Oxaloacetate Research Articles

Development of a Fatty Liver Model by Restricted Feeding of Lactating Sheep

Background: As a frequent subclinical disease, fatty liver disease (FLD) is associated with a severe negative energy balance (NEB) during the early lactation period, and usually cause of economic loss to dairy farmers. Liver biopsy is the gold standard for the assessment of FLD. However, as an invasive procedure, liver biopsy has several limitations and such procedures are not readily available to dairy farmers. To further evaluate FLD in dairy cows, a FLD model of lactating sheep was developed by simulation of the state of negative energy balance (NEB).Materials, Methods & Results: Fourteen pregnancy thin-tail ewes were divided into control group (CG, n = 4), non-lamb restrained feeding group (NRG, n = 4) and single birth restrained feeding group (SRG, n = 6). After lambing, NRG and SRG ewe were received a feed restrained diet for 16 days. Liver biopsies and blood was collected on days 1, 4, 7, 10, 13, and 16, and biochemical parameters were analyzed. With restricted feeding and lactation administration, ewes in SRG showed increased liver fat concentrations (LFC) from days 4 post-administration and severe LFC was detected at day 13. Compared with CG, SRG sheep showed significant lower concentration of serum glucose (Glu) from days 7-13 and higher non-esterified fatty acid (NEFA) from days 4-16, β-hydroxybutyric acid (BHBA) from days 4-16, triglyceride from days 4-16, low-density lipoprotein cholesterol from days 4-16, lactate dehydrogenase (LDH) from days 13-16, aspartate aminotransferase (AST) at days 16. While, ewes in NRG showed normal LFC levels, and high concentration of serum Glu and insulin from days 4-16 were detected than CG and SRG ewes. With restricted feeding, ewes in NRG and SRG showed significant low level of revised quantitative insulin sensitivity check index from days 4-16 and high level of liver total cholesterol (TC) at day 16. Liver pathological characteristics showed LFC of NEB sheep was first detected around the liver portal area.Discussion: In this study, a model of FLD in lactating thin-tail sheep was developed by restricted feeding. Serum glucose concentrations were sharply decreased in SRG sheep，that was due to the large energy requirements for lactation and low energy provided by a restricted diet. While non-lactating NRG sheep demonstrated lower fat mobilization, which was considered to contribute to the high concentrations of serum glucose, as compared to SRG sheep. Meanwhile, in a state of NEB, oxaloacetic acid, which is generated by glycolysis and glycogenic amino acids, tends to be used for gluconeogenesis, that a generous amount of NEFA is incompletely oxidized to generate ketone body in SRG sheep, which is a major component of BHBA. Liver TC concentrations were significantly higher in NRG sheep than those in the SRG sheep, while liver triglyceride was significantly lower. The high level of liver TC in NRG sheep was considered to induce removal of triglyceride from the liver in the form of VLDL. Compared with CG sheep, although higher levels of liver TC were detected in SRG sheep on postpartum day 16, these levels were considered too low to induce significant depletion of triglycerides from the liver. In this study, the increase in serum AST and LDH was considered to cause by oxidative stress in mitochondria, and LDH concentrations was considered more sensitively than AST for LFC caused by NEB. Liver pathological characteristics showed that FLD caused by NEB had a major impact on reduced LFC, although no significant liver fibrosis was detected. While different from FLD caused by high-fat diet, TG was first accumulates around the hepatic lobules and LFC of NEB sheep was first detected around the liver portal area. It was considered that high concentrations of NEFA are prioritized for oxygenation in the liver portal area, which results in triglyceride accumulation.

Oxaloacetate Ameliorates Chemical Liver Injury via Oxidative Stress Reduction and Enhancement of Bioenergetic Fluxes.

Chemical injury is partly due to free radical lipid peroxidation, which can induce oxidative stress and produce a large number of reactive oxygen species (ROS). Oxaloacetic acid is an important intermediary in the tricarboxylic acid cycle (TCA cycle) and participates in metabolism and energy production. In our study, we found that oxaloacetate (OA) effectively alleviated liver injury which was induced by hydrogen peroxide (H2O2) in vitro and carbon tetrachloride (CCl4) in vivo. OA scavenged ROS, prevented oxidative damage and maintained the normal structure of mitochondria. We further confirmed that OA increased adenosine triphosphate (ATP) by promoting the TCA production cycle and oxidative phosphorylation (OXPHOS). Finally, OA inhibited the mitogen-activated protein kinase (MAPK) and apoptotic pathways by suppressing tumor necrosis factor-α (TNF-α). Our findings reveal a mechanism for OA ameliorating chemical liver injury and suggest a possible implementation for preventing the chemical liver injury.

