Intracellular Pyruvate Concentration Research Articles

Balancing Pyruvate Node Based on a Dual-Layered Dynamic Regulation System to Improve the Biosynthesis of Caffeic Acid in Candida glycerinogenes.

Caffeic acid is a phenolic acid compound widely applied in the food and pharmaceutical fields. Currently, one of the reasons for the low yield of caffeic acid biosynthesis is that the carbon flow enters mainly into the TCA cycle via pyruvate, which leads to low concentrations of erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP), the precursors of caffeic acid synthesis. Here, we developed a growth-coupled dual-layered dynamic regulation system. This system controls intracellular pyruvate supply in real time by responding to intracellular pyruvate and p-coumaric acid concentrations, autonomously coordinates pathway gene expression, and redirects carbon metabolism to balance cell growth and caffeic acid synthesis. Finally, our constructed engineered strain based on the dual-layered dynamic regulation system achieved a caffeic acid titer of 559.7 mg/L in a 5 L bioreactor. Thus, this study demonstrated the efficiency and potential of this system in boosting the yield of aromatic compounds.

Dynamic fluctuations in a bacterial metabolic network

The operation of the central metabolism is typically assumed to be deterministic, but dynamics and high connectivity of the metabolic network make it potentially prone to generating fluctuations. However, time-resolved measurements of metabolite levels in individual cells that are required to characterize such fluctuations remained a challenge, particularly in small bacterial cells. Here we use single-cell metabolite measurements based on Förster resonance energy transfer, combined with computer simulations, to explore the real-time dynamics of the metabolic network of Escherichia coli. We observe that steplike exposure of starved E. coli to glycolytic carbon sources elicits large periodic fluctuations in the intracellular concentration of pyruvate in individual cells. These fluctuations are consistent with predicted oscillatory dynamics of E. coli metabolic network, and they are primarily controlled by biochemical reactions around the pyruvate node. Our results further indicate that fluctuations in glycolysis propagate to other cellular processes, possibly leading to temporal heterogeneity of cellular states within a population.

Intracellular pyruvate levels positively correlate with cytokine production capacity in tolerant monocytes from patients with pneumonia

BackgroundCommunity-acquired pneumonia (CAP) is responsible for a high morbidity and mortality worldwide. Monocytes are essential for pathogen recognition and the initiation of an innate immune response. Immune cells induce intracellular glycolysis upon activation to support several functions. ObjectiveTo obtain insight in the metabolic profile of blood monocytes during CAP, with a focus on glycolysis and branching metabolic pathways, and to determine a possible association between intracellular metabolite levels and monocyte function. MethodsMonocytes were isolated from blood of patients with CAP within 24 h of hospital admission and from control subjects matched for age, sex and chronic comorbidities. Changes in glycolysis, oxidative phosphorylation (OXPHOS), tricarboxylic acid (TCA) cycle and the pentose phosphate pathway were investigated through RNA sequencing and metabolomics measurements. Monocytes were stimulated ex vivo with lipopolysaccharide (LPS) to determine their capacity to produce tumor necrosis factor (TNF), interleukin (IL)-1β and IL-10. Results50 patients with CAP and 25 non-infectious control subjects were studied. When compared with control monocytes, monocytes from patients showed upregulation of many genes involved in glycolysis, including PKM, the gene encoding pyruvate kinase, the rate limiting enzyme for pyruvate production. Gene set enrichment analysis of OXPHOS, the TCA cycle and the pentose phosphate pathway did not reveal differences between monocytes from patients and controls. Patients' monocytes had elevated intracellular levels of pyruvate and the TCA cycle intermediate α-ketoglutarate. Monocytes from patients were less capable of producing cytokines upon LPS stimulation. Intracellular pyruvate (but not α-ketoglutarate) concentrations positively correlated with IL-1β and IL-10 levels released by patients' (but not control) monocytes upon exposure to LPS. ConclusionThese results suggest that elevated intracellular pyruvate levels may partially maintain cytokine production capacity of hyporesponsive monocytes from patients with CAP.

