Effects Of Several Metabolites Research Articles

Mendelian randomization with incomplete measurements on the exposure in the Hispanic Community Health Study/Study of Latinos

Mendelian randomization has been widely used to assess the causal effect of a heritable exposure variable on an outcome of interest, using genetic variants as instrumental variables. In practice, data on the exposure variable can be incomplete due to high cost of measurement and technical limits of detection. In this paper, we propose a valid and efficient method to handle both unmeasured and undetectable values of the exposure variable in one-sample Mendelian randomization analysis with individual-level data. We estimate the causal effect of the exposure variable on the outcome using maximum likelihood estimation and develop an expectation maximization algorithm for the computation of the estimator. Simulation studies show that the proposed method performs well in making inference on the causal effect. We apply our method to the Hispanic Community Health Study/Study of Latinos, a community-based prospective cohort study, and estimate the causal effect of several metabolites on phenotypes of interest.

Crosstalk between Pseudomonas aeruginosa antibiotic resistance and virulence mediated by phenylethylamine.

Multidrug efflux pumps are among the main Pseudomonas aeruginosa antibiotic-resistance determinants. Besides, efflux pumps are also involved in other relevant activities of bacterial physiology, including the quorum sensing-mediated regulation of bacterial virulence. Nevertheless, despite the relevance of efflux pumps in bacterial physiology, their interconnection with bacterial metabolism remains obscure. The effect of several metabolites on the expression of P. aeruginosa efflux pumps, and on the virulence and antibiotic resistance of this bacterium, was studied. Phenylethylamine was found to be both inducer and substrate of MexCD-OprJ, an efflux pump involved in P. aeruginosa antibiotic resistance and in extrusion of precursors of quorum-sensing signals. Phenylethylamine did not increase antibiotic resistance; however, the production of the toxin pyocyanin, the tissue-damaging protease LasB and swarming motility were reduced in the presence of this metabolite. This decrease in virulence potential was mediated by a reduction of lasI and pqsABCDE expression, which encode the proteins that synthesise the signalling molecules of two quorum-sensing regulatory pathways. This work sheds light on the interconnection between virulence and antibiotic-resistance determinants, mediated by bacterial metabolism, and points to phenylethylamine as an anti-virulence metabolite to be considered in the study of therapies against P. aeruginosa infections.

Systematic evaluation for the causal effects of blood metabolites on osteoporosis: Genetic risk score and Mendelian randomization.

PurposeOsteoporosis is associated with metabolic alterations, but the causal roles of serum metabolites on osteoporosis have not been identified.MethodsBased on the large individual-level datasets from UK Biobank as well as GWAS summary datasets, we first constructed genetic risk scores (GRSs) for 308 of 486 human serum metabolites and evaluated the effect of each GRS on 2 major osteoporosis phenotypes, i.e., estimated bone miner density (eBMD) and fracture, respectively. Then, two-sample Mendelian Randomization (MR) was performed to validate the casual metabolites on osteoporosis. Multivariable MR analysis tested whether the effects of metabolites on osteoporosis are independent of possible confounders. Finally, we conducted metabolic pathway analysis for the metabolites involved in bone metabolism.ResultsWe identified causal effects of 18 metabolites on eBMD and 1 metabolite on fracture with the GRS method after adjusting for multiple tests. Then, 9 of them were further validated with MR as replication, where comprehensive sensitive analyses proved robust of the causal associations. Although not identified in GRS, 3 metabolites were associated with at least three osteoporosis traits in MR results. Multivariable MR analysis determined the independent causal effect of several metabolites on osteoporosis. Besides, 23 bone metabolic pathways were detected, such as valine, leucine, isoleucine biosynthesis (p = 0.053), and Aminoacyl-tRNA biosynthesis (p = 0.076), and D-glutamine and D-glutamate metabolism (p = 0.004).ConclusionsThe systematic causal analyses strongly suggested that blood metabolites have causal effects on osteoporosis risk.

Relative activity of triclabendazole metabolites against the liver fluke, Fasciola hepatica

A study has been carried out to determine the relative activity of triclabendazole (TCBZ) and its sulphoxide (TCBZ SO) and sulphone (TCBZ SO 2) metabolites against the adult stage of the liver fluke, Fasciola hepatica. Flukes were incubated for 24 h in vitro in 15 μg/ml of each of the compounds and prepared for scanning and transmission electron microscopy. All three compounds induced changes to the surface morphology of the fluke, the changes comprising swelling and blebbing to a greater or lesser extent in different regions of the fluke. TCBZ SO 2 was more disruptive anteriorly and TCBZ SO posteriorly. Internal ultrastructural changes were evident following incubation with each of the compounds, with an order of severity TCBZ SO 2 > TCBZ SO > TCBZ. Swelling of the basal infolds and mitochondria were observed in the tegumental syncytium. In the tegumental cell bodies, there was a reduction in the number of secretory bodies, disruption of the Golgi complexes and swelling of the mitochondria. Severe flooding of the internal tissues was observed with TCBZ SO 2 and, to a lesser extent, with TCBZ SO and TCBZ. The results demonstrate that both TCBZ and TCBZ SO 2 are capable of disrupting the fluke in vitro and are not the inactive compounds they were assumed to be previously. They may well contribute to drug action in vivo as well, indicating that drug action is due to the additive effects of several metabolites, rather than being due to a single active metabolite, namely, TCBZ SO.

