Targeted metabolomics combined with network pharmacology to reveal the protective role of luteolin in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease that significantly endangers human health, where metabolism may drive pathogenesis: a shift from mitochondrial oxidation to glycolysis occurs in diseased pulmonary vessels and the right ventricle. An increase in pulmonary vascular resistance in patients with heart failure with a preserved ejection fraction portends a poor prognosis. Luteolin exists in numerous foods and is marketed as a dietary supplement assisting in many disease treatments. However, little is known about the protective effect of luteolin on metabolism disorders in diseased pulmonary vessels. In this study, we found that luteolin apparently reversed the pulmonary vascular remodeling of PAH rats by inhibiting the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). Moreover, network pharmacology and metabolomics results revealed that the arachidonic acid pathway, amino acid pathway and TCA cycle were dysregulated in PAH. A total of 14 differential metabolites were significantly changed during the PAH, including DHA, PGE2, PGD2, LTB4, 12-HETE, 15-HETE, PGF2α, and 8-iso-PGF2α metabolites in the arachidonic acid pathway, and L-asparagine, oxaloacetate, N-acetyl-L-ornithine, butane diacid, ornithine, glutamic acid metabolites in amino acid and TCA pathways. However, treatment with luteolin recovered the LTB4, PGE2, PGD2, 12-HETE, 15-HETE, PGF2α and 8-iso-PGF2α levels close to normal. Meanwhile, we showed that luteolin also downregulated the gene and protein levels of COX 1, 5-LOX, 12-LOX, and 15-LOX in the arachidonic acid pathway. Collectively, this work highlighted the metabolic mechanism of luteolin-protected PAH and showed that luteolin would hold great potential in PAH prevention.

Long noncoding RNA OVAAL enhances nucleotide synthesis through pyruvate carboxylase to promote 5-fluorouracil resistance in gastric cancer.

5-Fluorouracil (5-FU) is widely used in gastric cancer treatment, yet 5-FU resistance remains an important clinical challenge. We established a model based on five long noncoding RNAs (lncRNA) to effectively assess the prognosis of gastric cancer patients; among them, lncRNA OVAAL was markedly upregulated in gastric cancer and associated with poor prognosis and 5-FU resistance. In vitro and in vivo assays confirmed that OVAAL promoted proliferation and 5-FU resistance of gastric cancer cells. Mechanistically, OVAAL bound with pyruvate carboxylase (PC) and stabilized PC from HSC70/CHIP-mediated ubiquitination and degradation. OVAAL knockdown reduced intracellular levels of oxaloacetate and aspartate, and the subsequent pyrimidine synthesis, which could be rescued by PC overexpression. Moreover, OVAAL knockdown increased sensitivity to 5-FU treatment, which could be reversed by PC overexpression or repletion of oxaloacetate, aspartate, or uridine. OVAAL overexpression enhanced pyrimidine synthesis to promote proliferation and 5-FU resistance of gastric cancer cells, which could be abolished by PC knockdown. Thus, OVAAL promoted gastric cancer cell proliferation and induced 5-FU resistance by enhancing pyrimidine biosynthesis to antagonize 5-FU induced thymidylate synthase dysfunction. Targeting OVAAL-mediated nucleotide metabolic reprograming would be a promising strategy to overcome chemoresistance in gastric cancer.

Oxaliplatin-Fluoropyrimidine Combination (XELOX) Therapy Does Not Affect Plasma Amino Acid Levels and Plasma Markers of Oxidative Stress in Colorectal Cancer Surgery Patients: A Pilot Study.

