mM). It was demonstrated that HIETS played a major part in the whole pyruvate metabolism at various pH and finally in the AL supersynthesis. For instance, at pH =7.0, the effect of supersynthesis failed to be observed due to the HIETS low activity, and the pyruvate metabolism proceeded by the homolactic pathway. However, at pH 5.3—6.5, the HIETS activity was so high that it permitted the system to take over a function of the major regulator of the redox NAD+/NADH balance leaving the minor part to LDH. The productivities of LDH and acetolactate-synthase pathways depended on the HIETS activity at various pH values, whereas that of the pyruvatedehydrogenase pathway was a straight function of external pH. The latter was rather high in the close to neutral pH area (6.0—6.5) and was significantly lower in the neutral (pH=6.0—6.5) and light-acidic (pH<6.0) zones.

The regulation of 2,3-butanediol synthesis in Klebsiella pneumoniae as revealed by gene over-expressions and metabolic flux analysis

A variety of microorganism species are able naturally to produce 2,3-butanediol (2,3-BDO), although only a few of them are suitable for consideration as having potential for mass production purposes. Klebsiella pneumoniae (K. pneumoniae) is one such strain which has been widely studied and used industrially to produce 2,3-BDO. In the central carbon metabolism of K. pneumoniae, the 2,3-BDO synthesis pathway is dominated by three essential enzymes, namely acetolactate decarboxylase, acetolactate synthase, and butanediol dehydrogenase, which are encoded by the budA, budB, and budC genes, respectively. The mechanisms of the three enzymes have been characterized with regard to their function and roles in 2,3-BDO synthesis and cell growth (Blomqvist et al. in J Bacteriol 175(5):1392-1404, 1993), while a few studies have focused on the cooperative mechanisms of the three enzymes and their mutual interactions. Therefore, the K. pneumoniae KCTC2242::ΔwabG wild-type strain was utilized to reconstruct seven new mutants by single, double, and triple overexpression of the three enzymes key to this study. Subsequently, continuous cultures were performed to obtain steady-state metabolism in the organisms and experimental data were analyzed by metabolic flux analysis (MFA) to determine the regulation mechanisms. The MFA results showed that the seven overexpressed mutants all exhibited enhanced 2,3-BDO production, and the strain overexpressing the budBA gene produced the highest yield. While the enzyme encoded by the budA gene produced branched-chain amino acids which were favorable for cell growth, the budB gene enzyme rapidly enhanced the conversion of acetolactate to acetoin in an oxygen-dependent manner, and the budC gene enzyme catalyzed the reversible conversion of acetoin to 2,3-BDO and regulated the intracellular NAD(+)/NADH balance.

Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression

Despite advances in clinical therapy, metastasis remains the leading cause of death in breast cancer patients. Mutations in mitochondrial DNA, including those affecting complex I and oxidative phosphorylation, are found in breast tumors and could facilitate metastasis. This study identifies mitochondrial complex I as critical for defining an aggressive phenotype in breast cancer cells. Specific enhancement of mitochondrial complex I activity inhibited tumor growth and metastasis through regulation of the tumor cell NAD+/NADH redox balance, mTORC1 activity, and autophagy. Conversely, nonlethal reduction of NAD+ levels by interfering with nicotinamide phosphoribosyltransferase expression rendered tumor cells more aggressive and increased metastasis. The results translate into a new therapeutic strategy: enhancement of the NAD+/NADH balance through treatment with NAD+ precursors inhibited metastasis in xenograft models, increased animal survival, and strongly interfered with oncogene-driven breast cancer progression in the MMTV-PyMT mouse model. Thus, aberration in mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, while therapeutic normalization of the NAD+/NADH balance can inhibit metastasis and prevent disease progression.

Abstract IA3: Normalizing tumor cell metabolism in breast cancer metastasis: A novel therapeutic approach

