Glycosphingolipids modulate renal phosphate transport in potassium deficiency

Background. Potassium (K) deficiency (KD) and/or hypokalemia have been associated with disturbances of phosphate metabolism The purpose of the present study was to determine the cellular mechanisms that mediate the impairment of renal proximal tubular Na/Pi cotransport in a model of K deficiency in the rat. Methods. K deficiency in the rat was achieved by feeding rats a K-deficient diet for seven days. which resulted in a marked decrease in serum and tissue K content. Results. K deficiency resulted in a marked increase in urinary Pi excretion and a decrease in the V-max of brush-border membrane (BBM) Na/Pi cotransport activity (1943 95 in control vs. 1183 +/- 99 pmol/5 sec/mg BBM protein in K deficiency. P < 0.02). Surprisingly. the decrease in Na/Pi cotransport activity was associated with increases in the abundance of type I (NaPi-1). and type II (NaPi-2) and type III (Glvr-1) Na/Pi protein. The decrease in Na/Pi transport was associated with significant alterations in BBM lipid composition, including increases in sphingomyelin. glucosylceramide. and ganglioside GM, content and a decrease in BBM lipid fluidity. Inhibition of glucosylceramide synthesis resulted in increases in BBM Na/Pi cotransport activity in control and K-deficient rats. The resultant Na/Pi cotransport activity in K-deficit nt rats was the same as in control rats (1148 +/- 52 in control + PDMP vs. 11.52 +/- 61 pmol/5 sec/mg BBM protein in K deficiency + PDMP). These changes in transport activity occurred independent of further changes in BBM NaPi-2 protein or renal cortical NaPi-2 mRNA abundance. Conclusion. K deficiency in the rat causes inhibition of renal Na/Pi cotransport activity by post-translational mechanisms that are mediated in part through alterations in glucosylceramide content and membrane lipid dynamics.

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [H-3]zeatin riboside ([H-3]ZR), a water-soluble blue dye or [H-3]H2O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within I h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [H-3]ZR and of blue dye in the same bud position was increased 3- to 10-fold relative to intact plants, whereas content of [H-3]H2O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [H-3]H2O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [H-3]ZR injected at physiological concentrations. Within 1 h, 80-95% of [H-3]ZR was converted to other active CKs (mainly zeatin riboside-5'phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O-acetylZR, O-acetylZRMP and a compound correlated with sites of high CK-concentrations) and inactive catabolites (adenosine, adenine, 5'AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis. (c) 2005 Wiley Periodicals, Inc.

Carbamyl phosphate synthase deficiency: Diagnosed during pregnancy in a 41-year-old

Carbamyl phosphate synthase deficiency (CPS) is a rare urea cycle defect. We present a case of a 41-year-old woman diagnosed with CPS deficiency during pregnancy. She is the oldest CPS-deficient patient, at diagnosis, reported to date and the first to be diagnosed during pregnancy. This case highlights the need for consideration of inborn errors of metabolism in adults presenting with unusual neurological and psychiatric conditions. Crown Copyright (c) 2006 Published by Elsevier Ltd. All rights reserved.

Cross-talk towards the response regulator NtrC controlling nitrogen metabolism in Rhodobacter capsulatus

Rhodobacter capsulatus NtrB/NtrC two-component regulatory system controls expression of genes involved in nitrogen metabolism including urease and nitrogen fixation genes. The ntrY-ntrX genes, which are located immediately downstream of the nifR3-ntrB-ntrC operon, code for a two-component system of unknown function. Transcription of ntrY starts within the ntrC-ntrY intergenic region as shown by primer extension analysis, but maximal transcription requires, in addition, the promoter of the nifR3-ntrB-ntrC operon. While ntrB and ntrY single mutant strains were able to grow with either urea or N-2 as sole nitrogen source, a ntrB/ntrY double mutant (like a ntrC-deficient strain) was no longer able to use urea or N-2. These findings suggest that the histidine kinases NtrB and NtrY can substitute for each other as phosphodonors towards the response regulator NtrC.