Photo-Oxidation of Bisphenol A in Aqueous Solutions at Near Neutral pH by a Fe(III)-Carboxylate Complex with Oxalacetic Acid as a Benign Molecule.

The photo-oxidation of organic pollutants as induced by ferric-carboxylate complexes was known to be a photo-Fenton-like process. The use of a carboxylate ligand with higher efficiency and lower toxicity at near neutral pH is of high interest to researchers. In this work, photo-oxidation of bisphenol A (BPA) induced by a ferric-oxalacetic acid complex in aqueous solutions was investigated under 395 nm LED lamps. The results showed that the rate of BPA degradation increased in the order pH 10.0 << 8.0 < 6.5 < 4.0 within the first 10 min. More than 90% of BPA was successfully oxidized with Fe(III)/oxalacetic acid with a ratio of 1:5 at pH 6.5, which was primarily attributed to the generated hydroxyl radical. Iron in the Fe(III)-oxalacetic acid system was reused by simple addition of oxalacetic acid to the reaction mixture. Compared to common carboxylate ligands (pyruvic acid, oxalic acid, and citric acid), oxalacetic acid is more efficient and environmentally friendly for the Fe(III)-carboxylate complex-based photo-Fenton-like process at near neutral pH.

Real-Time Monitoring of Tricarboxylic Acid Metabolites in Exhaled Breath.

The tricarboxylic acid (TCA) cycle is one of the most important metabolic pathway for cellular respiration in aerobic organisms. It provides and collects intermediates for many other interconnecting pathways and acts as a hub connecting metabolism of carbohydrates, fatty acids, and amino acids. Alteration in intracellular levels of its intermediates has been linked with a wide range of illnesses ranging from cancer to cellular necrosis or liver cirrhosis. Therefore, there exists an intrinsic interest in monitoring such metabolites. Our goal in this study was to evaluate whether, at least the most volatile metabolites of the TCA cycle, could be detected in breath in vivo and in real time. We used secondary electrospray ionization coupled with high-resolution mass spectrometry (SESI-HRMS) to conduct this targeted analysis. We enrolled six healthy individuals who provided full exhalations into the SESI-HRMS system at different times during 3 days. For the first time, we observed exhaled compounds that appertain to the TCA cycle: fumaric, succinic, malic, keto-glutaric, oxaloacetic, and aconitic acids. We found high intraindividual variability and a significant overall difference between morning and afternoon levels for malic acid, oxaloacetic acid, and aconitic acid, supporting previous studies suggesting circadian fluctuations of these metabolites in humans. This study provides first evidence that TCA cycle could conveniently be monitored in breath, opening new opportunities to study in vivo this important metabolic pathway.

A high-throughput screening assay for pyruvate carboxylase

Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potent small molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC.

Discovery of Novel Small Molecule Inhibitors of Bacterial Pyruvate Carboxylase

Staphylococcus aureus and Listeria monocytogenes are pathogenic bacteria that can cause serious infections for immunocompromised individuals1, 2. Bacterial metabolism has been linked to pathogenicity and, in particular, the metabolic enzyme pyruvate carboxylase (PC) has been shown to be an important contributor to L. monocytogenes virulence1, 2. Small molecules that could inhibit bacterial PC activity would better reveal the role of this enzyme in a wide range of bacterial infections and could have the potential to be developed into antimicrobial agents. However, there are currently no known specific or potent small molecule inhibitors of PC activity3. We aim to find, develop, and characterize novel small molecule inhibitors of PC that will aid in understanding the structure, mechanism, and regulation of this enzyme. PC catalyzes the conversion of pyruvate to oxaloacetate (OAA) and a novel fixed‐time assay has been developed to detect OAA with a diazonium salt, Fast Violet B (FVB). The validated assay has been used to screen specific subsets of rationally designed inhibitors as well as compounds in an unbiased high‐throughput screen. Using both approaches, we have discovered novel small molecule inhibitors of bacterial PC that have the potential to be developed into valuable research tools and future antimicrobial agents.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A High‐Throughput Assay to Measure Phosphoenolpyruvate Carboxykinase

