How strict is galactose restriction in adults with galactosaemia? International practice.

Dietary management of 418 adult patients with galactosaemia (from 39 centres/12 countries) was compared. All centres advised lactose restriction, 6 restricted galactose from galactosides ± fruits and vegetables and 12 offal. 38% (n=15) relaxed diet by: 1) allowing traces of lactose in manufactured foods (n=13) or 2) giving fruits, vegetables and galactosides (n=2). Only 15% (n=6) calculated dietary galactose. 32% of patients were lost to dietetic follow-up. In adult galactosaemia, there is limited diet relaxation.

Dietary practices in pyridoxine non-responsive homocystinuria: a European survey.

BACKGROUND: Within Europe, the management of pyridoxine (B6) non-responsive homocystinuria (HCU) may vary but there is limited knowledge about treatment practice. AIM: A comparison of dietetic management practices of patients with B6 non-responsive HCU in European centres. METHODS: A cross-sectional audit by questionnaire was completed by 29 inherited metabolic disorder (IMD) centres: (14 UK, 5 Germany, 3 Netherlands, 2 Switzerland, 2 Portugal, 1 France, 1 Norway, 1 Belgium). RESULTS: 181 patients (73% >16 years of age) with HCU were identified. The majority (66%; n=119) were on dietary treatment (1-10 years, 90%; 11-16 years, 82%; and >16 years, 58%) with or without betaine and 34% (n=62) were on betaine alone. The median natural protein intake (g/day) on diet only was, by age: 1-10 years, 12 g; 11-16 years, 11 g; and >16 years, 45 g. With diet and betaine, median natural protein intake (g/day) by age was: 1-10 years, 13 g; 11-16 years, 20 g; and >16 years, 38 g. Fifty-two percent (n=15) of centres allocated natural protein by calculating methionine rather than a protein exchange system. A methionine-free l-amino acid supplement was prescribed for 86% of diet treated patients. Fifty-two percent of centres recommended cystine supplements for low plasma concentrations. Target treatment concentrations for homocystine/homocysteine (free/total) and frequency of biochemical monitoring varied. CONCLUSION: In B6 non-responsive HCU the prescription of dietary restriction by IMD centres declined with age, potentially associated with poor adherence in older patients. Inconsistencies in biochemical monitoring and treatment indicate the need for international consensus guidelines.

Dietary management of urea cycle disorders: European practice.

BACKGROUND: There is no published data comparing dietary management of urea cycle disorders (UCD) in different countries. METHODS: Cross-sectional data from 41 European Inherited Metabolic Disorder (IMD) centres (17 UK, 6 France, 5 Germany, 4 Belgium, 4 Portugal, 2 Netherlands, 1 Denmark, 1 Italy, 1 Sweden) was collected by questionnaire describing management of patients with UCD on prescribed protein restricted diets. RESULTS: Data for 464 patients: N-acetylglutamate synthase (NAGS) deficiency, n=10; carbamoyl phosphate synthetase (CPS1) deficiency, n=29; ornithine transcarbamoylase (OTC) deficiency, n=214; citrullinaemia, n=108; argininosuccinic aciduria (ASA), n=80; arginase deficiency, n=23 was reported. The majority of patients (70%; n=327) were aged 0-16y and 30% (n=137) >16y. Prescribed median protein intake/kg body weight decreased with age with little variation between disorders. The UK tended to give more total protein than other European countries particularly in infancy. Supplements of essential amino acids (EAA) were prescribed for 38% [n=174] of the patients overall, but were given more commonly in arginase deficiency (74%), CPS (48%) and citrullinaemia (46%). Patients in Germany (64%), Portugal (67%) and Sweden (100%) were the most frequent users of EAA. Only 18% [n=84] of patients were prescribed tube feeds, most commonly for CPS (41%); and 21% [n=97] were prescribed oral energy supplements. CONCLUSIONS: Dietary treatment for UCD varies significantly between different conditions, and between and within European IMD centres. Further studies examining the outcome of treatment compared with the type of dietary therapy and nutritional support received are required.

The capacity to transport potassium influences sodium tolerance in Saccharomyces cerevisiae.

The capacity to transport potassium and to discriminate between the different alkali cations has been found to affect sodium tolerance in Saccharomyces cerevisiae. Mutants with a defective capacity to transport K+ were more sensitive to high concentrations of Na+ because they accumulated more Na+ and less K+ than wild-type cells which showed high discrimination between K+ and Na+.

The byp1-3 allele of the Saccharomyces cerevisiae GGS1/TPS1 gene and its multi-copy suppressor tRNA(GLN) (CAG): Ggs1/Tps1 protein levels restraining growth on fermentable sugars and trehalose accumulation.