Titanium phosphate for Fuel Cell Proton Conduction Membranes

Environmental issues due to increases in emissions of air pollutants and greenhouse gases are driving the development of clean energy delivery technologies such as fuel cells. Low temperature Proton Exchange Membrane Fuel Cells (PEMFC) use hydrogen as a fuel and their only emission is water. While significant advances have been made in recent years, a major limitation of the current technology is the cost and materials limitations of the proton conduction membrane. The proton exchange membrane performs three critical functions in the PEMFC membrane electrode assembly (MEA): (i) conduction of protons with minimal resistance from the anode (where they are generated from hydrogen) to the cathode (where they combine with oxygen and electrons, from the external circuit or load), (ii) providing electrical insulation between the anode and cathode to prevent shorting, and (iii) providing a gas impermeable barrier to prevent mixing of the fuel (hydrogen) and oxidant. The PFSA (perfluorosulphonic acid) family of membranes is currently the best developed proton conduction membrane commercially available, but these materials are limited to operation below 100oC (typically 80oC, or lower) due to the thermochemical limitations of this polymer. For both mobile and stationary applications, fuel cell companies require more durable, cost effective membrane technologies capable of delivering enhanced performance at higher temperatures (typically 120oC, or higher. This is driving research into a wide range of novel organic and inorganic materials with the potential to be good proton conductors and form coherent membranes. There are several research efforts recently reported in the literature employing inorganic nanomaterials. These include functionalised silica phosphates [1,2], fullerene [3] titania phosphates [4], zirconium pyrophosphate [5]. This work addresses the functionalisation of titania particles with phosphoric acid. Proton conductivity measurements are given together with structural properties.

Muscle metabolites and performance during high-intensity, intermittent exercise

Six men were studied during four 30-s all-out exercise bouts on an air-braked cycle ergometer. The first three exercise bouts were separated by 4 min of passive recovery; after the third bout, subjects rested for 4 min, exercised for 30 min at 30-35% peak O-2 consumption, and rested for a further 60 min before completing the fourth exercise bout. Peak power and total work were reduced (P < 0.05) during bout 3 [765 +/- 60 (SE) W; 15.8 +/- 1.0 kJ] compared with bout 1 (1,168 +/- 55 mT, 23.8 +/- 1.2 kJ), but no difference in exercise performance was observed between bouts 1 and 4 (1,094 +/- 64 W, 23.2 +/- 1.4 kJ). Before bout 3, muscle ATP, creatine phosphate (CP), glycogen, pH, and sarcoplasmic reticulum (SR) Ca2+ uptake were reduced, while muscle lactate and inosine 5'-monophosphate were increased. Muscle ATP and glycogen before bout 4 remained lower than values before bout I (P < 0.05), but there were no differences in muscle inosine 5'-monophosphate, lactate, pH, and SR Ca2+ uptake. Muscle CP levels before bout 4 had increased above resting levels. Consistent with the decline in muscle ATP were increases in hypoxanthine and inosine before bouts 3 and 4. The decline in exercise performance does not appear to be related to a reduction in muscle glycogen. Instead, it may be caused by reduced CP availability, increased H+ concentration, impairment in SR function, or some other fatigue-inducing agent.

Effect of oral creatine supplementation on near-maximal strength and repeated sets of high-intensity bench press exercise

This study examined the effects of 26 days of oral creatine monohydrate (Cr) supplementation on near-maximal muscular strength, high-intensity bench press performance, and body composition. Eighteen male powerlifters with at least 2 years resistance training experience took part in this 28-day experiment. Pre and postmeasurements (Days 1 and 28) were taken of near-maximal muscular strength, body mass, and % body fat. There were two periods of supplementation Days 2 to 6 and Days 7 to 27. ANOVA and t-tests revealed that Cr supplementation significantly increased body mass and lean body mass with no changes in % body fat. Significant increases in 3-RM strength occurred in both groups, both absolute and relative to body mass; the increases were greater in the Cr group. The change in total repetitions also increased significantly with Cr supplementation both in absolute terms and relative to body mass, while no significant change was seen in the placebo (P) group. Creatine supplementation caused significant changes in the number of BP reps in Sets 1, 4, and 5. No changes occurred in the P group. It appears that 26 days of Cr supplementation significantly improves muscular strength and repeated near-maximal BP performance, and induces changes in body composition.

Glycine metabolism by plant roots and its occurrence in Australian plant communities

