The utilisation of nitrogenous compounds by commercial non-Saccharomyces yeasts associated with wine.

Commercial wine fermentation is commonly conducted by inoculated strains of Saccharomyces cerevisiae. However, other non-Saccharomyces yeast species have recently become popular co-inoculants. Co-inoculated yeast species compete with each other for nutrients, and such competition may impact fermentation kinetics and aroma production. Understanding the specific nutrient requirements of non-Saccharomyces strains therefore is essential to better characterize the competitive potential of each strain, and to support rational decision making for nutrient supplementation during wine making. This study investigated the nitrogen source preference of commercial non-Saccharomyces yeasts by conducting pure culture and sequential culture fermentations in synthetic grape musts with adjusted nitrogen contents. Amino acid and ammonium uptake varied between yeast species. Lachancea thermotolerans and Torulaspora delbrueckii assimilated more nitrogen at a faster rate than Pichia kluyveri and Metschnikowia pulcherrima. Significant variation in amino acid preference between species was observed. Sequential fermentations confirmed the more competitive behaviour of L. thermotolerans and T. delbrueckii, with consequences for fermentation kinetics and aroma production. Furthermore, the data suggest that declining populations of non-Saccharomyces yeasts release nitrogen and supports the activity of S. cerevisiae. The data provide the most detailed assessment of nitrogen utilisation by the investigated yeast strains in a wine environment.

Carnitine Requires Choline to Exert Physiological Effects in Saccharomyces cerevisiae.

L-Carnitine is a key metabolite in the energy metabolism of eukaryotic cells, functioning as a shuttling molecule for activated acyl-residues between cellular compartments. In higher eukaryotes this function is essential, and defects in carnitine metabolism has severe effects on fatty acid and carbon metabolism. Carnitine supplementation has been associated with an array of mostly beneficial impacts in higher eukaryotic cells, including stress protection and regulation of redox metabolism in diseased cells. Some of these phenotypes have no obvious link to the carnitine shuttle, and suggest that carnitine has as yet unknown shuttle-independent functions. The existence of shuttle-independent functions has also been suggested in Saccharomyces cerevisiae, including a beneficial effect during hydrogen peroxide stress and a detrimental impact when carnitine is co-supplemented with the reducing agent dithiothreitol (DTT). Here we used these two distinct yeast phenotypes to screen for potential genetic factors that suppress the shuttle independent physiological effects of carnitine. Two deletion strains, Δcho2 and Δopi3, coding for enzymes that catalyze the sequential conversion of phosphatidylethanolamine to phosphatidylcholine were identified for suppressing the phenotypic effects of carnitine. Additional characterisation indicated that the suppression cannot be explained by differences in phospholipid homeostasis. The phenotypes could be reinstated by addition of extracellular choline, but show that the requirement for choline is not based on some overlapping function or the structural similarities of the two molecules. This is the first study to suggest a molecular link between a specific metabolite and carnitine-dependent, but shuttle-independent phenotypes in eukaryotes.

Synthesis and in vivo evaluation of the putative breast cancer resistance protein inhibitor [11C]methyl 4-((4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl)phenyl)amino-carbonyl)-2-(quinoline-2-carbonylamino)benzoate.

INTRODUCTION: The multidrug efflux transporter breast cancer resistance protein (BCRP) is highly expressed in the blood-brain barrier (BBB), where it limits brain entry of a broad range of endogenous and exogenous substrates. Methyl is a recently discovered BCRP-selective inhibitor, which is structurally derived from the potent P-glycoprotein (P-gp) inhibitor tariquidar. The aim of this study was to develop a new PET tracer based on 1 to map BCRP expression levels in vivo. METHODS: Compound 1 was labelled with (11)C in its methyl ester function by reaction of the corresponding carboxylic acid 2 with [(11)C]methyl triflate. Positron emission tomography (PET) imaging of [(11)C]-1 was performed in wild-type, Mdr1a/b((-/-)), Bcrp1((-/-)) and Mdr1a/b((-/-))Bcrp1((-/-)) mice (n=3 per mouse type) and radiotracer metabolism was assessed in plasma and brain. RESULTS: Brain-to-plasma ratios of unchanged [(11)C]-1 were 4.8- and 10.3-fold higher in Mdr1a/b((-/-)) and in Mdr1a/b((-/-))Bcrp1((-/-)) mice, respectively, as compared to wild-type animals, but only modestly increased in Bcrp1((-/-)) mice. [(11)C]-1 was rapidly metabolized in vivo giving rise to a polar radiometabolite which was taken up into brain tissue. CONCLUSION: Our data suggest that [(11)C]-1 preferably interacts with P-gp rather than BCRP at the murine BBB which questions its reported in vitro BCRP selectivity. Consequently, [(11)C]-1 appears to be unsuitable as a PET tracer to map cerebral BCRP expression.

Carnitine supplementation has protective and detrimental effects in Saccharomyces cerevisiae that are genetically mediated.

l-Carnitine plays a well-documented role in eukaryotic energy homeostasis by acting as a shuttling molecule for activated acyl residues across intracellular membranes. This activity, supported by carnitine acyl-transferases and transporters, is referred to as the carnitine shuttle. However, several pleiotropic and often beneficial effects of carnitine in humans have been reported that appear to be unrelated to shuttling activity, but little conclusive evidence regarding molecular mechanisms exists. We have recently demonstrated a role of carnitine, independent of the carnitine shuttle, in yeast stress protection. Here, we show that carnitine specifically protects against oxidative stress caused by H(2)O(2) and the superoxide-generating agent menadione. Surprisingly, carnitine has a detrimental effect on survival when combined with thiol-modifying agents. Central elements of the oxidative stress response, specifically the transcription factors Yap1p and Skn7p, are shown to be required for carnitine's protective effect, but several downstream effectors are dispensable. A DNA microarray-based analysis identifies Cyc3p, a cytochrome c heme lyase, as being important for carnitine's impact during oxidative stress. These findings establish a direct genetic link to a carnitine-related phenotype that is independent of the shuttle system and suggests that Saccharomyces cerevisiae should provide a useful model for further elucidation of carnitine's physiological roles.

Carnitine biosynthesis in Neurospora crassa: identification of a cDNA coding for epsilon-N-trimethyllysine hydroxylase and its functional expression in Saccharomyces cerevisiae.

The biosynthesis of L-carnitine in eukaryotic organisms was first elucidated in the ascomycete Neurospora crassa. The first step of the pathway is catalysed by epsilon-N-trimethyllysine hydroxylase (TMLH), which converts epsilon-N-trimethyllysine into beta-hydroxy-N-epsilon-trimethyllysine in a reaction dependent on alpha-ketoglutarate, Fe2+ and oxygen. Here we report on the cloning of the N. crassa TMLH cDNA and its functional expression in Saccharomyces cerevisiae. The TMLH cDNA contains an open reading frame of 1413 base pairs encoding a predicted polypeptide of 471 amino acids. The Michaelis-Menten constants of the heterologously expressed enzyme were determined for epsilon-N-trimethyllysine, alpha-ketoglutarate, Fe2+ and correspond to 0.33 mM, 133 microM and 46 microM, respectively.