Chemotherapy for colorectal cancer may lower muscle protein synthesis and increase oxidative stress. We hypothesize that chemotherapy may worsen plasma amino acids (AAs) and markers of oxidative stress (MOS). Therefore, this study aimed to document plasma AAs and MOS before, during and after chemotherapy in colorectal cancer (CRC) surgery patients. Fourteen normal-weight CRC patients were enrolled one month after surgery and scheduled for oxaliplatin-fluoropyrimidine combination (XELOX) therapy. Venous blood samples for AA and MOS (malondialdehyde, MDA; 8-hydroxy-2'-deoxyguanosine, 8-OHdG) measurements were drawn in fasting patients before each oxaliplatin infusion at initiation (A), 1 month (B) and 3 months (C) of the therapy, and after XELOX had finished (6 months, D). The results showed that during XELOX therapy (from phase B to phase D), in comparison to baseline (phase A), the branched chain amino acid/essential amino acid ratio, branched chain amino acids expressed as a percentage of total AAs, and arginine expressed as a percentage of total AAs significantly decreased (p = 0.017, p = 0.028, p = 0.028, respectively). Plasma levels of MOS did not change significantly. This study indicates that XELOX therapy does not affect plasma AA levels or worsen oxidative stress.

Purification and kinetic characteristics of strombine dehydrogenase from the foot muscle of the hard clam (Meretrix lusoria).

Strombine dehydrogenase (SDH, EC 1.5.1.22) from the foot of the hard clam Meretrix lusoria was purified over 470-fold to apparent homogeneity. It has a monomeric structure with a relative molecular mass of 46,000. Two isoenzymes were identified with isoelectric points of 6.83 and 6.88. SDH is heat labile, and has pH and temperature optima of 7.4-7.6 and 45-46°C, respectively. l-Alanine, glycine, and pyruvate are the preferred substrates. l-Serine is the third preferred amino acid. Iminodiacetate with the lowest K(i) of SDH at both pH 6.5 and 7.5 was the strongest inhibitor among succinate, acetate, iminodiacetate, oxaloacetate, and l-/d-lactate. The inhibitory activities of succinate at pH 6.5, and iminodiacetate and oxaloacetate at pH 7.5 on the SDH were higher. These inhibitors are either competitive or mixed-competitive inhibitors. Half of the enzymatic activity of SDH was inhibited by 0.2mM Fe(3+) and 0.6mM Zn(2+).

Use of some novel alternative electron sinks to inhibit ruminal methanogenesis.

Several compounds were evaluated in vitro as alternative electron sinks to ruminal methanogenesis. They were incubated with ruminal fluid, buffer mixture, and finely ground alfalfa hay for 24 h, at 0, 6, 12, and 18 mM initial concentrations. The propionate enhancer oxaloacetic acid, the butyrate enhancer beta-hydroxybutyrate, and the butyrate unsaturated analog 3-butenoic acid were ineffective in decreasing methanogenesis. Nevertheless, beta-hydroxybutyrate increased apparent fermentation of the alfalfa hay substrate from 58.0 to 63.4%, and 3-butenoic acid seemed to increase it from 62.0 to 73.7%. Almost all of added oxaloacetic acid disappeared during the incubation, while only between 30.3 and 53.4% of beta-hydroxybutyrate disappeared. The butyrate enhancers acetoacetate and crotonic acid, and the butyrate unsaturated analog 2-butynoic acid, decreased methanogenesis by a maximum of 18,9 and 9%, respectively. Crotonic acid at 18 mM initial concentration seemed to increase the substrate apparent fermentation from 57.0 to 68.2%. Between 78.6 and 100% of acetoacetate disappeared during the incubation. The propionate unsaturated analog propynoic acid, and the unsaturated ester ethyl 2-butynoate, decreased methanogenesis by a maximum of 76 and 79%, respectively. Less than 5% of propynoic acid disappeared. The substrate apparent fermentation was decreased by propynoic acid from 62.0 to 57.4%, and seemed to have been decreased by ethyl 2-butynoate from 62.0 to 29.3%. More accurate measurements of the disappearance of some of the compounds studied are needed to better understand how they are metabolized and how they affect fermentation.

Regulation and roles of phosphoenolpyruvate carboxykinase in plants.