Byp1-3 is an amber nonsense allele of the Saccharomyces cerevisiae GGS1/TPS1 gene which encodes the small subunit of the trehalose synthase complex. Mutations in this gene confer an inability to grow on glucose or fructose but the phenotype of byp1-3 mutants is leaky in a strain-dependent manner. Overexpression of the isolated byp1-3 allele suppressed the growth defect of a ggs1/tps1 delta mutant. Expression of an in-vitro-generated mutant allele of GGS1/TPS1 that lacks all the coding sequences downstream from the byp1-3 mutation led to the production of a shortened protein that did not complement the ggs1/tps1 delta mutant. We have isolated, as an allele-specific multi-copy suppressor of the growth defect of the byp1-3 mutant on fructose, the gene for tRNA(GLN) (CAG). Thus the leaky phenotype of byp1-3 mutants is due to a low level of read through of the internal nonsense codon by tRNA(GLN) (CAG). Using overexpression of the isolated byp1-3 allele, as well as of the tRNA(GLN) (CAG) gene, we were able to demonstrate that as little as about 10% of the normal Ggs1/Tps1 protein level is sufficient for slow growth on fructose. We also show a correlation between the level of Ggs1/Tps1, the ability to accumulate trehalose in stationary phase and the ability to grow on fermentable sugars. Sequence analysis of the cloned tRNA(GLN) (CAG) gene showed that it is located 700 bp upstream of URA10. However, we found considerable differences to the reported sequence of URA10, in particular in the non-coding region.

Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress.

The Saccharomyces cerevisiae FPS1 gene, which encodes a channel protein belonging to the MIP family, has been isolated previously as a multicopy suppressor of the growth defect of the fdp1 mutant (allelic to GGS1/TPS1) on fermentable sugars. Here we show that overexpression of FPS1 enhances glycerol production. Enhanced glycerol production caused by overexpression of GPD1 encoding glycerol-3-phosphate dehydrogenase also suppressed the growth defect of ggs1/tps1 delta mutants, suggesting a novel role for glycerol production in the control of glycolysis. The suppression of ggs1/tps1 delta mutants by GPD1 depends on the presence of Fps1. Mutants lacking Fps1 accumulate a greater part of the glycerol intracellularly, indicating that Fps1 is involved in glycerol efflux. Glycerol-uptake experiments showed that the permeability of the yeast plasma membrane for glycerol consists of an Fps1-independent component probably due to simple diffusion and of an Fps1-dependent component representing facilitated diffusion. The Escherichia coli glycerol facilitator expressed in a yeast fps1 delta mutant can restore the characteristics of glycerol uptake, production and distribution fully, but restores only partially growth of a ggs1/tps1 delta fps1 delta double mutant on glucose. Fps1 appears to be closed under hyperosmotic stress when survival depends on intracellular accumulation of glycerol and apparently opens rapidly when osmostress is lifted. The osmostress-induced High Osmolarity Glycerol (HOG) response pathway is not required for inactivation of Fps1. We conclude that Fps1 is a regulated yeast glycerol facilitator controlling glycerol production and cytosolic concentration, and might have additional functions.

Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis.

The GGS1/TPS1 gene of the yeast Saccharomyces cerevisiae encodes the trehalose-6-phosphate synthase subunit of the trehalose synthase complex. Mutants defective in GGS1/TPS1 have been isolated repeatedly and they showed variable pleiotropic phenotypes, in particular with respect to trehalose content, ability to grow on fermentable sugars, glucose-induced signaling and sporulation capacity. We have introduced the fdp1, cif1, byp1 and glc6 alleles and the ggs1/tps1 deletion into three different wild-type strains, M5, SP1 and W303-1A. This set of strains will aid further studies on the molecular basis of the complex pleiotropic phenotypes of ggs1/tps1 mutants. The phenotypes conferred by specific alleles were clearly dependent on the genetic background and also differed for some of the alleles. Our results show that the lethality caused by single gene deletion in one genetic background can become undetectable in another background. The sporulation defect of ggs1/tps1 diploids was neither due to a deficiency in G1 arrest, nor to the inability to accumulate trehalose. Ggs1/tps1 delta mutants were very sensitive to glucose and fructose, even in the presence of a 100-fold higher galactose concentration. Fifty-percent inhibition occurred at concentrations similar to the Km values of glucose and fructose transport. The inhibitory effect of glucose in the presence of a large excess of galactose argues against an overactive glycolytic flux as the cause of the growth defect. Deletion of genes of the glucose carrier family shifted the 50% growth inhibition to higher sugar concentrations. This finding allows for a novel approach to estimate the relevance of the many putative glucose carrier genes in S. cerevisiae. We also show that the GGS1/TPS1 gene product is not only required for the transition from respirative to fermentative metabolism but continuously during logarithmic growth on glucose, in spite of the absence of trehalose under such conditions.

Disruption of the Kluyveromyces lactis GGS1 gene causes inability to grow on glucose and fructose and is suppressed by mutations that reduce sugar uptake.

