The aim of this study was to characterize nanoparticles (NPs) composed of chitosan (CS) and evaluate their potential for brain delivery of the neurotransmitter Dopamine (DA). For this purpose, CS based NPs were incubated with DA at two different concentrations giving rise to nanocarriers denoted as DA/CSNPs (1) and DA/CSNPs (5), respectively. X-ray Photoelectron Spectroscopy (XPS) analysis confirmed that DA was adsorbed onto the external surface of such NPs. The cytotoxic effect of the CSNPs and DA/CSNPs was assessed using the MTT test and it was found that the nanovectors are less cytotoxic than the neurotransmitter DA after 3 h of incubation time. Transport studies across MDCKII-MDR1 cell line showed that DA/CSNPs (5) give rise to a significant transport enhancing effect compared with the control and greater than the corresponding DA/CSNPs (1). Measurement of reactive oxygen species (ROS) suggested a low DA/CSNPs neurotoxicity after 3 h. In vivo brain microdialysis experiments in rat showed that intraperitoneal acute administration of DA/CSNPs (5) (6-12 mg/kg) induced a dose-dependent increase in striatal DA output. Thus, these CS nanoparticles represent an interesting technological platform for DA brain delivery and, hence, may be useful for Parkinson's disease treatment. (C) 2011 Elsevier B.V. All rights reserved.

Grape berries (Vitis vinifera L fruit) exhibit a double-sigmoid pattern of development that results from two successive periods of vacuolar swelling during which the nature of accumulated solutes changes significantly. Throughout the first period, called green or herbaceous stage, berries accumulate high levels of organic acids, mainly malate and tartrate. At the cellular level fruit acidity comprises both metabolism and vacuolar storage. Malic acid compartmentation is critical for optimal functioning of cytosolic enzymes. Therefore, the identification and characterization of the carriers involved in malate transport across sub-cellular compartments is of great importance. The decrease in acid content during grape berry ripening has been mainly associated to mitochondrial malate oxidation. However, no Vitis vinifera mitochondrial carrier involved in malate transport has been reported to date. Here we describe the identification of three V. vinifera mitochondrial dicarboxylate/tricarboxylate carriers (VvDTC1-3) putatively involved in mitochondrial malate, citrate and other di/tricarboxylates transport. The three VvDTCs are very similar, sharing a percentage of identical residues of at least 83 %. Expression analysis of the encoding VvDTC genes in grape berries shows that they are differentially regulated exhibiting a developmental pattern of expression. The simultaneous high expression of both VvDTC2 and VvDTC3 in grape berry mesocarp close to the onset of ripening suggests that these carriers might be involved in the transport of malate into mitochondria.

The modification of enzyme cofactor concentrations can be used as a method for both studying and engineering metabolism. We varied Saccharomyces cerevisiae mitochondrial NAD levels by altering expression of its specific mitochondrial carriers. Changes in mitochondrial NAD levels affected the overall cellular concentration of this coenzyme and the cellular metabolism. In batch culture, a strain with a severe NAD depletion in mitochondria succeeded in growing, albeit at a low rate, on fully respiratory media. Although the strain increased the efficiency of its oxidative phosphorylation, the ATP concentration was low. Under the same growth conditions, a strain with a mitochondrial NAD concentration higher than that of the wild type similarly displayed a low cellular ATP level, but its growth rate was not affected. In chemostat cultures, when cellular metabolism was fully respiratory, both mutants showed low biomass yields, indicative of impaired energetic efficiency. The two mutants increased their glycolytic fluxes, and as a consequence, the Crabtree effect was triggered at lower dilution rates. Strikingly, the mutants switched from a fully respiratory metabolism to a respirofermentative one at the same specific glucose flux as that of the wild type. This result seems to indicate that the specific glucose uptake rate and/or glycolytic flux should be considered one of the most important independent variables for establishing the long-term Crabtree effect. In cells growing under oxidative conditions, bioenergetic efficiency was affected by both low and high mitochondrial NAD availability, which suggests the existence of a critical mitochondrial NAD concentration in order to achieve optimal mitochondrial functionality.