Phosphoenolpyruvate carboxykinase (PCK) is probably ubiquitous in flowering plants, but is confined to certain cells or tissues. It is regulated by phosphorylation, which renders it less active by altering both its substrate affinities and its sensitivity to regulation by adenylates. In the leaves of some C4 plants, such as Panicum maximum, dephosphorylation increases its activity in the light. In other tissues such regulation probably avoids futile cycling between phosphoenolpyruvate and oxaloacetate. Although PCK generally acts as a decarboxylase in plants, its affinity for CO2 measured at physiological concentrations of metal ions is high and would allow it to be freely reversible in vivo. While its function in gluconeogenesis in seeds postgermination and in leaves of C4 and crassulacean acid metabolism plants is clearly established, the possible functions of PCK in other plant cells are discussed, drawing parallels with those in animals, including its integrated function in cataplerosis, nitrogen metabolism, pH regulation, and gluconeogenesis.

Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data.

The concentration of an inhibitor that decreases the rate of an enzyme-catalysed reaction by 50%, symbolized i(0.5), is often used in pharmacological studies to characterize inhibitors. It can be estimated from the common inhibition plots used in biochemistry by means of the fact that the extrapolated inhibitor concentration at which the rate becomes infinite is equal to -i(0.5). This method is, in principle, more accurate than comparing the rates at various different inhibitor concentrations, and inferring the value of i(0.5) by interpolation. Its reciprocal, 1/i(0.5), is linearly dependent on v(0)/V, the uninhibited rate divided by the limiting rate, and the extrapolated value of v(0)/V at which 1/i(0.5) is zero allows the type of inhibition to be characterized: this value is 1 if the inhibition is strictly competitive; greater than 1 if the inhibition is mixed with a predominantly competitive component; infinite (i.e. 1/i(0.5) does not vary with v(0)/V) if the inhibition is pure non-competitive (i.e. mixed with competitive and uncompetitive components equal); negative if the inhibition is mixed with a predominantly uncompetitive component; and zero if it is strictly uncompetitive. The type of analysis proposed has been tested experimentally by examining inhibition of lactate dehydrogenase by oxalate (an uncompetitive inhibitor with respect to pyruvate) and oxamate (a competitive inhibitor with respect to pyruvate), and of cytosolic malate dehydrogenase by hydroxymalonate (a mixed inhibitor with respect to oxaloacetate). In all cases there is excellent agreement between theory and experiment.

Beta-poly(L-malate) production by non-growing microplasmodia of Physarum polycephalum. Effects of metabolic intermediates and inhibitors.

The production of beta-poly(L-malate) (PMLA) by non-growing microplasmodia of Physarum polycephalum was investigated. Growth was minimal in culture medium devoid of nitrogen source, but PMLA production occurred at a substantial rate. The addition of metabolic intermediates, malate, fumarate, succinate, and oxaloacetate, and the omission of hematin showed considerable growth inhibition in the presence of the nitrogen source, while PMLA production per unit biomass increased significantly. The results indicated that PMLA production was dissociated from biomass production under these conditions. The stimulating effect of carbonate on PMLA production was independent on growth. Cultivation in the absence of the nitrogen source and hematin or in the presence of the metabolites may be a useful technique for efficient PMLA production at a minimum of biomass production.

Potent inhibition of macrophomate synthase by reaction intermediate analogs.

Potent inhibitors for macrophomate synthase, which has recently been found to catalyze a highly unusual five-step chemical transformation, were explored. Among 11 oxalacetate analogs tested, only three analogs had moderate to relatively strong inhibitory activities (I50 1.3-8.1 mM). On the other hand, among 35 bicyclic intermediate analogs synthesized, two diacids were found to be the most potent inhibitors (I50 0.80, 0.84 mM) which had a much higher affinity than that of the natural substrate 2-pyrone. (-)-Enantiomers of the diacids showed 30 times stronger activity (I50 0.34, 0.41 mM) than (+)-ones. The I50/Km values (0.20, 0.24) showed their potent inhibitions. Competitive inhibitions were observed in two representative inhibitors.

Citrate promotes attachment of Prevotella nigrescens (intermedia) ATCC 25261 to hydroxyapatite.

