A single mutation in enzyme I of the sugar phosphotransferase system confers penicillin tolerance to Streptococcus gordonii.

Tolerance is a poorly understood phenomenon that allows bacteria exposed to a bactericidal antibiotic to stop their growth and withstand drug-induced killing. This survival ability has been implicated in antibiotic treatment failures. Here, we describe a single nucleotide mutation (tol1) in a tolerant Streptococcus gordonii strain (Tol1) that is sufficient to provide tolerance in vitro and in vivo. It induces a proline-to-arginine substitution (P483R) in the homodimerization interface of enzyme I of the sugar phosphotransferase system, resulting in diminished sugar uptake. In vitro, the susceptible wild-type (WT) and Tol1 cultures lost 4.5 and 0.6 log(10) CFU/ml, respectively, after 24 h of penicillin exposure. The introduction of tol1 into the WT (WT P483R) conferred tolerance (a loss of 0.7 log(10) CFU/ml/24 h), whereas restitution of the parent sequence in Tol1 (Tol1 R483P) restored antibiotic susceptibility. Moreover, penicillin treatment of rats in an experimental model of endocarditis showed a complete inversion in the outcome, with a failure of therapy in rats infected with WT P483R and the complete disappearance of bacteria in animals infected with Tol1 R483P.

Simple-sugar meals target GLUT2 at enterocyte apical membranes to improve sugar absorption: a study in GLUT2-null mice.

The physiological significance of the presence of GLUT2 at the food-facing pole of intestinal cells is addressed by a study of fructose absorption in GLUT2-null and control mice submitted to different sugar diets. Confocal microscopy localization, protein and mRNA abundance, as well as tissue and membrane vesicle uptakes of fructose were assayed. GLUT2 was located in the basolateral membrane of mice fed a meal devoid of sugar or containing complex carbohydrates. In addition, the ingestion of a simple sugar meal promoted the massive recruitment of GLUT2 to the food-facing membrane. Fructose uptake in brush-border membrane vesicles from GLUT2-null mice was half that of wild-type mice and was similar to the cytochalasin B-insensitive component, i.e. GLUT5-mediated uptake. A 5 day consumption of sugar-rich diets increased fructose uptake fivefold in wild-type tissue rings when it only doubled in GLUT2-null tissue. GLUT5 was estimated to contribute to 100 % of total uptake in wild-type mice fed low-sugar diets, falling to 60 and 40 % with glucose and fructose diets respectively; the complement was ensured by GLUT2 activity. The results indicate that basal sugar uptake is mediated by the resident food-facing SGLT1 and GLUT5 transporters, whose mRNA abundances double in long-term dietary adaptation. We also observe that a large improvement of intestinal absorption is promoted by the transient recruitment of food-facing GLUT2, induced by the ingestion of a simple-sugar meal. Thus, GLUT2 and GLUT5 could exert complementary roles in adapting the absorption capacity of the intestine to occasional or repeated loads of dietary sugars.

Expression of asparagine synthetase in response to carbohydrate supply in model callus cultures and shoot tips of asparagus (Asparagus officinalis L.)

Sugar uptake and metabolism were studied in callus cultures and shoot tips of asparagus. Asparagus callus cultures were used to model senescence in shoot tips. Callus cultures absorbed glucose from a nutrient medium, and accumulated sucrose, glucose and fructose. This uptake of glucose by the callus cultures down-regulated expression of asparagine synthetase and beta -galactosidase transcripts that otherwise accumulated when sugar was withheld. When 80 mm-long asparagus shoots were excised from growing plants and placed in 2% and 8% sucrose solutions, endogenous concentrations of sucrose, glucose, fructose, UDPglucose, and glucose-6-phosphate declined in the 30mm-long meristematic tip regions. At the same time, asparagine and asparagine synthetase gene transcripts began to accumulate in these tips. When 10 mm-long asparagus shoot tips were placed on glucose- or fructose-containing agar, the tips accumulated sucrose, glucose and fructose, and asparagine accumulation and expression of asparagine synthetase were marginally reduced. We concluded that in callus cultures, asparagine synthetase expression was sugar regulated, but that sugar regulation was not as pronounced in asparagus shoot tips. This may be due in part to slower rates of sugar uptake into shoot tips and in part to compartmentation of sugars in the tips. We suggest that callus cultures are not a suitable model for metabolic studies in asparagus shoot tips.