Phosphoenolpyruvate Carboxykinase (PEPCK EC 4.1.1.32) catalyzes the conversion of oxaloacetate (OAA) into phosphoenolpyruvate (PEP) in the presence of GTP. In humans, two isoforms of PEPCK are found: cytosolic form (PEPCK‐C, also called PCK1) and mitochondrial form (PEPCK‐M, also called PCK2). PEPCK‐C (PCK1) is a rate‐controlling enzyme in gluconeogenesis. Recent studies found abnormal concentrations of PEPCK in diabetic mice. Overexpression of PEPCK attenuates insulin signaling and decreases hepatic insulin sensitivity in transgenic mice. Our high throughput assay for Phosphoenolpyruvate Carboxykinase activity provides a valuable tool for both mechanistic and therapeutic studies of diabetes as well as avenue for targeting PEPCK activity.The Phosphoenolpyruvate Carboxykinase activity assay is developed by using a set of specific enzymes that convert Phosphoenolpyruvate (PEP) and carbonate into a series of intermediates and hydrogen peroxide, which in turn, reacts with a probe generating a colorimetric signal (OD570 nm). The color intensity is directly proportional to the amount of active Phosphoenolpyruvate Carboxykinase. This assay is simple, sensitive, high‐throughput adaptable and can detect less than 10 μU of Phosphoenolpyruvate Carboxykinase activity per sample.In conclusion, a Phosphoenolpyruvate Carboxykinase (PEPCK) Activity Assay kit is developed. The kit provides easy and robust tool for measuring PEPCK activity in biological and purified samples (BioVision, Inc. Cat. # K359‐100).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA interference of GhPEPC2 enhanced seed oil accumulation and salt tolerance in Upland cotton

Phosphoenolpyruvate carboxylase (PEPCase) mainly produces oxaloacetic acid for tricarboxylic acid (TCA) cycle. Here we reported that GhPEPC2 silencing with PEPC2-RNAi vector could regulate oil and protein accumulation in cottonseeds. In GhPEPC2 transgenic plants, PEPCase activities in immature embryos were significantly reduced, and the oil content in seed kernel was increased 7.3 percentages, whereas total proteins decreased 5.65 percentages. Compared to wild type, agronomical traits of transgenic plant were obviously unaffected. Furthermore, gene expression profile of GhPEPC2 transgenic seeds were investigated using RNA-seq, most lipid synthesis related genes were up-regulated, but amino acid metabolic related genes were down-regulated. In addition, the GhPEPC2 transgenic cotton seedlings were stressed using sodium salts at seedling stage, and the salt tolerance was significantly enhanced. Our observations of GhPEPC2 in cotton would shade light on understanding the regulation of oil content, protein accumulation and salt tolerance enhancement in other plants.

Oxaloacetate induces apoptosis in HepG2 cells via inhibition of glycolysis.

Most cancer cells perform glycolysis despite having sufficient oxygen. The specific metabolic pathways of cancer cells have become the focus of cancer treatment. Recently, accumulating evidence indicates oxidative phosphorylation (OXPHOS) and glycolysis can be regulated with each other. Thus, we suggest that the glycolysis of cancer cells is inhibited by restoring or improving OXPHOS in cancer cells. In our study, we found that oxaloacetate (OA) induced apoptosis in HepG2 cells in vivo and in vitro. Meanwhile, we found that OA induced a decrease in the energy metabolism of HepG2 cells. Further results showed that the expression and activity of glycolytic enzymes were decreased with OA treatment. Conversely, the expression and activity of enzymes involved in the TCA cycle and OXPHOS were increased with OA treatment. The results indicate that OA can inhibit glycolysis through enhancement of OXPHOS. In addition, OA‐mediated suppression of HIF1α, p‐Akt, and c‐myc led to a decrease in glycolysis level. Therefore, OA has the potential to be a novel anticancer drug.