Large quantities of Prevotella nigrescens (intermedia) ATCC 25261 (P. nigrescens) cells adhere to hydroxyapatite (HA) treated with citrate, but do not adhere experimental pellicle prepared from human whole saliva. To determine the nature of the citrate responsible for promoting P. nigrescens cell adhesion, the duration and frequency of citrate treatment of HA and the inhibitory effect of other carboxylates were tested. The citrate rapidly adhered to HA beads in less than 15 min. With a lower concentration (0.4 mM) of citrate, four treatments of HA were required to promote the maximum adherence to P. nigrescens cells. Citrate-enhanced P. nigrescens cell adherence to HA beads was also inhibited in the presence of cis-aconitate, oxaloacetate and oxalsuccinate. It was also found that P. nigrescens cells heated to 65 degrees C or higher for 5 min could no longer become attached to citrate-treated HA. These data suggest that citrate is one of the essential factors responsible for P. nigrescens cell attachment to apatitic surfaces, and that P. nigrescens' adhesion to citrate is extremely heat-sensitive.

Homotropic activation via the subunit interaction and allosteric symmetry revealed on analysis of hybrid enzymes of L-lactate dehydrogenase.

L-Lactate dehydrogenase from Bifidobacterium longum shows homotropic activation by pyruvate as well as heterotropic activation by fructose 1,6-bisphosphate. Hybrid enzymes were produced from the wild-type subunit and a mutant subunit, whose substrate specificity was altered to that of malate dehydrogenase, and separated to analyze the substrate-induced homotropic activation mechanism. Oxamate, a competitive inhibitor of L-lactate dehydrogenase, was used to mimic the substrate-induced activation of the wild-type subunit as "a regulatory subunit." The malate dehydrogenase activity of the mutant subunit as "the catalytic subunit" of the hybrid enzymes was measured, and the activity of the mutant subunit was activated on the addition of oxamate. Thus, we directly observed the inter-subunit homotropic activation transmitted from the wild-type to the mutant subunit. Moreover, "isomeric" hybrid enzymes that have different structural subunit arrangements but identical subunit compositions showed identical kinetic natures. This indicates that the enzyme maintains its subunit symmetry during the allosteric transition.

Flavin adenine dinucleotide and flavin mononucleotide metabolism in rat liver--the occurrence of FAD pyrophosphatase and FMN phosphohydrolase in isolated mitochondria.

In order to gain some insight into mitochondrial flavin biochemistry, rat liver mitochondria essentially free of lysosomal and microsomal contamination were prepared and their capability to metabolise externally added and endogenous FAD and FMN tested both spectroscopically and via HPLC. The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD-->FMN and FMN-->riboflavin conversion, respectively, is shown. They differ from each other and from extramitochondrial enzymes, as judged by their pH profile and inhibitor sensitivity, and can be separated in a partial FAD pyrophosphatase purification. Digitonin titration and subfractionation experiments show that FAD pyrophosphatase is located in the outer mitochondrial membrane and FMN phosphohydrolase in the intermembrane space. Since these enzymes can metabolise endogenous FAD and FMN, which are made available by using both Triton X-100 and the effector oxaloacetate, a proposal is made that FAD pyrophosphatase and FMN phosphohydrolase play a major role in mitochondrial flavoprotein turnover.

Oscillations in activities of enzymes in pancreatic islet subcellular fractions induced by physiological concentrations of effectors.