Hexose and pentose transport inascomycetousyeasts: an overview

FEMS Yeast Research, Vol. 9, nº 4

Facilitated glucose transporters in epithelial cells.

The molecular cloning of facilitated sugar transporters has led to the identification of a family of transport molecules having similar functions, but possessing specific kinetic and regulatory properties. These transporter isoforms are characterized by different primary structures, specific tissue localization, and polarized expression within the same epithelial cells. The use of Xenopus oocytes for the functional expression of different members of this transporter family has been of considerable value in defining the kinetic properties and sugar specificities of the different isoforms. The expression of chimeric or variously mutated transporters should, in the near future, permit the determination of the structural basis for their kinetic properties and sugar specificities. cDNA probes and antipeptide antibodies specific for each isoform are now being used to determine their specific regulation during development and in different states of altered glucose homeostasis. The variety of molecular forms implicated in the apparently simple task of sugar uptake or transepithelial transport has been surprising. With the available molecular tools now in hand, it will be possible to study these mechanisms in much greater detail.

Molecular analysis of maltotriose active transport and fermentation by Saccharomyces cerevisiae reveals a determinant role for the AGT1 permease

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known alpha-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and AM inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high-and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K-m, 36 +/- 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.

T-3 Rapidly Increases SLC2A4 Gene Expression and GLUT4 Trafficking to the Plasma Membrane in Skeletal Muscle of Rat and Improves Glucose Homeostasis

Background: Glucose transporter 4 (GLUT4) is highly expressed in muscle and fat tissue, where triiodothyronine (T-3) induces solute carrier family 2 facilitated glucose transporter member 4 (SLC2A4) gene transcription. T-3 was also shown to rapidly increase glucose uptake in myocytes exposed to cycloheximide, indicating that it might act nongenomically to regulate GLUT4 availability. We tested this hypothesis by evaluating, in thyroidectomized rats (Tx rats), the acute and/or chronic T-3 effects on GLUT4 mRNA expression and polyadenylation, protein content, and trafficking to the plasma membrane (PM) in skeletal muscle, as well as on blood glucose disappearance rate (kITT) after insulin administration. Methods: Rats were surgically thyroidectomized and treated with T-3 (0.3 to 100 mu g/100 g body weight) from 10 minutes to 5 days, and killed thereafter. Sham-operated (SO) rats were used as controls. Total RNA was extracted from the skeletal muscles (soleus [SOL] and extensorum digitalis longus [EDL]) and subjected to Northern blotting analysis using rat GLUT4 cDNA probe. Total protein was extracted and subjected to specific centrifugations for subcellular fractionation, and PM as well as microsomal (M) fractions were subjected to Western blotting analysis, using anti-GLUT4 antiserum as a probe. GLUT4 mRNA polyadenylation was examined by a rapid amplification of cDNA ends-poly(A) test (RACE-PAT). Results: Thyroidectomy reduced skeletal muscle GLUT4 mRNA, mRNA poly(A) tail length, protein content, and trafficking to the PM, as well as the kITT. The acute T-3 treatment rapidly (30 minutes) increased all these parameters compared with Tx rats. The 5-day T-3 treatment increased GLUT4 mRNA and protein expression, and restored GLUT4 trafficking to the PM and kITT to SO values. Conclusions: The results presented here show for the first time that, in parallel to its transcriptional action on the SLC2A4 gene, T-3 exerts a rapid post-transcriptional effect on GLUT4 mRNA polyadenylation, which might increase transcript stability and translation efficiency, leading to the increased GLUT4 content and availability to skeletal muscle, as well as on GLUT4 translocation to the PM, improving the insulin sensitivity, as shown by the kITT.