Effect of Anaplerotic Pathways Activation on CO2-dependent Anaerobic Glucose Utilization by Escherichia coli Strains Deficient in the Main Pathways of Mixed Acid Fermentation

The effect of anaplerotic pathways activation on CO2-dependent anaerobic glucose utilization by Escherichia coli strains deficient in the main fermentation pathways and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO2 generation in the strains was ensured resulting from oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Sodium bicarbonate dissolved in the medium was used as an external source of CO2. The genes of heterologous pyruvate carboxylase and native NADH-dependent malic enzyme were overexpressed in the strains to allow anaplerotic carboxylation of pyruvic acid to oxaloacetic or malic acid. The ability of the strains to reoxidize NADH utilizing carboxylation products was additionally increased due to enhanced expression of malate dehydrogenase gene. In the case of endogenous CO2 formation, the activation of anaplerotic pathways did not cause a notable increase in the anaerobic glucose consumption by the constructed strains. At the same time, the expression of pyruvate carboxylase led to a pronounced decrease in the secretion of pyruvic acid with the concomitant increase in the yield of four-carbon metabolites. Further enhancement of NADH-dependent malic enzyme expression provoked activation of a pyruvate–oxaloacetate–malate–pyruvate futile cycle in the strains. The availability in the medium of the external CO2 source sharply increased the anaerobic utilization of glucose by strains expressing pyruvate carboxylase. The activity of the futile cycle has raised with the increased malic enzyme expression and dropped upon enhancement of malate dehydrogenase expression. As a result, the efficiency of CO2-dependent anaerobic glucose utilization coupled to the formation of four-carbon carboxylation products increased in the studied strains resulting from the primary anaplerotic conversion of pyruvic acid into oxaloacetic acid followed by the involvement of the precursor formed in NADH-consuming biosynthetic reactions dominating over the reactions of the revealed futile cycle.

Seasonal variations of low molecular weight hydroxy-dicarboxylic acids and oxaloacetic acid in remote marine aerosols from Chichijima Island in the western North Pacific (December 2010–November 2011)

Concentrations of homologous hydroxy-dicarboxylic acids (diacids) (hC3-hC6) and keto-diacid (oxaloacetic acid) were measured in the atmospheric aerosols collected at Chichijima Island (27.04° N, 142.13° E) in the western North Pacific from December 2010 to November 2011. The monthly averaged concentrations of hydroxy-diacids and oxaloacetic acid were significantly higher in spring followed by winter and autumn. Molecular distributions of hydroxy-diacids demonstrated that malic acid was the most abundant species in all four seasons, followed by tartronic acid in winter and spring and 3- and 2-hydroxyglutaric acids in summer and autumn. Hydroxy-diacids and keto-diacid maximized in spring and winter when air masses originated from the Asian continent with westerly winds. The concentrations of total hydroxy-diacids and oxaloacetic acid ranged from 0.1 to 27.3 ng m−3 and <0.005 to 2 ng m−3, respectively. The enhanced concentrations of diacids and their intermediates in winter and spring are associated with a long-range atmospheric transport of pollutants from East Asia to remote Chichijima Island followed by photochemical processing of organic aerosols. Seasonal molecular distribution of hydroxy-diacids and oxaloacetic acid was found to be dependent on the source strengths and plausible photochemical processing to form smaller diacids. Moderate to strong correlations among hydroxy-diacids, oxaloacetic acid and low molecular weight (LMW) diacids suggest that hydroxy-diacids and oxaloacetic acid are the intermediates in the photochemical oxidation of LMW diacid. Hence, photochemical formation of the most abundant LMW diacids, i.e., oxalic acid, could be produced from hydroxy- and keto-diacid as intermediates.

Integrated Analysis of the Transcriptome and Metabolome of Corynebacterium glutamicum during Penicillin-Induced Glutamic Acid Production.

Corynebacterium glutamicum is known for its ability to produce glutamic acid and has been utilized for the fermentative production of various amino acids. Glutamic acid production in C. glutamicum is induced by penicillin. In this study, the transcriptome and metabolome of C. glutamicum is analyzed to understand the mechanism of penicillin-induced glutamic acid production. Transcriptomic analysis with DNA microarray revealed that expression of some glycolysis- and TCA cycle-related genes, which include those encoding the enzymes involved in conversion of glucose to 2-oxoglutaric acid, is upregulated after penicillin addition. Meanwhile, expression of some TCA cycle-related genes, encoding the enzymes for conversion of 2-oxoglutaric acid to oxaloacetic acid, and the anaplerotic reactions decreased. In addition, expression of NCgl1221 and odhI, encoding proteins involved in glutamic acid excretion and inhibition of the 2-oxoglutarate dehydrogenase, respectively, is upregulated. Functional category enrichment analysis of genes upregulated and downregulated after penicillin addition revealed that genes for signal transduction systems are enriched among upregulated genes, whereas those for energy production and carbohydrate and amino acid metabolisms are enriched among the downregulated genes. As for the metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry, the intracellular content of most metabolites of the glycolysis and the TCA cycle decreased dramatically after penicillin addition. Overall, these results indicate that the cellular metabolism and glutamic acid excretion are mainly optimized at the transcription level during penicillin-induced glutamic acid production by C. glutamicum.