Glucose, the most potent insulin secretagogue, stimulates insulin secretion by aerobic glycolysis, but other secretagogues stimulate insulin release exclusively by mitochondrial metabolism. It is well known that in the intact pancreatic beta-cell, either kind of secretagogue can induce oscillations in metabolism (e.g., glycolysis, ATP/ADP, NAD(P)/NAD(P)H ratios) that occur with a periodicity similar to oscillations in membrane electrical potential and insulin secretion. In this study, pancreatic islet cytosol or mitochondrial fractions were incubated in the presence of physiological concentrations of substrates. Repeated additions of physiological effectors caused oscillations in the activities of the three enzymes studied. Succinate dehydrogenase activity in islet mitochondrial extracts was made to oscillate by adding oxaloacetate (5 micromol/l) to inhibit the enzyme. The enzyme was reactivated by adding acetyl-CoA (3 micromol/l), which combines with oxaloacetate in the citrate synthase reaction and lowers the concentration of oxaloacetate, thus beginning another oscillation. Pyruvate kinase activity was made to oscillate by adding fructose bisphosphate (10 micromol/l). Fructose bisphosphate was degraded to triose phosphates fairly rapidly, and, as it was degraded, there was a parallel decrease in pyruvate kinase activity. The enzyme was reactivated and made to oscillate with subsequent additions of fructose bisphosphate. The mitochondrial glycerol phosphate dehydrogenase was made to oscillate by adding EGTA to chelate calcium, which activates the enzyme. When the concentration of free calcium was raised to >0.1 micromol/l by adding more calcium, the activity of the enzyme increased. Repeated additions of chelator and calcium caused the enzyme activity to oscillate. The results with these three enzymes and physiological concentrations of naturally occurring effectors raise the possibility that the activities of not only these enzymes but of numerous enzymes oscillate in vivo in response to levels of allosteric effectors and substrates. If this is the case, pacemaker activity may result from complex effects distributed across multiple regulatory sites in both the cytosol and mitochondria, rather than from a single enzyme acting as a primary pacemaker.

Pyruvate protects neurons against hydrogen peroxide-induced toxicity.

Hydrogen peroxide (H2O2) is suspected to be involved in numerous brain pathologies such as neurodegenerative diseases or in acute injury such as ischemia or trauma. In this study, we examined the ability of pyruvate to improve the survival of cultured striatal neurons exposed for 30 min to H2O2, as estimated 24 hr later by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay. Pyruvate strongly protected neurons against both H2O2 added to the external medium and H2O2 endogenously produced through the redox cycling of the experimental quinone menadione. The neuroprotective effect of pyruvate appeared to result rather from the ability of alpha-ketoacids to undergo nonenzymatic decarboxylation in the presence of H2O2 than from an improvement of energy metabolism. Indeed, several other alpha-ketoacids, including alpha-ketobutyrate, which is not an energy substrate, reproduced the neuroprotective effect of pyruvate. In contrast, lactate, a neuronal energy substrate, did not protect neurons from H2O2. Optimal neuroprotection was achieved with relatively low concentrations of pyruvate (

L-aspartate-evoked inhibition of melatonin production in rat pineal glands.

Our previous studies in rat indicated that pinealocytes secrete L-glutamate through microvesicle-mediated exocytosis to regulate negatively melatonin production. Recently, we further found that pinealocytes secrete L-aspartate through microvesicle-mediated exocytosis. In the present study, we investigated the role of L-aspartate in the melatonin production in isolated rat pineal glands. It was found that L-aspartate inhibits norepinephrine-stimulated melatonin production as well as serotonin N-acetyltransferase activity reversibly and dose-dependently, the concentrations required for 50% inhibition being 150 and 175 microM, respectively. L-Asparagine and oxaloacetate, metabolites of L-aspartate, had no effect on the melatonin production. These results suggest that pinealocytes use L-aspartate, as well as L-glutamate, as a negative regulator for melatonin production.

Isolation and characterization of cytosolic malate dehydrogenase from Trichomonas vaginalis.

Malate dehydrogenase (EC 1.1.1.37.) (MDH) was purified to apparent homogeneity from the cytosolic fraction of the protozoan Trichomonas vaginalis Donné. The four step purification included ion-exchange chromatography (DEAE-Sephacel and Q-Sepharose, elution with NaCl) and affinity chromatography (Reactive Red Agarose, elution with NADH and NaCl). The enzyme was purified about 132-fold (30.6% yield) to a specific activity of 352 units mg-1. The Km values determined at pH 7.8 (pH optimum from 7.5 to 8.3) for oxaloacetate and NADH were 16.2 microM and 10.6 microM, respectively. The MDH activity was inhibited by the substrate, decreasing to 50% at about 1 microM concentration of oxaloacetate. The reverse reaction from malate to oxaloacetate showed a pH optimum around pH 9.5. The Km for malate and NAD+ (determined at pH 7.8) were 1220 microM and 69.9 microM, respectively. SDS-PAGE analysis of the purified MDH revealed a single band with an apparent size of 34.5 kDa. The native molecular weight was estimated by HPLC gel filtration to be 60 kDa, which indicates that the T. vaginalis MDH exists as a dimer.