Integrating a dynamic central metabolism model of cancer cells with a hybrid 3D multiscale model for vascular hepatocellular carcinoma growth

We develop here a novel modelling approach with the aim of closing the conceptual gap between tumour-level metabolic processes and the metabolic processes occurring in individual cancer cells. In particular, the metabolism in hepatocellular carcinoma derived cell lines (HEPG2 cells) has been well characterized but implementations of multiscale models integrating this known metabolism have not been previously reported. We therefore extend a previously published multiscale model of vascular tumour growth, and integrate it with an experimentally verified network of central metabolism in HEPG2 cells. This resultant combined model links spatially heterogeneous vascular tumour growth with known metabolic networks within tumour cells and accounts for blood flow, angiogenesis, vascular remodelling and nutrient/growth factor transport within a growing tumour, as well as the movement of, and interactions between normal and cancer cells. Model simulations report for the first time, predictions of spatially resolved time courses of core metabolites in HEPG2 cells. These simulations can be performed at a sufficient scale to incorporate clinically relevant features of different tumour systems using reasonable computational resources. Our results predict larger than expected temporal and spatial heterogeneity in the intracellular concentrations of glucose, oxygen, lactate pyruvate, f16bp and Acetyl-CoA. The integrated multiscale model developed here provides an ideal quantitative framework in which to study the relationship between dosage, timing, and scheduling of anti-neoplastic agents and the physiological effects of tumour metabolism at the cellular level. Such models, therefore, have the potential to inform treatment decisions when drug response is dependent on the metabolic state of individual cancer cells.

Potentials of multi-stress tolerant yeasts, Saccharomyces cerevisiae and Pichia kudriavzevii for fuel ethanol production from industrial cassava wastes

Cassava processing wastes are suitable substrates for bioethanol production due to their high starch contents. However, these cassava wastes often contain various substances that inhibit cell growth and ethanol production. The abilities of multi-stress tolerant Saccharomyces cerevisiae LC269108 and Pichia kudriavzevii LC375240 to produce ethanol from cassava industrial wastes were evaluated. Analysis of cassava peels and pulps from two cassava starch factories showed that the cassava peels contained high cyanide (KCN) concentrations while cassava pulps contained high concentrations of sodium dodecyl benzene sulfonate (SDBS) used to improve starch extraction in the factory. Although S. cerevisiae LC269108 produced ethanol efficiently from the cassava peel, its growth and ethanol production were inhibited in the cassava pulp. On the other hand, the growth and ethanol production by P. kudriavzevii LC375240 were inhibited slightly in the cassava peel but not in the cassava pulp. Based on the intracellular pyruvate concentrations in the presence of KCN and SDBS, it was inferred that their inhibition site is on the pyruvate synthetic pathways. With a co-culture of the two strains, ethanol production from the cassava peel increased significantly but there was no significant improvement in ethanol production from the cassava pulp.

Pyruvate-responsive genetic circuits for dynamic control of central metabolism.

Dynamic regulation is a promising strategy for fine-tuning metabolic fluxes in microbial cell factories. However, few of these synthetic regulatory systems have been developed for central carbon metabolites. Here we created a set of programmable and bifunctional pyruvate-responsive genetic circuits for dynamic dual control (activation and inhibition) of central metabolism in Bacillus subtilis. We used these genetic circuits to design a feedback loop control system that relies on the intracellular concentration of pyruvate to fine-tune the target metabolic modules, leading to the glucaric acid titer increasing from 207 to 527 mg l-1. The designed logic gate-based circuits were enabled by the characterization of a new antisense transcription mechanism in B. subtilis. In addition, a further increase to 802 mg l-1 was achieved by blocking the formation of by-products. Here, the constructed pyruvate-responsive genetic circuits are presented as effective tools for the dynamic control of central metabolism of microbial cell factories.