Analysis of the Mycoplasma bovis lactate dehydrogenase reveals typical enzymatic activity despite the presence of an atypical catalytic site motif.

The lactate dehydrogenase (LDH) of Mycoplasma genitalium has been predicted to also act as a malate dehydrogenase (MDH), but there has been no experimental validation of this hypothesized dual function for any mollicute. Our analysis of the metabolite profile of Mycoplasma bovis using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) detected malate, suggesting that there may be MDH activity in M. bovis. To investigate whether the putative l-LDH enzyme of M. bovis has a dual function (MDH and LDH), we performed bioinformatic and functional biochemical analyses. Although the amino acid sequence and predicted structural analysis of M. bovisl-LDH revealed unusual residues within the catalytic site, suggesting that it may have the flexibility to possess a dual function, our biochemical studies using recombinant M. bovis -LDH did not detect any MDH activity. However, we did show that the enzyme has typical LDH activity that could be inhibited by both MDH substrates oxaloacetate (OAA) and malate, suggesting that these substrates may be able to bind to M. bovis LDH. Inhibition of the conversion of pyruvate to lactate by OAA may be one method the mycoplasma cell uses to reduce the potential for accumulation of intracellular lactate.

Removal of Feedback Inhibition of Corynebacterium glutamicum Phosphoenolpyruvate Carboxylase by Addition of a Short Terminal Peptide

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the carboxylation of phosphoenolpyruvate (PEP) in the presence of bicarbonate to form oxaloacetate (OAA), and it plays an important role in high-efficient production of OAA-derived metabolites such as lysine, glutamate and succinate. However, PEPCs often suffered from serious feedback inhibition by various metabolic effectors like aspartate. Here, the feedback inhibition of PEPC from Corynebacterium glutamicum was removed by adding a short terminal peptide like His-tag. The effect of His-tag location on the structure and important properties such as activity, thermostability and feedback inhibition of PEPC has been investigated. The purified untagged PEPC, Nterminal His-tagged PEPC (PEPC-N-His) and C-terminal His-tagged PEPC (PEPC-C-His) were characterized. PEPCN- His (439.71/sec/mM) showed a 1.26 and 186-fold higher catalytic efficiency than untagged PEPC (348.59/sec/mM) and PEPC-C-His (2.36/sec/mM), respectively. Both PEPCN- His and untagged PEPC were significantly inhibited by aspartate at the concentrations above 4 mM (residual activities < 10%), while PEPC-C-His was almost desensitized to aspartate within 10 mM (around 90% of residual activity). Structural analysis showed that the extension of C-terminus may cause steric hindrance for aspartate binding with enzymes, leading to the deregulation of feedback inhibition of PEPC-C-His. This study provides a deeper understanding of the effect of terminal fragments on the structure and function of PEPCs, and helps to engineer the feedback inhibition of PEPCs and structurally similar enzymes.

Urinary Excretion of 2-Oxo Acids Is Greater in Rats with Streptozotocin-Induced Diabetes.

