Key amino acid residues of the AGT1 permease required for maltotriose consumption and fermentation by Saccharomyces cerevisiae.

AIMS: The AGT1 gene encodes for a general α-glucoside-H+ symporter required for efficient maltotriose fermentation by Saccharomyces cerevisiae. In the present study, we analysed the involvement of four charged amino acid residues present in this transporter that are required for maltotriose consumption and fermentation by yeast cells. METHODS AND RESULTS: By using a knowledge-driven approach based on charge, conservation, location, three-dimensional (3D) structural modelling and molecular docking analysis, we identified four amino acid residues (Glu-120, Asp-123, Glu-167 and Arg-504) in the AGT1 permease that could mediate substrate binding and translocation. Mutant permeases were generated by site-directed mutagenesis of these charged residues, and expressed in a yeast strain lacking this permease (agt1∆). While mutating the Arg-504 or Glu-120 residues into alanine totally abolished (R504A mutant) or greatly reduced (E120A mutant) maltotriose consumption by yeast cells, as well as impaired the active transport of several other α-glucosides, in the case of the Asp-123 and Glu-167 amino acids, it was necessary to mutate both residues (D123G/E167A mutant) in order to impair maltotriose consumption and fermentation. CONCLUSIONS: Based on the results obtained with mutant proteins, molecular docking and the localization of amino acid residues, we propose a transport mechanism for the AGT1 permease. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results present new insights into the structural basis for active α-glucoside-H+ symport activity by yeast transporters, providing the molecular bases for improving the catalytic properties of this type of sugar transporters.

Extracellular maltotriose hydrolysis by Saccharomyces cerevisiae cells lacking the AGT1 permease.

In brewing, maltotriose is the least preferred sugar for uptake by Saccharomyces cerevisiae cells. Although the AGT1 permease is required for efficient maltotriose fermentation, we have described a new phenotype in some agt1Δ strains of which the cells do not grow on maltotriose during the first 3-4 days of incubation, but after that, they start to grow on the sugar aerobically. Aiming to characterize this new phenotype, we performed microarray gene expression analysis which indicated upregulation of high-affinity glucose transporters (HXT4, HXT6 and HXT7) and α-glucosidases (MAL12 and IMA5) during this delayed cellular growth. Since these results suggested that this phenotype might be due to extracellular hydrolysis of maltotriose, we attempted to detect glucose in the media during growth. When an hxt-null agt1Δ strain was grown on maltotriose, it also showed the delayed growth on this carbon source, and glucose accumulated in the medium during maltotriose consumption. Considering that the poorly characterized α-glucosidase encoded by IMA5 was among the overexpressed genes, we deleted this gene from an agt1Δ strain that showed delayed growth on maltotriose. The ima5Δ agt1Δ strain showed no maltotriose utilization even after 200 h of incubation, suggesting that IMA5 is likely responsible for the extracellular maltotriose hydrolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Maltotriose is the second most abundant sugar present in brewing. However, many yeast strains have difficulties to consume maltotriose, mainly because of its low uptake rate by the yeast cells when compared to glucose and maltose uptake. The AGT1 permease is required for efficient maltotriose fermentation, but some strains deleted in this gene are still able to grow on maltotriose after an extensive lag phase. This manuscript shows that such delayed growth on maltotriose is a consequence of extracellular hydrolysis of the sugar. Our results also indicate that the IMA5-encoded α-glucosidase is likely the enzyme responsible for this phenotype.

Characterization of maltotriose transporters from the Saccharomyces eubayanus subgenome of the hybrid Saccharomyces pastorianus lager brewing yeast strain Weihenstephan 34/70.

The genome from the Saccharomyces pastorianus industrial lager brewing strain Weihenstephan 34/70, a natural Saccharomyces cerevisiae/Saccharomyces eubayanus hybrid, indicated the presence of two different maltotriose transporter genes: a new gene in the S. eubayanus subgenome with 81% of homology to the AGT1 permease from S. cerevisiae, and an amplification of the S. eubayanus MTY1 maltotriose permease previously identified in S. pastorianus yeasts. To characterize these S. eubayanus transporter genes, we used a S. cerevisiae strain deleted in the AGT1 permease and introduced the desired permease gene(s) into this locus through homologous recombination. Our results indicate that both the MTY1 and AGT1 genes from the S. eubayanus subgenome encode functional maltotriose transporters that allow fermentation of this sugar by yeast cells, despite their apparent differences in the kinetics of maltotriose-H(+) symport activity. The presence of two maltotriose transporters in the S. eubayanus subgenome not only highlights the importance of sugar transport for efficient maltotriose utilization by industrial yeasts, but these new genes can be used in breeding and/or selection programs aimed at increasing yeast fitness for the efficient fermentation of brewer's wort.