Bedeutung von allelen Varianten des Hexose-Transporters Hxt3p und der Hexokinasen Hxk1p und Hxk2p für Aufnahme und Verwertung von Glucose und Fructose durch Weinhefen

In Hinsicht darauf, dass sich S. cerevisiae-Stämme im Laufe der Domestizierung und Anpassung an verschiedene Habitate genetisch verändert haben, wurde in dieser Arbeit eine repräsentative Auswahl von Labor-, kommerziellen und in der Natur vorkommenden Saccharomyces-Stämmen und ihren Interspezies-Hybriden auf die Verbreitung alleler Varianten der Hexokinase-Gene HXK1 und HXK2 getestet. Von den Hexose-Transportern stand Hxt3p im Mittelpunkt, da seine essentielle Rolle bei der Vergärung von Glucose und Fructose bereits belegt wurde.rnIn dieser Arbeit wurde gezeigt, dass es bedeutende Unterschiede in der Vergärung von Glucose und Fructose zwischen Weinhefen der Gattung Saccharomyces gibt, die z.T. mit Struktur-Varianten des Hexose-Transporter Hxt3p korrelieren. rnInsgesamt 51 Hefestämme wurden auf ihre allele Variante des HXT3-Gens untersucht. Dabei haben sich drei Hauptgruppen (die Fermichamp®-Typ Gruppe, Bierhefen und Hybrid-Stämme) mit unterschiedlichem HXT3-Allel ergeben. Im Zusammenhang mit der Weinherstellung wurden signifikante Nukleotid-Substitutionen innerhalb des HXT3-Gens der robusten S. cerevisiae-Stämme (wie z.B. Sekthefen, kommerzielle Starterkulturen) und Hybrid-Stämmen festgestellt. Diese Hefen zeichneten sich durch die Fähigkeit aus, den Most trotz stressigen Umwelt-Bedingungen (wie hohe Ethanol-Konzentration, reduzierter Ammonium-Gehalt, ungünstiges Glucose:Fructose-Verhältnis) zu vergären. rnDie Experimente deuten darauf hin, dass die HXT3-Allel-Variante des als Starterkultur verwendbaren Stammes Fermichamp®, für den verstärkten Fructose-Abbau verantwortlich ist. Ein gleiches Verhalten der Stämme mit dieser Allel-Variante wurde ebenfalls beobachtet. Getestet wurden die S. cerevisiae-Stämme Fermichamp® und 54.41, die bezüglich Hxt3p-Aminosäuresequenz gleich sind, gegenüber zwei S. cerevisiae-Stämmen mit dem HXT3-Standard-Alleltyp Fermivin® und 33. Der Unterschied in der Hexose-Verwertung zwischen Stämmen mit Fermichamp®- und Standard-Alleltyp war in der Mitte des Gärverlaufs am deutlichsten zu beobachten. Beide Gruppen, sowohl mit HXT3 Fermichamp®- als auch Fermivin®-Alleltyp vergoren die Glucose schneller als die Fructose. Der Unterschied aber zwischen diesen HXT3-Alleltypen bei der Zucker-Verwertung lag darin, dass der Fermichamp®-Typ eine kleinere Differenz in der Abbau-Geschwindigkeit der beiden Hexosen zeigte als der Fermivin®-Typ. Die Zuckeraufnahme-Messungen haben die relativ gute Fructose-Aufnahme dieser Stämme bestätigt.rnEbenfalls korrelierte der fructophile Charakter des Triple-Hybrides S. cerevisiae x S. kudriavzevii x S. bayanus-Stamm HL78 in Transportexperimenten mit verstärkter Aufnahme von Fructose im Vergleich zu Glucose. Insgesamt zeigte dieser Stamm ähnliches Verhalten wie die S. cerevisiae-Stämme Fermichamp® und 54.41. rnIn dieser Arbeit wurde ein Struktur-Modell des Hexose-Transporters Hxt3p erstellt. Als Basis diente die zu 30 % homologe Struktur des Proton/Xylose-Symporters XylE aus Escherichia coli. Anhand des Hxt3p-Modells konnten Sequenzbereiche mit hoher Variabilität (Hotspots) in drei Hxt3p-Isoformen der Hauptgruppen (die Fermichamp®-Typ Gruppe, Bierhefen und Hybrid-Stämme) detektiert werden. Diese signifikanten Aminosäure-Substitutionen, die eine mögliche Veränderung der physikalischen und chemischen Eigenschaften des Carriers mit sich bringen, konzentrieren sich auf drei Bereiche. Dazu gehören die Region zwischen den N- und C-terminalen Domänen, die cytosolische Domäne und der Outside-Loop zwischen Transmembranregion 9 und Transmembranregion 10. rnObwohl die Transportmessungen keinen Zusammenhang zwischen Stämmen mit unterschiedlichen HXT3-Allelen und ihrer Toleranz gegenüber Ethanol ergaben, wurde ein signifikanter Anstieg in der Zuckeraufnahme nach vorheriger 24-stündiger Inkubation mit 4 Vol% Ethanol bei den Teststämmen beobachtet. rnInsgesamt könnten allele Varianten von HXT3-Gen ein nützliches Kriterium bei der Suche nach robusten Hefen für die Weinherstellung oder für andere industrielle Anwendungen sein. Die Auswirkung dieser Modifikationen auf die Struktur und Effizienz des Hexose-Transporters, sowie der mögliche Zusammenhang mit Ethanol-Resistenz müssen weiter ausführlich untersucht werden. rnEin Zusammenhang zwischen den niedrig variablen Allel-Varianten der Hexokinase-Gene HXK1 und HXK2 und dem Zucker-Metabolismus wurde nicht gefunden. Die Hexokinasen der untersuchten Stämme wiesen allerdings generell eine signifikante geringere Affinität zu Fructose im Vergleich zu Glucose auf. Hier liegt sicherlich eine Hauptursache für den Anstieg des Fructose:Glucose-Verhältnisses im Laufe der Vergärung von Traubenmosten.rn