Pharmacological actions of isoprostane metabolites and phytoprostanes in human and bovine pulmonary smooth muscles

We examined the responses to various isoprostane derivatives in bovine/human airway and pulmonary arteries. All biological activity of 15-F 2t-IsoP was lost in its two major metabolites (15-keto-15-F 2t-IsoP and 13,14-dihydro-15-keto-15-F 2t-IsoP). We also examined the effects of several metabolites of 15-F 2t-IsoP synthesized within our own laboratory—both epimers of 2,3-dinor-15-F 2t-IsoP and of 2,3-dinor-5,6-dihydro-15-F 2t-IsoP, as well as 20-carboxy-2,3,4,5-tetranor-15 oxo-5,6,13,14-tetrahydro-15-F 2t-isoP)—finding none of these to have any substantial excitatory effect. Finally, several plant-derived isoprostanes (“phytoprostanes”) synthesized within our laboratory elicited little or no excitatory response in these three pulmonary smooth muscle preparations. We conclude that, although isoprostane exhibit powerful constrictor effects on airway and pulmonary vascular smooth muscles, metabolic processing of those isoprostanes essentially abolishes those biological actions; also, the phytoprostanes lack any appreciable pharmacological activity on those smooth muscle preparations.

Purification and characterization of NADP-dependent glutamate dehydrogenase from the commercial mushroom Agaricus bisporus.

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent glutamate dehydrogenase (NADP-GDH) of Agaricus bisporus, a key enzyme in ammonia assimilation, was purified to apparent electrophoretic homogeneity with 27% recovery of the initial activity. The molecular weight of the native enzyme was 330 kDa. The enzyme is probably a hexamer, composed of identical subunits of 48 kDa. The isoelectric point of the enzyme was found at pH 4.8. The N-terminus appeared to be blocked. The enzyme was specific for NADP(H). The Km-values were 2.1, 3.2, 0.074, 27.0, and 0.117 mM for ammonia, 2-oxoglutarate, NADPH, L-glutamate, and NADP respectively. The pH optima for the amination and deamination reactions were found to be 7.6 and 9.0, respectively. The temperature optimum was 33 degrees C. The effect of several metabolites on the enzyme's activity was tested. Pyruvate, oxaloacetate, ADP, and ATP showed some inhibitory effect. Divalent cations slightly stimulated the aminating reaction. Antibodies raised against the purified enzyme were able to precipitate NADP-GDH activity from a cell-free extract in an anticatalytic immunoprecipitation test. Analysis of a Western blot showed the antibodies to be specific for NADP-GDH.

Kinetic properties of phosphoenolpyruvate carboxylase from lupin nodules and roots

The kinetic properties of two forms of phosphoenolpyruvate carboxylase (PEPC I and PEPC II, EC 4.1, 1.31) from lupin (Lupinus luteus L. cv. Ventus) nodules and one enzyme form (PEPC III) from roots were studied. The Michaelis constant (Km) values for PEP, Mg2+ and especially HCO3−were lower for PEPC I. Kinetic studies showed that aspartate is a competitive inhibitor at pH 7.2 and inhibitor constant (Ki) values are different for the three forms of PEPC. Malate is a competitive inhibitor for PEPC I and PEPC III and shows mixed‐type inhibition for PEPC II. Malate inhibition is dependent upon the pH of the assay. Different effect of several metabolites was also observed. The temperature optimum was near 39°C for PEPC I and around 43°C for PEPC II and PEPC III. PEPC I appeared to be the most thermolabile. It is suggested that PEPC I from lupin nodules is closely associated with N2 fixation.

Proton Transporting Enzymes at the Tonoplast of Leaf Cells of the CAM Plant Kalanchoe daigremontiana III. Regulation of the ATPase

Purified tonoplast vesicles of the CAM plant Kalanchoe daigremontiana were obtained by a simplified sucrose gradient centrifugation method. To investigate the regulation of the H + -translocating ATPase, the effects of several metabolites were studied. ATP hydrolysis was not affected by pyrophosphate (PP i ) and inorganic phosphate (P i ). ADP exhibited competitive inhibition (K i = 0.31 mM). Changing ATP:Mg ratios at constant substrate (MgATP 2- ) concentrations resulted in severe inhibition of the ATPase by low concentrations of free Mg 2+ ions (K i ≈ 0.50 mM). The relevance of these data in regulation of CAM is discussed.