Microarray karyotyping of maltose-fermenting Saccharomyces yeasts with differing maltotriose utilization profiles reveals copy number variation in genes involved in maltose and maltotriose utilization.

AIMS: We performed an analysis of maltotriose utilization by 52 Saccharomyces yeast strains able to ferment maltose efficiently and correlated the observed phenotypes with differences in the copy number of genes possibly involved in maltotriose utilization by yeast cells. METHODS AND RESULTS: The analysis of maltose and maltotriose utilization by laboratory and industrial strains of the species Saccharomyces cerevisiae and Saccharomyces pastorianus (a natural S. cerevisiae/Saccharomyces bayanus hybrid) was carried out using microscale liquid cultivation, as well as in aerobic batch cultures. All strains utilize maltose efficiently as a carbon source, but three different phenotypes were observed for maltotriose utilization: efficient growth, slow/delayed growth and no growth. Through microarray karyotyping and pulsed-field gel electrophoresis blots, we analysed the copy number and localization of several maltose-related genes in selected S. cerevisiae strains. While most strains lacked the MPH2 and MPH3 transporter genes, almost all strains analysed had the AGT1 gene and increased copy number of MALx1 permeases. CONCLUSIONS: Our results showed that S. pastorianus yeast strains utilized maltotriose more efficiently than S. cerevisiae strains and highlighted the importance of the AGT1 gene for efficient maltotriose utilization by S. cerevisiae yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results revealed new maltotriose utilization phenotypes, contributing to a better understanding of the metabolism of this carbon source for improved fermentation by Saccharomyces yeasts.

Expression of high-affinity trehalose-H+ symport in Saccharomyces cerevisiae.

The expression of the high-affinity trehalose-H+ symport was investigated in various Saccharomyces cerevisiae strains and culture conditions. Previous kinetic studies of trehalose transport in yeast have revealed the existence of at least two different uptake mechanisms: a high-affinity trehalose-H+ symport activity repressed by glucose, and a constitutive low-affinity transport activity, a putative facilitated diffusion process. Exogenously added trehalose was not an inducer of the high-affinity transport activity, and a correlation between trehalose and maltose uptake by yeast cells was found. Our results indicate that the maltose-H+ symporters encoded by MAL11, MAL21, and MAL41 are not responsible for the trehalose transport activity. The analysis of both trehalose and maltose transport activities in wild-type and in laboratory strains with defined MAL genes showed that the trehalose-H+ symporter was under control of MAL regulatory genes. Our results also suggest that the recently characterized AGT1 gene of S. cerevisiae may encode the high-affinity trehalose-H+ symporter. During diauxic growth on glucose the transport activity was low during the first exponential phase of growth, increased as glucose was exhausted from the medium, and decreased again as the cells reached the late stationary phase. This pattern was coincident with that of the intracellular levels of trehalose. The strong correlation between these two parameters may be of physiological significance during adaptation of yeast cells to stress conditions.

Secretion of the acid trehalase encoded by the CgATH1 gene allows trehalose fermentation by Candida glabrata.

The emergent pathogen Candida glabrata differs from other yeasts because it assimilates only two sugars, glucose and the disaccharide trehalose. Since rapid identification tests are based on the ability of this yeast to rapidly hydrolyze trehalose, in this work a biochemical and molecular characterization of trehalose catabolism by this yeast was performed. Our results show that C. glabrata consumes and ferments trehalose, with parameters similar to those observed during glucose fermentation. The presence of glucose in the medium during exponential growth on trehalose revealed extracellular hydrolysis of the sugar by a cell surface acid trehalase with a pH optimum of 4.4. Approximately ∼30% of the total enzymatic activity is secreted into the medium during growth on trehalose or glycerol. The secreted enzyme shows an apparent molecular mass of 275 kDa in its native form, but denaturant gel electrophoresis revealed a protein with ∼130 kDa, which due to its migration pattern and strong binding to concanavalin A, indicates that it is probably a dimeric glycoprotein. The secreted acid trehalase shows high affinity and activity for trehalose, with Km and Vmax values of 3.4 mM and 80 U (mg protein)(-1), respectively. Cloning of the CgATH1 gene (CAGLOK05137g) from de C. glabrata genome, a gene showing high homology to fungal acid trehalases, allowed trehalose fermentation after heterologous expression in Saccharomyces cerevisiae.