2-Oxo acids derived from amino acids, glucose, and fatty acids are key intermediates in energy production. During diabetes, energy production is known to be lower than in healthy individuals. However, it was unknown whether the production of 2-oxo acids is impacted by diabetes. In the present study, I compared the quantities of 2-oxo acids (pyruvic acid, oxaloacetic acid, 2-oxoglutaric acid, 2-oxoadipic acid, 2-oxoisovaleric acid, 2-oxo-3-methylvaleric acid, and 2-oxo-4-methylvaleric acid) excreted in the urine of normoglycemic control rats and rats with streptozotocin-induced diabetes, which reflect the quantities of unused 2-oxo acids in the body. Greater urinary excretion of unused 2-oxo acids thus implies an impairment in energy production. The respective quantities of urinary pyruvic acid + oxaloacetic acid (measured together), 2-oxoglutaric acid, 2-oxoadipic acid, 2-oxoisovaleric acid, 2-oxo-3-methylvaleric acid, and 2-oxo-4-methylvaleric acid in the diabetic rats were 2.0- (p<0.0001), 2.5- (p<0.0001), 1.5- (p=0.008), 7.6- (p<0.0001), 6.1- (p<0.0001), and 2.1-fold (p<0.0001) greater than in the control rats per 1 g food intake. Thus, the biggest differences were observed in 2-oxoisovaleric acid (a catabolite of valine) and 2-oxo-3-methylvaleric acid (a catabolite of isoleucine). These findings indicate that energy production in the body is suppressed under diabetic conditions.

Immunogenicity of Low Dose Recombinant DNA Hepatitis B Vaccine in Children Ten Years Old and Younger

To evaluate the usefulness of low dose hepatitis-B vaccine, we recruited 70 healthy children of both sexes aged ten years or less. To be included in the study a child must have been clinically healthy and gave negative results for hepatitis-B virus infection (HBsAg, anti-HBs, anti-HBc), and with seerum glutamic oxaloacetic acid transaminase less than 50 mU/1. Subjects with intercurrent infection and those with chronic disease or moderate to severe malnutrition were excluded from the study. They were given 3 injections of 2,5 ml rDNA hepatitis B vaccine intramuscularly on month 0, month 1, and month 6. The seroconversion rate after the third injection in all subjects was 92.3%; this was slightly different between the well-nourished and under-nourished children (93.9% vs. 87,5%), and between girls and boys (96.7% vs 88.6%). The only side effect noted was fever encountered in 1 subject. We conclude that three intramuscular injections of 2.5 mg of rDNA hepatitis B vaccine give good anti-HBs titer, so that this method may be considered to be used in the mass immunization program.

Biouptake, toxicity and biotransformation of triclosan in diatom Cyclotella sp. and the influence of humic acid.

Triclosan is one of the most frequently detected emerging contaminants in aquatic environment. In this study, we investigated the biouptake, toxicity and biotransformation of triclosan in freshwater algae Cyclotella sp. The influence of humic acid, as a representative of dissolved organic matter, was also explored. Results from this study showed that triclosan was toxic to Cyclotella sp. with 72h EC50 of 324.9μgL-1. Humic acid significantly reduced the toxicity and accumulation of triclosan in Cyclotella sp. SEM analysis showed that Cyclotella sp. were enormously damaged under 1mgL-1 triclosan exposure and repaired after the addition of 20mgL-1 humic acid. Triclosan can be significantly taken up by Cyclotella sp. The toxicity of triclosan is related to bioaccumulated triclosan as the algal cell numbers decreased when intracellular triclosan increased. A total of 11 metabolites were identified in diatom cells and degradation pathways are proposed. Hydroxylation, methylation, dechlorination, amino acids conjunction and glucuronidation contributed to the transformative reactions of triclosan in Cyclotella sp., producing biologically active products (e.g., methyl triclosan) and conjugation products (e.g., glucuronide or oxaloacetic acid conjugated triclosan), which may be included in the detoxification mechanism of triclosan.

Brownian dynamic study of an enzyme metabolon in the TCA cycle: Substrate kinetics and channeling.

Malate dehydrogenase (MDH) and citrate synthase (CS) are two pacemaking enzymes involved in the tricarboxylic acid (TCA) cycle. Oxaloacetate (OAA) molecules are the intermediate substrates that are transferred from the MDH to CS to carry out sequential catalysis. It is known that, to achieve a high flux of intermediate transport and reduce the probability of substrate leaking, a MDH-CS metabolon forms to enhance the OAA substrate channeling. In this study, we aim to understand the OAA channeling within possible MDH-CS metabolons that have different structural orientations in their complexes. Three MDH-CS metabolons from native bovine, wild-type porcine, and recombinant sources, published in recent work, were selected to calculate OAA transfer efficiency by Brownian dynamics (BD) simulations and to study, through electrostatic potential calculations, a possible role of charges that drive the substrate channeling. Our results show that an electrostatic channel is formed in the metabolons of native bovine and recombinant porcine enzymes, which guides the oppositely charged OAA molecules passing through the channel and enhances the transfer efficiency. However, the channeling probability in a suggested wild-type porcine metabolon conformation is reduced due to an extended diffusion length between the MDH and CS active sites, implying that the corresponding arrangements of MDH and CS result in the decrease of electrostatic steering between substrates and protein surface and then reduce the substrate transfer efficiency from one active site to another.