Reaction rate of pyruvate and hydrogen peroxide: assessing antioxidant capacity of pyruvate under biological conditions

Pyruvate, a pivotal glucose metabolite, is an α-ketoacid that reacts with hydrogen peroxide (H2O2). Its pharmacological precursor, ethyl pyruvate, has shown anti-inflammatory/anti-tissue injury effects in various animal models of disease, but failed in a multicenter clinical trial. Since rodents, but not humans, can convert ethyl pyruvate to pyruvate in blood plasma, this additional source of extracellular pyruvate may have contributed to the discrepancy between the species. To examine this possibility, we investigated the kinetics of the reaction under biological conditions and determined the second order rate constant k as 2.360 ± 0.198 M−1 s−1. We then calculated the time required for H2O2 elimination by pyruvate. The results show that, with an average intracellular concentration of pyruvate (150 µM), elimination of 95% H2O2 at normal to pathological concentrations (0.01–50 µM) requires 141–185 min (2.4–3 hour). With 1,000 µM pyruvate, a concentration that can only exist extracellularly or in cell culture media, 95% elimination of H2O2 at 5–200 µM requires 21–25 min. We conclude that intracellular pyruvate, or other α-ketoacids, whose endogenous concentration is controlled by metabolism, have little role in H2O2 clearance. An increased extracellular concentration of pyruvate, however, does have remarkable peroxide scavenging effects, considering minimal peroxidase activity in this space.

Exogenous pyruvate represses histone gene expression and inhibits cancer cell proliferation via the NAMPT-NAD+-SIRT1 pathway.

Pyruvate is a glycolytic metabolite used for energy production and macromolecule biosynthesis. However, little is known about its functions in tumorigenesis. Here, we report that exogenous pyruvate inhibits the proliferation of different types of cancer cells. This inhibitory effect of pyruvate on cell growth is primarily attributed to its function as a signal molecule to repress histone gene expression, which leads to less compact chromatin and misregulation of genome-wide gene expression. Pyruvate represses histone gene expression by inducing the expression of NAD+ biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT) via myocyte enhancer factor 2C (MEF2C), which then increases NAD+ levels and activates the histone deacetylase activity of SIRT1. Chromatin immunoprecipitation analysis indicates that pyruvate enhances SIRT1 binding at histone gene promoters where it reduces histone acetylation. Although pyruvate delays cell entry into S phase, pyruvate represses histone gene expression independent of cell cycle progression. Moreover, we find that administration of pyruvate reduces histone expression and retards tumor growth in xenograft mice without significant side effects. Using tissues from cervical and lung cancer patients, we find intracellular pyruvate concentrations inversely correlate with histone protein levels. Together, we uncover a previously unknown function of pyruvate in regulating histone gene expression and cancer cell proliferation.

Pyruvate produced by Brugia spp. via glycolysis is essential for maintaining the mutualistic association between the parasite and its endosymbiont, Wolbachia.

Human parasitic nematodes are the causative agents of lymphatic filariasis (elephantiasis) and onchocerciasis (river blindness), diseases that are endemic to more than 80 countries and that consistently rank in the top ten for the highest number of years lived with disability. These filarial nematodes have evolved an obligate mutualistic association with an intracellular bacterium, Wolbachia, a symbiont that is essential for the successful development, reproduction, and survival of adult filarial worms. Elimination of the bacteria causes adult worms to die, making Wolbachia a primary target for developing new interventional tools to combat filariases. To further explore Wolbachia as a promising indirect macrofilaricidal drug target, the essential cellular processes that define the symbiotic Wolbachia-host interactions need to be identified. Genomic analyses revealed that while filarial nematodes encode all the enzymes necessary for glycolysis, Wolbachia does not encode the genes for three glycolytic enzymes: hexokinase, 6-phosphofructokinase, and pyruvate kinase. These enzymes are necessary for converting glucose into pyruvate. Wolbachia, however, has the full complement of genes required for gluconeogenesis starting with pyruvate, and for energy metabolism via the tricarboxylic acid cycle. Therefore, we hypothesized that Wolbachia might depend on host glycolysis to maintain a mutualistic association with their parasitic host. We did conditional experiments in vitro that confirmed that glycolysis and its end-product, pyruvate, sustain this symbiotic relationship. Analysis of alternative sources of pyruvate within the worm indicated that the filarial lactate dehydrogenase could also regulate the local intracellular concentration of pyruvate in proximity to Wolbachia and thus help control bacterial growth via molecular interactions with the bacteria. Lastly, we have shown that the parasite’s pyruvate kinase, the enzyme that performs the last step in glycolysis, could be a potential novel anti-filarial drug target. Establishing that glycolysis is an essential component of symbiosis in filarial worms could have a broader impact on research focused on other intracellular bacteria-host interactions where the role of glycolysis in supporting intracellular survival of bacteria has been reported.