Active alpha-glucoside transport in Saccharomyces cerevisiae.

The AGT1 permease is a alpha-glucoside-H+ symporter responsible for the active transport of maltose, trehalose, maltotriose, alpha-methylglucoside, melezitose and sucrose. In wild-type as well as in MAL constitutive strains, alpha-methylglucoside seemed to be the best inducer of transport activity, while trehalose had no inducing effect. Based on the initial rates of transport it seems that the sugar preferentially transported by this permease is trehalose, followed by sucrose.

Colorimetric determination of active alpha-glucoside transport in Saccharomyces cerevisiae.

Fermentation of alpha-glucosides (maltose, maltotriose) by Saccharomyces cerevisiae cells is a critical phase in the processes of brewing and breadmaking. Utilization of alpha-glucosides requires the active transport of the sugar across the cell membrane and, subsequently, its hydrolysis by cytoplasmic glucosidases. Although transport activities are usually assayed using radiolabeled substrates, we have developed a simple, cheap and reliable colorimetric assay for the determination of alpha-glucoside uptake using p-nitrophenyl-alpha-D-glucopyranoside (pNPalphaG) as substrate. Our results show that pNPalphaG is actively transported by S. cerevisiae cells by a H+-symport mechanism, which depends on the electrochemical proton gradient across the plasma membrane. pNPalphaG uptake is mediated by the AGT1 alpha-glucoside permease, which has a high affinity (Km=3 mM) for this chromogenic substrate. This simple colorimetric uptake assay can be used to analyze the expression and regulation of the AGT1 permease in S. cerevisiae cells.

An assay for glycosylphosphatidylinositol-anchor degrading phospholipases.

This paper describes a new approach to assay phospholipases which cleave glycosylphosphatidylinositol using a biotinylated protein substrate coupled to 125I-streptavidin and Triton X-114 phase separation. Substrate preparation with variant surface glycoprotein of Trypamosoma brucei, its characterization and solubilization by glycosylphosphatidylinositol-specific phospholipase C and D are reported. Hydrolysis of substrate exhibited first-order kinetics with respect to enzyme concentration, and the rate constant of the reaction is independent both from substrate concentration and reaction time. This assay was compared with the one using 3H-myristoylated variant surface glycoprotein and proved to be equally suitable to quantitate glycosylphosphatidylinositol-specific phospholipases, with the advantage that avoids biosynthetic labeling. Furthermore, it introduces a basic methodology which can be easily adapted to use other glycosylphosphatidylinositol-anchored proteins as substrates.

Further characterization of the acidic GPI-hydrolyzing phospholipase present in human sera.

A phospholipase from human serum capable of hydrolyzing glycosylphosphatidylinositol membrane anchors was described and partially characterized by our group some years ago. This activity presented a pH optimum between 5.0 and 6.0 and was inhibited by EDTA, EGTA and 1,10-phenanthroline. Partial purification showed that the enzyme was a glycoprotein with an apparent molecular weight of 140 kDa as judged by gel filtration. Other investigators characterized at the same time a phospholipase D with similar properties but with a pH optimum near 7.5. We now confirm that the human serum enzyme is indeed a phospholipase D capable of hydrolyzing mfVSG and glycolipids A and C from T. brucei. Isoelectric focusing of whole sera suggests the presence of two isoforms, one with a pI of 4.7 which was the form previously purified by our group, and others with pI from 6.2 to 7.4.

Kinetics of active sucrose transport in Saccharomyces cerevisiae.

The kinetic analysis of active sucrose-H+ uptake by Saccharomyces cerevisiae revealed the presence of two transport systems with high and low affinity for sucrose. The MAL2T permease has a low affinity (K(m) = 120 +/-20 mM) for sucrose, while the alpha-glucoside transporter encoded by the AGT1 gene is a high affinity sucrose-H+ symporter (K(m) = 7.9+/-0.8 mM) that increases the specific growth rate of cells growing on sucrose.