Soluble organic nitrogen, including protein and amino acids, was found to be a ubiquitous form of soil N in diverse Australian environments. Fine roots of species representative of these environments were found to be active in the metabolism of glycine. The ability to incorporate [N-15]glycine was widespread among plant species from subantarctic to tropical communities. In species from subantarctic herbfield, subtropical coral cay, subtropical rainforest and wet heathland, [N-15]glycine incorporation ranged from 26 to 45% of (NH4+)-N-15 incorporation and was 2- to 3-fold greater than (NO3-)-N-15 incorporation. Most semiarid mulga and tropical savanna woodland species incorporated [N-15]glycine and (NO3-)-N-15 in similar amounts, 18-26% of (NH4+)-N-15 incorporation. We conclude that the potential to utilise amino acids as N sources is of widespread occurrence in plant communities and is not restricted to those from low temperature regimes or where N mineralisation is limited. Seedlings of Hakea (Proteaceae) were shown to metabolise glycine, with a rapid transfer of N-15 from glycine to serine and other amino compounds. The ability to take up and metabolise glycine was unaffected by the presence of equimolar concentrations of NO3- and NH4+. Isonicotinic acid hydrazide (INH) did not inhibit the transfer of N-15-label from glycine to serine indicating that serine hydroxymethyltransferase was not active in glycine catabolism. In contrast aminooxyacetate (AOA) strongly inhibited transfer of N-15 from glycine to serine and labelling of other amino compounds, suggesting that glycine is metabolised in roots and cluster roots of Hakea via an aminotransferase.

Enzymology and molecular biology of prokaryotic sulfite oxidation

Despite its toxicity, sulfite plays a key role in oxidative sulfur metabolism and there are even some microorganisms which can use it as sole electron source. Sulfite is the main intermediate in the oxidation of sulfur compounds to sulfate, the major product of most dissimilatory sulfur-oxidizing prokaryotes. Two pathways of sulfite oxidation are known: (1) direct oxidation to sulfate catalyzed by a sulfite: acceptor oxidoreductase, which is thought to be a molybdenum-containing enzyme; (2) indirect oxidation under the involvement of the enzymes adenylylsulfate (APS) reductase and ATP sulfurylase and/or adenylylsulfate phosphate adenylyltransferase with APS as an intermediate. The latter pathway allows substrate phosphorylation and occurs in the bacterial cytoplasm. Direct oxidation appears to have a wider distribution; however, a redundancy of pathways has been described for diverse photo- or chemotrophic, sulfite-oxidizing prokaryotes. In many pro- and also eukaryotes sulfite is formed as a degradative product from molecules containing sulfur as a heteroatom. In these organisms detoxification of sulfite is generally achieved by direct oxidation to sulfate. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Directed evolution of mammalian cytochrome P450 enzymes involved in xenobiotic metabolism

Directed evolution of cytochrome P450 enzymes represents an attractive means of generating novel catalysts for specialized applications. Xenobiotic-metabolizing P450s are particularly well suited to this approach due to their inherent wide substrate specificity. In the present study, a novel method for DNA shuffling was developed using an initial restriction enzyme digestion step, followed by elimination of long parental sequences by size-selective filtration. P450 2C forms were subjected to a single round of shuffling then coexpressed with reductase in E. coli. A sample (54 clones) of the resultant library was assessed for sequence diversity, hemo- and apoprotein expression, and activity towards the substrate indole. All mutants showed a different RFLP pattern compared to all parents, suggesting that the library was free from contamination by parental forms. Haemoprotein expression was detectable in 45/54 (83%) of the mutants sampled. Indigo production was less than or comparable to the activities of one or more of the parental P450s, but three mutants showed indirubin production in excess of that seen with any parental form, representing a gain of function. In conclusion, a method is presented for the effective shuffling of P450 sequences to generate diverse libraries of mutant P450s containing a high proportion of correctly folded hemoprotein, and minimal contamination with parental forms.

Haemonchus contortus: The uptake and metabolism of closantel

Closantel is an anthe lmintic which associates with plasma albumin and is useful for the control of sheep parasites, such as Haemonchus contortus, that ingest blood. However, the utility of closantel for parasite control has been threatened by the emergence of resistance. The mechanisms of resistance are unknown. A closantel-resistant and a closantel-susceptible isolate of H. contortus were compared with respect to the distribution and metabolism of closantel. Neither strain appeared to metabolise closantel in vitro or in vivo. Following treatment of infected sheep with radioactively labelled closantel, isotope levels in closantel-resistant adult H. contortus were significantly lower than in susceptible worms. This reduced accumulation of drug could contribute to closantel resistance by mechanisms such as reduced feeding, failure to dissociate the drug-albumin complex in the gut or increased efflux of closantel from resistant worms. (C) 1997 Australian Society for Parasitology.

Increasing relevance of pharmacogenetics of drug metabolism in clinical practice

Much of the individual variation in drug response is due to genetic drug metabolic polymorphisms. Clinically relevant examples include acetylator status; cytochrome P450 2D6, 2C9 and 2C19 polymorphisms; and thiopurine methyltransferase deficiency. It is important to be aware of which drugs are subject to pharmacogenetic variability. In the future, population-based pharmacogenetic testing will allow more individualized drug treatment and will avoid the current empiricism.