Metabolic adaptation to glycolysis is a basic defense mechanism of macrophages for Mycobacterium tuberculosis infection.

Macrophages are major components of tuberculosis (TB) granulomas and are responsible for host defenses against the intracellular pathogen, Mycobacterium tuberculosis. We herein showed the strong expression of hypoxia-inducible factor-1α (HIF-1α) in TB granulomas and more rapid death of HIF-1α-conditional knockout mice than wild-type (WT) mice after M. tuberculosis infection. Although interferon-γ (IFN-γ) is a critical host-protective cytokine against intracellular pathogens, HIF-1-deficient macrophages permitted M. tuberculosis growth even after activation with IFN-γ. These results prompted us to investigate the role of HIF-1α in host defenses against infection. We found that the expression of lactate dehydrogenase-A (LDH-A) was controlled by HIF-1α in M. tuberculosis-infected macrophages IFN-γ independently. LDH-A is an enzyme that converts pyruvate to lactate and we found that the intracellular level of pyruvate in HIF-1α-deficient bone marrow-derived macrophages (BMDMs) was significantly higher than in WT BMDMs. Intracellular bacillus replication was enhanced by an increase in intracellular pyruvate concentrations, which were decreased by LDH-A. Mycobacteria in phagosomes took up exogenous pyruvate more efficiently than glucose, and used it as the feasible carbon source for intracellular growth. These results demonstrate that HIF-1α prevents the hijacking of pyruvate in macrophages, making it a fundamental host-protective mechanism against M. tuberculosis.

Peptide Transporter CstA Imports Pyruvate in Escherichia coli K-12

ABSTRACTPyruvate is an important intermediate of central carbon metabolism and connects a variety of metabolic pathways in Escherichia coli. Although the intracellular pyruvate concentration is dynamically altered and tightly balanced during cell growth, the pyruvate transport system remains unclear. Here, we identified a pyruvate transporter in E. coli using high-throughput transposon sequencing. The transposon mutant library (a total of 5 × 105 mutants) was serially grown with a toxic pyruvate analog (3-fluoropyruvate [3FP]) to enrich for transposon mutants lacking pyruvate transport function. A total of 52 candidates were selected on the basis of a stringent enrichment level of transposon insertion frequency in response to 3FP treatment. Subsequently, their pyruvate transporter function was examined by conventional functional assays, such as those measuring growth inhibition by the toxic pyruvate analog and pyruvate uptake activity. The pyruvate transporter system comprises CstA and YbdD, which are known as a peptide transporter and a conserved protein, respectively, whose functions are associated with carbon starvation conditions. In addition to the presence of more than one endogenous pyruvate importer, it has been suggested that the E. coli genome encodes constitutive and inducible pyruvate transporters. Our results demonstrated that CstA and YbdD comprise the constitutive pyruvate transporter system in E. coli, which is consistent with the tentative genomic locus previously suggested and the functional relationship with the extracellular pyruvate sensing system. The identification of this pyruvate transporter system provides valuable genetic information for understanding the complex process of pyruvate metabolism in E. coli.IMPORTANCE Pyruvate is an important metabolite as a central node in bacterial metabolism, and its intracellular levels are tightly regulated to maintain its functional roles in highly interconnected metabolic pathways. However, an understanding of the mechanism of how bacterial cells excrete and transport pyruvate remains elusive. Using high-throughput transposon sequencing followed by pyruvate uptake activity testing of the selected candidate genes, we found that a pyruvate transporter system comprising CstA and YbdD, currently annotated as a peptide transporter and a conserved protein, respectively, constitutively transports pyruvate. The identification of the physiological role of the pyruvate transporter system provides valuable genetic information for understanding the complex pyruvate metabolism in Escherichia coli.

Pyruvate Protects Giardia Trophozoites from Cysteine-Ascorbate Deprived Medium Induced Cytotoxicity.