Integration between Glycolysis and Glutamate-Glutamine Cycle Flux May Explain Preferential Glycolytic Increase during Brain Activation, Requiring Glutamate.

The 1988 observation by Fox et al. (1988) that brief intense brain activation increases glycolysis (pyruvate formation from glucose) much more than oxidative metabolism has been abundantly confirmed. Specifically glycolytic increase was unexpected because the amount of ATP it generates is much smaller than that formed by subsequent oxidative metabolism of pyruvate. The present article shows that preferential glycolysis can be explained by metabolic processes associated with activation of the glutamate-glutamine cycle. The flux in this cycle, which is essential for production of transmitter glutamate and GABA, equals 75% of brain glucose utilization and each turn is associated with utilization of ~1 glucose molecule. About one half of the association between cycle flux and glucose metabolism occurs during neuronal conversion of glutamine to glutamate in a process similar to the malate-aspartate shuttle (MAS) except that glutamate is supplied from glutamine, not formed from α-ketoglutarate (αKG) as during operation of conventional MAS. Regular MAS function is triggered by one oxidative process in the cytosol during glycolysis causing NAD+ reduction to NADH. Since NADH cannot cross the mitochondrial membrane (MEM) for oxidation NAD+ is re-generated by conversion of cytosolic oxaloacetate (OAA) to malate, which enters the mitochondria for oxidation and in a cyclic process regenerates cytosolic OAA. Therefore MAS as well as the “pseudo-MAS” necessary for neuronal glutamate formation can only operate together with cytosolic reduction of NAD+ to NADH. The major process causing NAD+ reduction is glycolysis which therefore also must occur during neuronal conversion of glutamine to glutamate and may energize vesicular glutamate uptake which preferentially uses glycolytically derived energy. Another major contributor to the association between glutamate-glutamine cycle and glucose utilization is the need for astrocytic pyruvate to generate glutamate. Although some oxidative metabolism occurs during glutamate formation it is only one half of that during normal tricarboxylic acid (TCA) cycle function. Glutamate’s receptor stimulation leads to potassium ion (K+) release and astrocytic uptake, preferentially fueled by glycolysis and followed by release and neuronal re-accumulation. The activation-induced preferential glycolysis diminishes with continued activation and is followed by an increased ratio between oxidative metabolism and glycolysis, reflecting oxidation of generated glutamate and accumulated lactate.

Enhanced biomass production by Phaeodactylum tricornutum overexpressing phosphoenolpyruvate carboxylase

Phaeodactylum tricornutum is an extensively studied model diatom and a promising candidate for biochemical and biotechnological engineering aimed at increasing biomass production. Phosphoenolpyruvate carboxylase (PEPCase), which converts phosphoenolpyruvate (PEP) to oxaloacetate (OAA) using bicarbonate, is a key metabolic enzyme of the tricarboxylic acid (TCA) cycle and the C4 photosynthetic pathway. Two transgenic P. tricornutum lines overexpressing PtPEPCase1 (PtPEPC1, JGI protein ID: 27976) were constructed and their photosynthetic productivity, cell growth, and biomass were characterized. The levels of PtPEPC1 mRNAs in the two transgenic lines were increased by 2.3 and 11.2-fold and the amounts of the PEPCase protein by 1.3 and 2.3-fold, respectively. PtPEPC1 was targeted to mitochondria. Addition of bicarbonate to the P. tricornutum culture increased biomass of the transformants by about 12% compared to wild type, and their maximum specific growth rate in exponential phase was about 10% greater than that of wild type. The transformants also exhibited higher photosynthetic productivity. We conclude that overexpression of mitochondrial PtPEPC1 enhanced both photosynthetic productivity and TCA cycle activity in P. tricornutum, thereby enhancing biomass production in the presence of dissolved inorganic carbon. Our findings suggest that P. tricornutum overexpressing PtPEPC1 can be used to mitigate rising atmospheric CO2 levels or for sustainable microalgal biomass production.