[Comparative study of the effects of auranofin and aurothiomalate on laboratory and clinical indicators in patients with rheumatoid arthritis]. / Komparativno istrazivanje ucinka auranofina i aurotiomalata na pojedine laboratorijske i klinicke pokazatelje u bolesnika s reumatoidnim artritisom.

Is there any significant difference in the effect and tolerance of the gold salts applied peroral and intramuscular in patients with rheumatoid arthritis (RA)? 97 patients with RA have been included in the research. Group used auranofin perorally comprised 30 patients with RA, 25 women and 5 men. Their average age was 53.4 years, the average disease course was 9.06 years. Group used aurothiomalate parenterally comprised 30 patients with RA, 23 women and 7 men. Their average age was 52.5 years, the average duration of their illness being 10.87 years. Control group comprised 37 patients with RA, 27 women and 10 men. Their average age was 58.2 years, the average disease course was 8.3 years. They did not use any "second line drug" or corticosteroids. During a six-month (26 week) continuous application of the gold salts (perorally and parenterally) the following parameters were observed in regular intervals: the erythrocyte sedimentation rate, the hemoglobin level in the serum, the C-reactive protein. Ritchie index, the PIP extent of the fist joints and the morning stiffness span of the small fist joints. The tolerance of the gold salts has also been controlled. The results have shown that there is no any significant difference between two forms of the gold salts in patients with RA. The statistical processing of data indicated that auranofin and aurothiomalate have significant effect on all controlled parameters. As regard of the side effects, patients accepted aurothiomalate better than auranofin.

[Use of non-steroidal antiinflammatory drugs in specialty polyclinic practice]. / Primjena nesteroidnih antiinflamatornih lijekova u specijalistickoj poliklinickoj praksi.

A retrospective investigation of the prescription of nonsteroidal antiinflammatory drugs (NSA) was performed in the Rheumatologic out-patient-institute in Zagreb, including 1000 patients of both sexes, aged 20-70 years. 500 outpatients were treated by NSA during 1987 and 1989 respectively for lumbosacral syndrome, rheumatoid arthritis, ankylosing spondylitis and coxarthrosis. The kind of NSA as well as the registered side-effects were analysed from case histories. During 1987, NSA were applied to 365 (73%) and during 1989 to 390 (78%) of the 500 patients. In both groups a phenyl-acetic acid derivative (diclophenac) was most often applied, followed by propionic acid derivatives and oxycams. The most rarely applied drugs were indol-acetic acid derivatives. Pyrazolones were given only to 2 patients with an acute flare of ankylosing spondylitis in 1987. A gastro-duodenal ulcer was the absolute counterindication for this kind of treatment. The number of side-effects in this investigation was relatively small (6.5% in 1987 and 5% in 1989), probably because this investigation was a retrospective one. The most common among them appeared in the gastro-intestinal tract.

Efectividad del tratamiento médico con misoprostol según dosis administrada en aborto retenido menor de 12 semanas / Effectiveness of medical treatment with misoprostol as abortion retained dose administered in less than 12 weeks

Antecedentes: El manejo terapéutico del aborto retenido consiste en evacuar la cavidad uterina espontáneamente o utilizando misoprostol previo al legrado quirúrgico. Objetivo: Evaluar la necesidad de dilatación mecánica post maduración cervical con misoprostol y la tasa de perforación uterina post legrado, utilizando diferentes dosis de misoprostol en pacientes con diagnóstico de aborto retenido menor a 12 semanas. Métodos: Se registraron datos demográficos y ginecológicos de una cohorte retrospectiva de pacientes con diagnóstico de aborto retenido menor a 12 semanas, entre enero de 2008 y diciembre de 2010. Se establecieron 3 grupos de trabajo según la dosis de misoprostol administrada vía vaginal, siendo de 100 (n=131), 200 (n=231) y 400 micrones (n=230), y se observaron las complicaciones asociadas al procedimiento. Resultados: La necesidad de dilatación mecánica fue significativamente mayor en el grupo que recibió 100 micrones de misoprostol al compararlo con el de 200 micrones y 400 micrones (p<0,01). No hubo diferencias estadísticamente significativas entre las que recibieron 200 versus 400 micrones de misoprostol. No hubo diferencias significativas respecto a perforación uterina. Conclusión: En el aborto retenido menor a 12 semanas, la necesidad de dilatación mecánica post maduración cervical, es menor si se utiliza 200 o 400 micrones de misoprostol, sin diferencias en la tasa de perforación uterina.