The mitochondrial aspartate-glutamate carrier isoform 1 (AGC1) catalyzes a Ca(2+)-stimulated export of aspartate to the cytosol in exchange for glutamate, and is a key component of the malate-aspartate shuttle which transfers NADH reducing equivalents from the cytosol to mitochondria. By sustaining the complete glucose oxidation, AGC1 is thought to be important in providing energy for cells, in particular in the CNS and muscle where this protein is mainly expressed. Defects in the AGC1 gene cause AGC1 deficiency, an infantile encephalopathy with delayed myelination and reduced brain N-acetylaspartate (NAA) levels, the precursor of myelin synthesis in the CNS. Here, we show that undifferentiated Neuro2A cells with down-regulated AGC1 display a significant proliferation deficit associated with reduced mitochondrial respiration, and are unable to synthesize NAA properly. In the presence of high glutamine oxidation, cells with reduced AGC1 restore cell proliferation, although oxidative stress increases and NAA synthesis deficit persists. Our data suggest that the cellular energetic deficit due to AGC1 impairment is associated with inappropriate aspartate levels to support neuronal proliferation when glutamine is not used as metabolic substrate, and we propose that delayed myelination in AGC1 deficiency patients could be attributable, at least in part, to neuronal loss combined with lack of NAA synthesis occurring during the nervous system development.

Peroxisomes are small organelles found in all eukaryotes, involved in a number of important metabolic pathways, including fatty acid α- and β-oxidation, biosynthesis of ether phospholipids and bile acids, and the degradation of purines, amino acids and polyamines. The functional role of the peroxisomal membrane as a permeability barrier to substrates and cofactors has been controversial for many years. The essential cofactors CoA, FAD and NAD+ are synthesized outside the peroxisomes and must be transported into the peroxisomal matrix where they are required for important processes. SLC25A17 (solute carrier family 25 member 17) is the only member of the mitochondrial carrier family that has been shown to be localized in the peroxisomal membrane. Recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN and AMP, and to a lesser extent of NAD+, PAP (adenosine 3',5'-diphosphate) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and was inhibited by pyridoxal 5'-phosphate and other mitochondrial carrier inhibitors. Moreover it was expressed to various degrees in all of the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD+ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP [1]. The plant homologue of SLC25A17 is the peroxisomal protein PXN encoded by the Arabidopsis gene At2g39970 which has recently been found to transport NAD+, NADH, AMP and ADP [2]. Upon heterologous expression of PXN in bacteria followed by purification and reconstitution in liposomes, uptake and efflux experiments revealed that PXN transports coenzyme A (CoA), dephospho-CoA, acetyl-CoA and adenosine 3', 5'-phosphate (PAP), besides NAD+, NADH, AMP and ADP. PXN catalyzed fast counter-exchange of substrates and much slower uniport. Transport was saturable with a submillimolar affinity for NAD+, CoA and other substrates. The physiological role of PXN is probably to provide the peroxisomes with the essential coenzymes NAD+ and CoA [3]. [1] G. Agrimi, A. Russo, P. Scarcia, F. Palmieri, The human gene SLC25A17 encodes a peroxisomal transporter of coenzyme A, FAD and NAD+, Biochem. J., 443 (2012) 241–247.

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP(+) and GSH/GSSG ratios in the cytosol of DeltaYHM2 cells as well as an increase in the NADPH/NADP(+) ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the DeltaYHM2 strain and more so by the DeltaYHM2DeltaZWF1 strain upon H(2)O(2) exposure, implying that Yhm2p has an antioxidant function.

In Saccharomyces cerevisiae there are 35 putative transport proteins which belong to the mitochondrial carriers family. The identified members of this family shuttle metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. We have functionally defined and characterized the mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae and its product was purified and reconstituted into liposomes. Its transport properties and kinetic parameters showed that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. It also transported oxaloacetate, succinate and fumarate to a lesser extent, but not malate and isocitrate. Yhm2p catalyzed a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate. By mass spectrometry analysis we observed a decrease in the NADPH/NADP+ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/ NADP+ ratio in their mitochondria compared to wild-type cells. Probably, Yhm2p acts as a key component of a citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol, and its physiological role is to increase the NADPH reducing power in the cytosol. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H2O2 exposure, implying that Yhm2p has an antioxidant function.