Regulation of acetohydroxy acid synthase activities in Escherichia coli K-12 by small metabolites

The effects of several metabolites (indole acetic acid, imidazole acetic acid and indole) on acetohydroxy acid synthase activities have been examined in both cya + and cya − strains. Specifically, indole acetic acid caused an increase in the rate of acetohydroxy acid synthase synthesis under both in vivo and in vitro conditions. Taken together, these data suggest that small metabolites, other than cAMP, can alter acetohydroxy acid synthase gene expression.

Metabolism of 4-pentenoic acid and inhibition of thiolase by metabolites of 4-pentenoic acid.

The metabolism of 4-pentenoic acid, a hypoglycemic agent and inhibitor of fatty acid oxidation, has been studied in rat heart mitochondria. Confirmed was the conversion of 4-pentenoic acid to 2,4-pentadienoyl coenzyme A (CoA), which either is directly degraded via beta-oxidation or is first reduced in a NADPH-dependent reaction before it is further degraded by beta-oxidation. At pH 6.9, the NADPH-dependent reduction of 2,4-pentadienoyl-CoA proceeds 10 times faster than its degradation by beta-oxidation. At pH 7.8, this ratio is only 2 to 1. The direct beta-oxidation of 2,4-pentadienoyl-CoA leads to the formation of 3-keto-4-pentenoyl-CoA, which is highly reactive and spontaneously converts to another 3-ketoacyl-CoA derivative (compound X). 3-Keto-4-pentenoyl-CoA is a poor substrate of 3-ketoacyl-CoA thiolase (EC 2.3..1.16) whereas compound X is not measurably acted upon by this enzyme. The effects of several metabolites of 4-pentenoic acid on the activity of 3-ketoacyl-CoA thiolase were studied. 3,4-Pentadienoyl-CoA is a weak inhibitor of this enzyme that is protected against the inhibition by acetoacetyl-CoA. The most effective inhibitor of 3-ketoacyl-CoA thiolase was found to be 3-keto-4-pentenoyl-CoA, which inhibits the enzyme in both a reversible and irreversible manner. The reversible inhibition is possibly a consequence of the inhibitor being a poor substrate of 3-ketoacyl-CoA thiolase. It is concluded that 4-pentenoic acid is metabolized in mitochondria by two pathways. The minor yields 3-keto-4-pentenoyl-CoA, which acts both as a reversible and as a irreversible inhibitor of 3-ketoacyl-CoA thiolase and consequently of fatty acid oxidation.

Studies on isocitrate lyase isolated from Lupinus cotyledons

Isocitrate lyase (threo-DS-isocitrate glyoxylate-lyase, EC 4.1.3.1) was isolated from cotyledons of Lupinus seedlings, purified 100-fold with respect to its initial specific activity and characterized (Km, pH optimum, Mg2+ requirement, sulfhydryl inhibitors, and synthase activity). The final purified preparation consisted of two homogeneous protein bands clearly separated by electrophoresis on polyacrylamide gel and chromatography on Sephadex G 200. Reducing agents are necessary for the maintenance of enzyme activity. The most effective reducing agent studied was 1,4-dithioerythreitol. The effect of several metabolites (oxalate, malonate, phosphoenolpyruvate, succinate, malate, tartrate, gluconate-6-phosphate, sorbose, sorbitol, and inositol) on the activity of purified preparations was tested. Oxalate proved to be the strongest inhibitor, seconded closely by phosphoenolpyruvate. The spectral characteristics of the purified enzyme are as follows: ultraviolet peak at 280 nm and fluorescence peak at 340 nm. The solid state infrared spectrum of the enzyme (lyophilized) showed that the enzyme was mostly in the alpha-helix conformation with very slight random orientation.

Polyribosome disaggregation and cell-free protein synthesis in preparations from cerebral cortex of hyperphenylalaninemic rats.

Abstract— Seven‐day‐old rats were injected intraperitoneally with l‐phenylalanine (1 g/kg) and the time course of brain polyribosome disaggregation and changes in brain levels of phenylalanine, tryptophan and tyrosine were determined. Disaggregation of brain polyribosomes preceded the increase in levels of phenylalanine in brain, and followed the same time course as depletion of tryptophan from brain.The effects of several metabolites of phenylalanine (which are formed in phenylketonuria) on protein synthesis in vitro was determined for brain and liver systems. None of the compounds tested was inhibitory at concentrations below 10 mM and in all cases hepatic protein synthesis was more sensitive to inhibition than was the corresponding system from brain.Ribosomal dimers, formed in brain after injection of phenylalanine, were incapable of supporting high levels of protein synthesis in vitro, a finding that suggested that the inhibition of protein synthesis in vitro in cell‐free systems of brain tissue after injection of phenylalanine into young rats was mediated by disaggregation of brain polyribosomes associated with tryptophan deficiency in brain.