Giardia lamblia, an anaerobic, amitochondriate protozoan parasite causes parasitic infection giardiasis in children and young adults. It produces pyruvate, a major metabolic product for its fermentative metabolism. The current study was undertaken to explore the effects of pyruvate as a physiological antioxidant during oxidative stress in Giardia by cysteine-ascorbate deprivation and further investigation upon the hypothesis that oxidative stress due to metabolism was the reason behind the cytotoxicity. We have estimated intracellular reactive oxygen species generation due to cysteine-ascorbate deprivation in Giardia. In the present study, we have examined the effects of extracellular addition of pyruvate, during oxidative stress generated from cysteine-ascorbate deprivation in culture media on DNA damage in Giardia. The intracellular pyruvate concentrations at several time points were measured in the trophozoites during stress. Trophozoites viability under cysteine-ascorbate deprived (CAD) medium in presence and absence of extracellular pyruvate has also been measured. The exogenous addition of a physiologically relevant concentration of pyruvate to trophozoites suspension was shown to attenuate the rate of ROS generation. We have demonstrated that Giardia protects itself from destructive consequences of ROS by maintaining the intracellular pyruvate concentration. Pyruvate recovers Giardia trophozoites from oxidative stress by decreasing the number of DNA breaks that might favor DNA repair.

The immunoregulatory role of alpha enolase in dendritic cell function during Chlamydia infection

BackgroundWe have previously reported that interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells that induced elevated protective immunity against Chlamydia. To further investigate the molecular and biochemical mechanism underlying the superior immunostimulatory property of IL-10 deficient DCs we performed proteomic analysis on protein profiles from Chlamydia-pulsed wild-type (WT) and IL-10−/− DCs to identify differentially expressed proteins with immunomodulatory properties.ResultsThe results showed that alpha enolase (ENO1), a metabolic enzyme involved in the last step of glycolysis was significantly upregulated in Chlamydia-pulsed IL-10−/− DCs compared to WT DCs. We further studied the immunoregulatory role of ENO1 in DC function by generating ENO1 knockdown DCs, using lentiviral siRNA technology. We analyzed the effect of the ENO1 knockdown on DC functions after pulsing with Chlamydia. Pyruvate assay, transmission electron microscopy, flow cytometry, confocal microscopy, cytokine, T-cell activation and adoptive transfer assays were also used to study DC function. The results showed that ENO1 knockdown DCs had impaired maturation and activation, with significant decrease in intracellular pyruvate concentration as compared with the Chlamydia-pulsed WT DCs. Adoptive transfer of Chlamydia-pulsed ENO1 knockdown DCs were poorly immunogenic in vitro and in vivo, especially the ability to induce protective immunity against genital chlamydia infection. The marked remodeling of the mitochondrial morphology of Chlamydia-pulsed ENO1 knockdown DCs compared to the Chlamydia-pulsed WT DCs was associated with the dysregulation of translocase of the outer membrane (TOM) 20 and adenine nucleotide translocator (ANT) 1/2/3/4 that regulate mitochondrial permeability. The results suggest that an enhanced glycolysis is required for efficient antigen processing and presentation by DCs to induce a robust immune response.ConclusionsThe upregulation of ENO1 contributes to the superior immunostimulatory function of IL-10 deficient DCs. Our studies indicated that ENO1 deficiency causes the reduced production of pyruvate, which then contributes to a dysfunction in mitochondrial homeostasis that may affect DC survival, maturation and antigen presenting properties. Modulation of ENO1 thus provides a potentially effective strategy to boost DC function and promote immunity against infectious and non-infectious diseases.

Overexpression of the truncated version of ILV2 enhances glycerol production in Saccharomyces cerevisiae.

Acetolactate synthase is a mitochondrial enzyme that catalyses the conversion of two pyruvate molecules to an acetolactate molecule with release of carbon dioxide. The overexpression of the truncated version of the corresponding gene, ILV2, that codes for presumably cytosolic acetolactate synthase in the yeast Saccharomyces cerevisiae, led to a decrease in intracellular pyruvate concentration. This recombinant strain was also characterized by a four-fold increase in glycerol production, with a concomitant 1.8-fold reduction in ethanol production, when compared to that of the wild-type strain under anaerobic conditions in a glucose alcoholic fermentation. Copyright © 2016 John Wiley & Sons, Ltd.

Time-dependent homeostasis between glucose uptake and consumption in astrocytes exposed to CoCl₂ treatment.