Plants make coenzyme A (CoA) in the cytoplasm but use it for reactions in mitochondria, chloroplasts, and peroxisomes, implying that these organelles have CoA transporters. A plant peroxisomal CoA transporter is already known, but plant mitochondrial or chloroplastic CoA transporters are not. Mitochondrial CoA transporters belonging to the mitochondrial carrier family, however, have been identified in yeast (Saccharomyces cerevisiae; Leu-5p) and mammals (SLC25A42). Comparative genomic analysis indicated that angiosperms have two distinct homologs of these mitochondrial CoA transporters, whereas nonflowering plants have only one. The homologs from maize (Zea mays; GRMZM2G161299 and GRMZM2G420119) and Arabidopsis (Arabidopsis thaliana; At1g14560 and At4g26180) all complemented the growth defect of the yeast leu5D mitochondrial CoA carrier mutant and substantially restored its mitochondrial CoA level, confirming that these proteins have CoA transport activity. Dual-import assays with purified pea (Pisum sativum) mitochondria and chloroplasts, and subcellular localization of green fluorescent protein fusions in transiently transformed tobacco (Nicotiana tabacum) Bright Yellow-2 cells, showed that the maize and Arabidopsis proteins are targeted to mitochondria. Consistent with the ubiquitous importance of CoA, the maize and Arabidopsis mitochondrial CoA transporter genes are expressed at similar levels throughout the plant. These data show that representatives of both monocotyledons and eudicotyledons have twin, mitochondrially located mitochondrial carrier family carriers for CoA. The highly conserved nature of these carriers makes possible their reliable annotation in other angiosperm genomes.

It is currently held that thiamin is made in chloroplasts and converted in the cytosol to the active cofactor thiamin diphosphate (ThDP), and that mitochondria and plastids import ThDP. The organellar transporters that mediate ThDP import in plants have not been identified. Comparative genomic analysis indicated that two members of the mitochondrial carrier family (MCF) in Arabidopsis (At5g48970 and At3g21390) and two in maize (GRMZM2G118515 and GRMZM2G124911) are related to the ThDP carriers of animals and Saccharomyces cerevisiae. Expression of each of these plant proteins in a S. cerevisiae ThDP carrier (TPC1) null mutant complemented the growth defect on fermentable carbon sources and restored the level of mitochondrial ThDP and the activity of the mitochondrial ThDP-dependent enzyme acetolactate synthase. The plant proteins were targeted to mitochondria as judged by dual import assays with purified pea mitochondria and chloroplasts, and by microscopic analysis of the subcellular localization of green fluorescent protein fusions in transiently transformed tobacco suspension cells. Both maize genes were shown to be expressed throughout the plant, which is consistent with the known ubiquity of mitochondrial ThDP-dependent enzymes. Collectively, these data establish that plants have mitochondrially located MCF carriers for ThDP, and indicate that these carriers are highly evolutionarily conserved. Our data provide a firm basis to propagate the functional annotation of mitochondrial ThDP carriers to other angiosperm genomes.

Whey generated in cheese manufacturing poses serious environmental issues that limit process profitability. The innovation in the dairy sector recognizes the "bio-refinery" as a key to successful handling of whey disposal and economic rise. Cheese whey valorisation is a complex process involving multiple technologies that might lead to value-added products (biomass, fine or bulk chemicals). This work focuses on the optimization of a fermentation process using whey as growth medium and carbon source. Lactose, which is abundant in whey, is a valuable carbon source. However, the microorganism more widely used in industrial fermentation processes, the yeast Saccharomyces cerevisiae, is not a lactose-fermenting yeast. We set up an innovative biotechnological process for the production on large scale of a not-genetically modified yeast biomass that can be used in different contexts, such as bread making, production of probiotics, nutraceuticals, bio-active molecules. In order to use the cheese whey as raw material for the cultivation of S. cerevisiae and to overcome the limitations in the use of lactose we used and externally added the enzyme β-galactosidase. The careful optimization of the amount of added enzyme allowed the gradual release by hydrolysis and the simultaneous consumption of glucose and galactose with a consequent decrease of ethanol and an increase of the biomass produced.

Although the decrease in pyruvate secretion by brewer’s yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl a-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and 13C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer’s yeasts.