Abstract Despite advances in clinical therapy, metastasis is still the leading cause of death in breast cancer patients. A better understanding of mechanisms that drive metastasis is a prerequisite for new approaches to effectively prevent and inhibit this most dangerous advancement of the disease. While alterations in the nuclear genome are pivotal in oncogenesis, a role of mitochondria in cancer progression has remained largely unexplored. Mutations in mitochondrial DNA are found in breast tumors and other cancers, however their involvement in driving the disease is unclear. Our study identifies mitochondrial complex I as critical for defining an aggressive phenotype in breast cancer cells. Complex I is the gate-keeper of the respiratory chain and catalyzes the first step of NADH oxidation. It elevates the cellular NAD+/NADH ratio and translocates protons across the inner mitochondrial membrane, which ultimately leads to energy production. We used a unique approach to define contributions of mitochondrial complex I activity to breast cancer progression, based on expression of yeast NADH dehydrogenase Ndi1. Ndi1 encodes a single protein that translocates to mitochondrial, faces the inner mitochondrial matrix and oxidizes NADH from the Krebs cycle. Specific enhancement of mitochondrial complex I activity by Ndi1 expression inhibited tumor growth and metastasis through regulation of the tumor cell NAD+/NADH redox balance, mTORC1 activity, and autophagy. Conversely, non-lethal reduction of NAD+ levels by interfering with nicotinamide phosphoribosyltransferase expression to disturb the NAD+ synthesis and recycling pathway, rendered tumor cells more aggressive and increased metastasis. Thus, the results indicate a cause-and-effect relationship between reduced NAD+/NADH ratios and metastatic activity. Having established that enhancement of NAD+/NADH levels by augmenting breast cancer cell complex I activity inhibits tumorigenicity and metastasis, we used this new concept therapeutically and hypothesized that supplementing tumor cell nutrients with NAD+ precursors, such as nicotinic acid (NIC) or nicotinamide (NAM), could interfere with breast cancer progression. We demonstrate that enhancing NAD+ levels through NAD+ precursor treatment effectively inhibits experimental metastasis of human breast cancer cells in xenograft models. Importantly, this treatment also inhibited spontaneous metastasis, and increased animal survival when the therapy was started after surgical removal of primary tumors. Furthermore, NAD+ precursor treatment strongly interferes with oncogene driven breast cancer development and progression in transgenic MMTV-PyMT mice. Thus, aberration in mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells while therapeutic normalization of the NAD+/NADH balance can inhibit metastasis and prevent disease progression. Our study demonstrates that mitochondrial complex I regulation of tumor cell NAD+/NADH levels impacts breast cancer growth and metastasis, and translates into a new therapeutic approach for preventing breast cancer progression. This is highly relevant as current standard of care for cancer patients relies primarily on chemo- and radiation therapies aimed at killing the tumor cells. Evolutionary models predict that selective pressure imposed by these approaches causes survival of resistant clones that eventually re-activate the disease. Based on the central involvement of metabolic tumor cell alterations in cancer, therapeutic normalization of tumor cell metabolism might interfere with the expansion of residual and break-through clones. Thus, a combination of standard therapy with NAD+ precursor treatment may halt breast cancer progression and prevent relapse. Citation Format: Antonio F. Santidrian, Akemi Matsuno-Yagi, Melissa Ritland, Byoung B. Seo1,2, Sarah E. LeBoeuf, Laurie J. Gay, Takao Yagi, Brunhilde Felding-Habermann. Normalizing tumor cell metabolism in breast cancer metastasis: A novel therapeutic approach. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA3.

Factors influencing the production of volatile phenols by wine lactic acid bacteria

This work aimed to evaluate the effect of certain factors on the production of volatile phenols from the metabolism of p-coumaric acid by lactic acid bacteria (LAB) ( Lactobacillus plantarum, L. collinoides and Pediococcus pentosaceus). The studied factors were: pH, l-malic acid concentration, glucose and fructose concentrations and aerobic/anaerobic conditions. It was found that, in the pH range of 3.5 to 4.5, the higher the pH the greater the production of volatile phenols. This behaviour is correlated with the effect of pH on bacterial growth. Increasing levels of l-malic acid in the medium diminished the production of 4-vinylphenol (4VP) and stimulated the production of 4-ethylphenol (4EP) by L. plantarum NCFB 1752 and L. collinoides ESB 99. The conversion of 4VP into 4EP by the activity of the vinylphenol reductase may be advantageous to the cells in the presence of l-malic acid, presumably due to the generation of NAD +, a cofactor required by the malolactic enzyme. Relatively high levels of glucose (20 g/L) led to an almost exclusive production of 4VP by L. plantarum NCFB 1752, while at low concentrations (≤ 5 g/L), 4EP is mainly or solely produced. Part of the glucose may be diverted to the production of mannitol as an alternative pathway to regenerate NAD +. This is corroborated by the experiments done with fructose, a compound that can be used as an electron acceptor by some bacteria becoming reduced to mannitol. In anaerobiosis, the reduction of 4VP into 4EP is clearly favoured, which is consistent with the need to increase the availability of NAD + in these conditions. This study shows that the amount and the ratio 4VP/4EP produced by LAB are greatly affected by certain environmental and medium composition factors. The behaviour of the bacteria seems to be driven by the intracellular NAD +/NADH balance.

Biological activity of three novel complexes with the ligand 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one against Leishmania spp.

We report on new 1,2,4-triazolo[1,5-a]pyrimidine complexes that have been developed and examined for both antiproliferative in vitro activity against Leishmania infantum and Leishmania braziliensis, and report their possible mechanism of action on L. infantum and L. braziliensis. Antileishmanial effects are described for newly synthesized Cu(II) and Co(II) complexes containing 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one (HmtpO) as a ligand. These complexes display a wide structural diversity: (i) mononuclear unit, [Cu(HmtpO)2(H2O)3](ClO4)2·H2O (1); (ii) two-dimensional framework, {[Cu(HmtpO)2(H2O)2](ClO4)(2)·2HmtpO}n (2); and (iii) chains, {[Co(HmtpO)(H2O)3](ClO4)(2)·2H2O}n (3). Compounds 1 and 2 appeared to be the most active against L. infantum (IC50 20.0 and 24.4 μM, respectively), and compounds 1 and 3 the most active against L. braziliensis (IC50 22.1 and 23.5 μM, respectively), with IC50s similar to those of the reference drug, glucantime (18.0 μM for L. infantum and 25.6 μM for L. braziliensis). These compounds were not toxic towards J774.2 macrophages. IC25 decreased infection capacity and severely reduced the multiplication of intracellular amastigotes, following the trend 1>3>2 for L. infantum and 3>1>2 for L. braziliensis. These complexes had an effect on the energy metabolism of the parasites at the level of the NAD+/NADH balance and the organelle membranes, causing their degradation and cell death. Cellular proliferation, metabolic and ultrastructural studies showed that the compounds 2>1>3 were highly active against L. infantum and L. braziliensis.