Inhibition of pigeon breast muscle alpha-ketoglutarate dehydrogenase by structural analogs of alpha-ketoglutarate.

Inhibition of alpha-ketoglutarate dehydrogenase (KGD) by dicarboxylates with (oxaloacetate and ketomalonate) and without (malonate, succinate, and glutarate) alpha-keto group was studied. Ketodicarboxylates at low concentrations inhibit KGD in competitive manner. Increase in their concentrations results in appearance of the noncompetitive component. The extent of KGD inhibition by keto dicarboxylates increases with structural similarity of the inhibitor and the substrate, irrespective of preliminary incubation of the enzyme with the inhibitor. This is indicative of blocking the substrate-binding site of KGD by dicarboxylates. In contrast, inhibitory effect of dicarboxylates which contain no keto group increases as their structural similarity with the substrate decreases. Saturation of KGD with dicarboxylates of this type does not completely suppress the enzymatic activity. Alternatively, these analogs display competitive mode of inhibition. Analysis of the data obtained suggests that these dicarboxylates produce catalytically active triple complex keto substrate-KGD-dicarboxylate and that KGD which enters the composition of such a complex inhibits a decreased affinity for the keto substrate as a result of the inhibitor binding.

Theileria annulata: in vitro cultivation of schizont-infected bovine lymphocytes.

Experimental parameters of optimization of the in vitro growth conditions for Theileria annulata schizont-infected bovine lymphoid cells are presented. Of the nine different media tested in the course of 14 successive passages, Leibovitz L-15 (L-15) and a combination of McCoy and Leibovitz L-15 (ML) were preferable to McCoy, Dulbecco (DMEM), RPMI 1640 or Eagles's minimum essential medium (MEM) based on either Hank's or Earle's salts. The lowest multiplication rate was obtained with M-199 medium based on Hank's or Earle's salts. The highest yield of cells was obtained when L-15 was supplemented with 20% newborn bovine serum, while lowering the serum concentration by half resulted in a 25% decrease in cell yield. There was no effect on multiplication rate observed during ten passages when 2-mercaptoethanol and a mixture of oxaloacetate, sodium pyruvate and insulin were added to the growth medium. On substitution of conditioned medium for 20 or 50% of the growth medium, the yield of cells decreased by 12 or 20%, respectively, a factor which might be considered in calculations of the cost of anti-T. annulata vaccine production. When cells were grown in stationary cultures in Roux bottles for 7 days without change of medium, the highest yield resulted from seeding at 10(7) cells per vessel (100 ml) in L-15 supplemented with 20% serum. In Roller bottles, with the same type and volume of medium and cultivation for 7 days without medium change, best yield resulted from seeding with the highest inoculum size of 4 x 10(7) cells per vessel.

Redox state of pyridine nucleotides, but not glutathione, regulate Ca2+ release by H2O2 from mitochondria of pulmonary smooth muscle.

Treatment of bovine pulmonary artery smooth muscle mitochondria with H2O2 stimulated oxidation of GSH and NAD(P)H along with an increase in Ca2+ release. Addition of oxaloacetate to mitochondrial suspension stimulated Ca2+ release and oxidation of NAD(P)H while GSH level remained unchanged. Subsequently, addition of beta-hydroxybutyrate which reduced mitochondrial pyridine nucleotides caused reuptake of the released Ca2+ without causing appreciable alteration of GSH level. Treatment of the mitochondria with 1,3-bis(2-dichloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, significantly decreased GSH level without producing discernible change in Ca2+ release and NAD(P)H oxidation.