Hypoxia has been implicated in the pathology of the central nervous system during stroke. It also has a significant effect on the regulation of glucose transporters (GLUTs), and homeostasis between glucose uptake and consumption. CoCl2 is a hypoxia‑mimetic agent, and thus stabilizes the hypoxia‑inducible factor 1α (HIF‑1α) subunit and regulates GLUT genes. GLUT‑1 and GLUT‑3 are the most common isoforms of the GLUT family present in the brain, with the former primarily expressed in astrocytes and the latter in neurons under physiological conditions. However, it remains controversial whether GLUT‑3 is expressed in astrocytes. Additionally, it is unclear whether the regulation of GLUT‑1 and GLUT‑3, and glucose homeostasis, are affected by CoCl2 treatment in a time‑dependent manner. In the present study, mRNA and protein levels of GLUT‑1, GLUT‑3 and HIF‑1α in astrocytes were examined by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. The intracellular glucose concentration, glycogen storage, ATP content, pyruvate concentration, lactate dehydrogenase (LDH) release activity and cell viability in astrocytes were also investigated. The observations of the current study confirmed that both protein and mRNA levels of GLUT‑1 and GLUT‑3 were elevated in a time‑dependent manner induced by CoCl2 treatment, followed by accumulation of HIF‑1α. Furthermore, in the early period of CoCl2 treatment (≤8 h at 100 µM), LDH release, ATP content, glycogen storage and cell viability remained unchanged, whereas intracellular pyruvate concentration increased and glucose concentration was reduced. However, in the later period of CoCl2 treatment (>8 h at 100 µM), LDH release and intracellular pyruvate concentration increased, while intracellular glucose concentration, ATP content and glycogen storage were reduced. This may be due to disruption of homeostasis and reduced cell viability. In conclusion, alteration in the expression levels of GLUT‑1 and GLUT‑3, and the homeostasis between glucose uptake and consumption were affected by CoCl2 treatment, in a time‑dependent manner, and may result in reduced energy production and cell viability in astrocytes.

Diacetyl Formation by Oenococcus oeni during Winemaking Induced by Exogenous Pyruvate

Pyruvate is the central metabolite in diacetyl synthesis by <i>Oenococcus oeni.</i> Therefore, any substrate that increases intracellular pyruvate concentration can induce diacetyl accumulation. This study evaluates the effect of exogenous pyruvate on diacetyl formation and the expression of diacetyl-related genes in <i>Oenococcus oeni</i> during winemaking. Diacetyl formation by <i>Oenococcus oeni</i> was induced by yeast-derived pyruvate in the early stage of winemaking. Furthermore, when additional pyruvate was added, α-acetolactate synthase (<i>alsS</i>) gene expression increased 1.6-fold and the diacetyl concentration increased from 0.4 mg/L to 2.3 mg/L. Although the highest <i>alsS</i> expression (a 10-fold increase) was found 24 hr after pyruvate addition, no further increase in diacetyl concentration was found. In addition, <i>alsD</i> was overexpressed (9-fold) at that point, indicating that α-acetolactate was converted into acetoin. Together, the results show that exogenous pyruvate induces diacetyl formation by <i>Oenococcus oeni</i> in the early stage of winemaking<i>.</i> Furthermore, pyruvate-derived diacetyl accumulates because of a delayed <i>alsD</i> response that prevents acetoin formation.

Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways

Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of L-lactate (Δldh) and/or acetate (Δpta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Δldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Δhpt Δspo0A). The Δpta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75 % more ethanol. A Δldh Δpta double-mutant strain evolved for faster growth had a growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to Δpta. Free amino acids were secreted by all examined strains, with both Δpta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Δldh Δpta reached 5 mM by the end of growth, or 2.7 % of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to sixfold in the Δpta and 16-fold in the Δldh Δpta strain. We hypothesize that the deletions in fermentation end-product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP⁺ through increased production of amino acids.