In the yeast Saccharomyces cerevisiae the GGS1 gene is essential for growth on glucose or other readily fermentable sugars. GGS1 is the same gene as TPS1 which was identified as encoding a subunit of the trehalose-6-phosphate synthase/phosphatase complex and it is allelic to the fdp1, byp1, glc6 and cif1 mutations. Its precise function in the regulation of sugar catabolism is unknown. We have cloned the GGS1 homologue from the distantly related yeast Kluyveromyces lactis. The KlGGS1 gene is 74% and 79% identical at the nucleotide and amino acid sequence level, respectively, to the S. cerevisiae counterpart. We also compared the sequence with the partly homologous products of the S. cerevisiae genes TPS2 and TSL1 which code for the larger subunits of the trehalose synthase complex and with a TSL1 homologue, TPS3, of unknown function. Multiple alignment of these sequences revealed several particularly well conserved elements. Disruption of GGS1 in K. lactis caused the same pleiotropic phenotype as in S. cerevisiae, i.e. inability to grow on glucose or fructose and strongly reduced trehalose content. We have also studied short-term glucose-induced regulatory effects related to cAMP and cAMP-dependent protein kinase, i.e. the cAMP signal, trehalase activation, trehalose mobilization and inactivation of fructose-1,6-bisphosphatase. These effects occur very rapidly in S. cerevisiae and are absent in the Scggs1 mutant. In K. lactis all these effects were much slower and largely unaffected by the Klggs1 mutation. On the other hand, glucose strongly induced pyruvate decarboxylase and activated the potassium transport system in K. lactis and both effects were absent in the Klggs1 mutant. Addition of glucose to galactose-grown cells of the Klggs1 mutant caused, as in S. cerevisiae, intracellular accumulation of free glucose and of sugar phosphates and a rapid drop of the ATP and inorganic phosphate levels. Glucose transport kinetics were the same for the wild type and the Klggs1 mutant in both derepressed cells and in cells incubated with glucose. We have isolated phenotypic revertants of the Klggs1 mutant for growth on fructose. The suppressors that we characterized had, to different extents, diminished glucose uptake in derepressed cells but cells incubated in glucose showed very different characteristics. The suppressor mutations prevented deregulation of glycolysis in the Klggs1 mutant but not the accumulation of free glucose. The mutants with higher residual uptake activity showed partially restored induction of pyruvate decarboxylase and activation of potassium transport.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae display a wide range of glucose-induced regulatory phenomena, including glucose-induced activation of the RAS-adenylate cyclase pathway and phosphatidylinositol turnover, rapid post-translational effects on the activity of different enzymes as well as long-term effects at the transcriptional level. A gene called GGS1 (for General Glucose Sensor) that is apparently required for the glucose-induced regulatory effects and several ggs1 alleles (fdp1, byp1 and cif1) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggs1 mutants are unable to grow on glucose or related readily fermentable sugars, apparently owing to unrestricted influx of sugar into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggs1 delta strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose-sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose-induced regulatory phenomena. Deregulation of these pathways in ggs1 mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggs1 diploids to sporulate.

Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae.

Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1delta mutant displayed only simple diffusion. Transformation of the fps1delta mutant with the glpF gene, which encodes glycerol transport in Escherichia coli, restored biphasic transport kinetics. Yeast extract-peptone-dextrose-grown wild-type cells had a higher passive diffusion constant than the fps1delta mutant, and ethanol enhanced the rate of proton diffusion to a greater extent in the wild type than in the fps1delta mutant. In addition, the lipid fraction of the fps1delta mutant contained a lower percentage of phospholipids and a higher percentage of glycolipids than that of the wild type. Fps1p, therefore, may be involved in the regulation of lipid metabolism in S. cerevisiae, affecting membrane permeability in addition to fulfilling its specific role in glycerol transport. Simultaneous uptake of glycerol and protons occurred in both glycerol- and ethanol-grown wild-type and fps1delta cells and resulted in the accumulation of glycerol at an inside-to-outside ratio of 12:1 to 15:1. Carbonyl cyanide m-chlorophenylhydrazone prevented glycerol accumulation in both strains and abolished transport in the fps1delta mutant grown on ethanol. Likewise, 2,4-dinitrophenol inhibited transport in glycerol-grown wild-type cells. These results indicate the presence of an Fps1p-dependent facilitated diffusion system in glucose-grown cells and an Fps1p-independent proton symport system in derepressed cells.

Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation.

The accumulation of compatible solutes, such as glycerol, in the yeast Saccharomyces cerevisiae, is a ubiquitous mechanism in cellular osmoregulation. Here, we demonstrate that yeast cells control glycerol accumulation in part via a regulated, Fps1p-mediated export of glycerol. Fps1p is a member of the MIP family of channel proteins most closely related to the bacterial glycerol facilitators. The protein is localized in the plasma membrane. The physiological role of Fps1p appears to be glycerol export rather than uptake. Fps1 delta mutants are sensitive to hypo-osmotic shock, demonstrating that osmolyte export is required for recovery from a sudden drop in external osmolarity. In wild-type cells, the glycerol transport rate is decreased by hyperosmotic shock and increased by hypo-osmotic shock on a subminute time scale. This regulation seems to be independent of the known yeast osmosensing HOG and PKC signalling pathways. Mutants lacking the unique hydrophilic N-terminal domain of Fps1p, or certain parts thereof, fail to reduce the glycerol transport rate after a hyperosmotic shock. Yeast cells carrying these constructs constitutively release glycerol and show a dominant hyperosmosensitivity, but compensate for glycerol loss after prolonged incubation by glycerol overproduction. Fps1p may be an example of a more widespread class of regulators of osmoadaptation, which control the cellular content and release of compatible solutes.