In yeast, pyruvate is placed at the crossroad of fermentation, oxidative metabolism and anabolic pathways. In this study we have characterized a previously developed pyruvate undersecreting sake yeast obtained by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, we investigated the mitochondrial activity of TCR7 by oxigraphy and 13C-metabolic flux analysis. The mutant strain (TCR7), displayed an higher mitochondrial pyruvate influx and oxidation, and a decreased glycerol production compared to the reference strain. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate extracellular secretion. Surprisingly mitochondrial activity resulted much higher in the sake yeast compared to CEN.PK 113-7D, the reference strain employied in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retained a branched mitochondrial structure for a longer time than laboratory strains. Further studies are needed to unveil the molecular mechanisms underlying these phenotypes.

Saccharomyces cerevisiae is the preferred microorganism in the ethanol fermentation industry. However its use for the fermentation of renewable resources such as lignocellulosic biomass is impaired by several metabolic bottlenecks and toxic by-products formed in the pre-treatment process. Furfural is the most abundant by-product of hemicellulose hydrolysis which inhibits yeast growth [1]. At low concentrations, S. cerevisiae can overcome furfural toxicity by converting it to the corresponding alcohol (furfuryl alcohol) by NAD(P)H-dependent reactions [2]. Unfortunately these mechanisms of detoxification compete for key enzymes and cofactors needed to branch carbon flow to respiration and to ethanol production. Several studies have reported that furfural at high concentrations decreases yeast viability, specific growth rate and volumetric fermentation rate. Recently it has been proposed that furfural induces reactive oxygen species (ROS) generation and cellular damage in S. cerevisiae (3) ever the mechanism of furfural toxicity in yeast is not yet fully understood. We have carried out a flow cytometry analysis of yeast CEN-PK cells in the presence of different concentrations of furfural. We did not observe any significant increase of ROS production even at concentrations that abolished growth. However, we found that furfural induced a strong membrane depolarization during the lag phase, followed by hyperpolarization and cell death after the cells had started growing. Our results shed light on the mechanisms of toxicity of furfural in yeast and pave the way to a rational approach to improvement of tolerance of S. cerevisiae.

La presente invenzione ha per oggetto un nuovo procedimento per la sintesi di alcoli chirali mediante l’impiego di Lactobacillus reuteri. L’invenzione ha anche per oggetto l’uso di tale microorganismo per la sintesi di detti alcoli chirali.

The essential cofactors coenzyme A (CoA), FAD and NAD+ are synthesized outside the peroxisomes and therefore must be transported into the peroxisomal matrix where they are required for important processes. In this work we have functionally identified and characterized SLC25A17, which is the only member of the mitochondrial carrier family that has previously been shown to be localized in the peroxisomal membrane. Herein, recombinant and purified SLC25A17 was reconstituted into liposomes. Its transport properties and kinetic parameters demonstrate that SLC25A17 is a transporter of CoA, FAD, FMN, AMP and to a lesser extent of NAD+, adenosine 3',5'-diphosphate (PAP) and ADP. SLC25A17 functioned almost exclusively by a counter-exchange mechanism, was saturable and inhibited by pyridoxal-5'-phosphate and other mitochondrial carrier inhibitors. It was expressed to various degrees in all the human tissues examined. Its main function is probably to transport free CoA, FAD and NAD+ into peroxisomes in exchange for intraperoxisomally generated PAP, FMN and AMP. This is the first report describing the identification and characterization of a transporter for multiple free cofactors in peroxisomes.

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.

The peroxisomal protein PXN encoded by the Arabidopsis gene At2g39970 has very recently been found to transport NAD(+), NADH, AMP and ADP. In this work we have reinvestigated the substrate specificity and the transport properties of PXN by using a wide range of potential substrates. Heterologous expression in bacteria followed by purification, reconstitution in liposomes, and uptake and efflux experiments revealed that PNX transports coenzyme A (CoA), dephospho-CoA, acetyl-CoA and adenosine 3', 5'-phosphate (PAP), besides NAD(+), NADH, AMP and ADP. PXN catalyzed fast counter-exchange of substrates and much slower uniport and was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline and tannic acid. Transport was saturable with a submillimolar affinity for NAD(+), CoA and other substrates. The physiological role of PXN is probably to provide the peroxisomes with the essential coenzymes NAD(+) and CoA.