PYRUVATE SUPPLEMENTATION ENHANCES VASCULAR ENDOTHELIAL GROWTH FACTOR PRODUCTION BY BONE MARROW-DERIVED MONONUCLEAR CELLS

1) Bone marrow‑derived mononuclear cells (BMMNC include endothelial progenitor cells (EPC which are character‑ ized by their secretion of angiogenic factors such as vascular endothelial growth factor (VEGF to recruit local endo‑ thelial cells thereby enabling the establishment of new blood vessels. Implantation of BMMNC has been clinically used for therapeutic purposes in the treatment of critical limb ischemia (CLI; results showed that it was ineffective in a substantial number of cases. To evaluate the appropriate concentration of pyruvate to achieve the highest VEGF gene expression cells were cultured with pyruvate at final concentrations up to 20 mM in 5% CO2 for 2‑4 days. The intracellular concentration of pyruvate was measured enzymatically and cell number and viability were determined. Expression levels of VEGF genes and numbers of CD31 + CD34 + cells were evaluated. Finally VEGF levels in the con‑ ditioned medium were examined in each condition. Pyruvate supplementation in murine BMMNC cultures success‑ fully increased intracellular pyruvate levels in a concentration‑dependent manner and 5 mM pyruvate was found to be the most appropriate to maintain viable cell number and up‑regulate VEGF gene after 2‑day culture. In addition VEGF in the conditioned medium was significantly elevated by the use of 5 mM pyruvate after 4‑day culture. From these results we suggest that preconditioning of BMMNC with 5 mM pyruvate for 2 days may be a useful way to safely and inexpensively enhance the angiogenic properties of BMMNC and the therapeutic effectiveness of cellular therapy for CLI.

Influence of lactose and lactate on growth and β-galactosidase activity of potential probiotic Propionibacterium acidipropionici

Dairy propionibacteria are microorganisms of interest for their role as starters in cheese technology and as well as their functions as probiotics. Previous studies have demonstrated that Propionibacterium acidipropionici metabolize lactose by a β-galactosidase that resists the gastrointestinal transit and the manufacture of a Swiss-type cheese, so that could be considered for their inclusion in a probiotic product assigned to intolerant individuals. In the present work we studied the effect of the sequential addition of lactose and lactate as first or second energy sources on the growth and β-galactosidase activity of P. acidipropionici Q4. The highest β-galactosidase activity was observed in a medium containing only lactate whereas higher final biomass was obtained in a medium with lactose. When lactate was used by this strain as a second energy source, a marked increase of the intracellular pyruvate level was observed, followed by lactate consumption and increase of specific β-galactosidase activity whereas lactose consumption became negligible. On the contrary, when lactose was provided as second energy source, lactic acid stopped to be metabolized, a decrease of the intracellular pyruvate concentration was observed and β-galactosidase activity sharply returned to a value that resembled the observed during the growth on lactose alone. Results suggest that the relative concentration of each substrate in the culture medium and the intracellular pyruvate level were decisive for both the choice of the energetic substrate and the β-galactosidase activity in propionibacteria. This information should be useful to decide the most appropriate vehicle to deliver propionibacteria to the host in order to obtain the highest β-galactosidase activity.

Leukocyte pyruvate kinase expression is reduced in normal human pregnancy but not in pre-eclampsia.

Emerging evidence suggests that metabolism influences immune cell signaling and immunoregulation. To examine the immunoregulatory role of glycolysis in pregnancy, we evaluated the properties of pyruvate kinase in leukocytes from non-pregnant women and those with normal pregnancy and pre-eclampsia. We evaluated pyruvate kinase expression in lymphocytes and neutrophils from non-pregnant, pregnant, and pre-eclampsia patients using fluorescence microscopy and flow cytometry. Leukocyte pyruvate kinase activity and pyruvate concentrations were also evaluated. To study pyruvate's effect on signaling, we labeled Jurkat T cells with Ca(2+) dyes and measured cell responses in the presence of agents influencing intracellular pyruvate. The expression of pyruvate kinase is reduced in lymphocytes and neutrophils from normal pregnant women in comparison with those of non-pregnant women and pre-eclampsia patients. Similarly, the activity of pyruvate kinase and the intracellular pyruvate concentration are reduced in leukocytes of normal pregnant women in comparison with non-pregnant women and women with pre-eclampsia. Using Jurkat cells as a model of leukocyte signaling, we have shown that perturbations of intracellular pyruvate influence Ca(2+) signals. Normal pregnancy is characterized by reduced pyruvate kinase expression within lymphocytes and neutrophils. We speculate that reduced pyruvate kinase expression modifies immune cell responses due to reduced pyruvate concentrations.