Background: The therapeutic management of missed abortion consists on evacuating the uterine cavity, spontaneously or by administration of misoprostol previous to curettage. Objectives: Evaluate the need of mechanical dilatation after cervical maturation with misoprostol and the rate of uterine perforation before curettage, using different doses of misoprostol in patients with diagnosis of missed abortion before 12 weeks. Methods: Demographic and gynecologic data were registered of a retrospective cohort of patients with the diagnosis of missed abortion before 12 weeks, between January 2008 and December 2010. Three groups were established according to the dose of misoprostol: 100 (n=131), 200 (n=231) and 400 microns (n=230). Complications associated to the procedure were observed. Results: The need of mechanical dilatation was significant higher for the group with 100 microns of misoprostol in comparison with 200 and 400 microns (p<0.001). There was no statistical significance among who received 200 versus 400 microns of misoprostol. No statistical significance was found for uterine perforation. Conclusion: In the missed abortion before 12 week, the need of mechanical dilatation is lower with 200 or 400 microns of misoprostol, without difference in uterine perforation rate.

Characterization of D-glucose transport in Trypanosoma rangeli.

Like in other trypanosomatids D-glucose is a crucial source of energy to Trypanosoma rangeli, a non-pathogenic parasite that in Central and South America infects triatomine vectors and different mammalian species, including humans. In several trypanosome species, D-glucose transporters were already described and cloned. In this study, we characterized the D-glucose transport activity present in 2 life-stage forms of T. rangeli (epimastigotes and trypomastigotes) using D-[U-14C]glucose as substrate. Our results indicate that T. rangeli transports D-glucose with high affinity in both epimastigote (Km 30 microM) and trypomastigotes (Km 80 microM) life-forms. Both transport activities were inhibited by Cytochalasin B and Phloretin, indicating that probably D-glucose uptake in T. rangeli is mediated by facilitated diffusion of the sugar. Significant differences were observed between epimastigotes and trypomastigotes in relation to their affinity for D-glucose analogues, and the predicted amino acid sequence of a putative D-glucose transporter from T. rangeli (TrHT1) showed a larger identity with the T. cruzi D-glucose transporter encoded by the TcrHT1 gene than with other transporters already characterized in trypanosomatids.

Improvement of maltotriose fermentation by Saccharomyces cerevisiae.

AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts.

The Kluyver effect for trehalose in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells are able to grow using trehalose as a sole source of carbon and energy. However, the biomass yield obtained with trehalose was higher, and the specific growth rate lower, than that obtained with glucose or maltose. The respiratory inhibitor antimycin A prevented cell growth on trehalose, and no ethanol or glycerol was formed during batch growth on this carbon source. Thus, S. cerevisiae exhibits the KLUYVER effect for trehalose: this disaccharide is assimilated and respired, but, in contrast to glucose or maltose, it cannot be fermented. The high-affinity trehalose-H+ symporter encoded by the AGT1 gene is required for growth on trehalose. Analysis of the differences in the metabolism of maltose and trehalose (both disaccharides of glucose transported by active transport systems) indicated that the absence of trehalose fermentation is a consequence of low sugar influx into the cells during growth on this carbon source.

Kinetics of active alpha-glucoside transport in Saccharomyces cerevisiae.

alpha-Glucosides are the most abundant fermentable sugars in the industrial applications of Saccharomyces cerevisiae, and the active transport across the plasma membrane is the rate-limiting step for their metabolism. In this report we performed a detailed kinetic analysis of the active alpha-glucoside transport system(s) present in a wild-type strain, and in strains with defined alpha-glucoside permeases. Our results indicate that the wild-type strain harbors active transporters with high and low affinity for maltose and trehalose, and low-affinity transport systems for maltotriose and alpha-methylglucoside. The maltose permease encoded by the MAL21 gene showed a high affinity (K(m) approximately 5 mM) for maltose, and a low affinity (K(m) approximately 90 mM) for trehalose. On the other hand, the alpha-glucoside permease encoded by the AGT1 gene had a high affinity (K(m) approximately 7 mM) for trehalose, a low affinity (K(m) approximately 18 mM) for maltose and maltotriose, and a very low affinity (K(m) approximately 35 mM) for alpha-methylglucoside.