Hexose Transport in Growing Petunia Pollen Tubes and Characterization of a Pollen-Specific, Putative Monosaccharide Transporter1

We investigated the molecular and physiological processes of sugar uptake and metabolism during pollen tube growth and plant fertilization. In vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only in medium containing sucrose (Suc) as a carbon source, but also in medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis demonstrated a rapid and complete conversion of Suc into equimolar amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petunia monosaccharide transporter 1), which is highly homologous to plant monosaccharide transporters, was isolated from petunia. Pmt1 belongs to a small gene family and is expressed specifically in the male gametophyte, but not in any other vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature and germinating pollen. A model describing the transport of sugars to the style, the conversion of Suc into Glc and Fru, and the active uptake by a monosaccharide transporter into the pollen tube is presented.

Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction. © 2004 European Molecular Biology Organization.

Anthocyanin synthesis, growth and nutrient uptake in suspension cultures of strawberry cells

Strawberry (Fragaria ananassa cv. Shikinari) cell suspension cultures carried out in shake flasks for 18 d were closely examined for cell growth, anthocyanin synthesis and the development of pigmented cells in relation to the uptake of carbohydrate, extracellular PO4, NO3, NH4, and calcium. Cell viability, extracellular anthocyanin content, pH and electrical conductivity of the broth were also monitored. The specific growth rate of strawberry cells at exponential phase was 0.27 and 0.28 d(-1) based on fresh and dry weight, respectively. Anthocyanin synthesis was observed to increase continuously to a maximum value of 0.86 mg/g fresh cell weight (FCW) at day 6, and was partially growth-associated. Anthocyanin synthesis was linearly related to the increase in pigmented cell ratio, which increased with time and reached a maximum value of ca. 70% at day 6 due to reduction in cell viability and depletion of substrate. Total carbohydrate uptake was closely associated with increase in cell growth, and glucose was utilized in preference to fructose. Nitrate and ammonia were consumed until 9 d of culture, but phosphate was completely absorbed within 4 d. Calcium was assimilated throughout the growth cycle. After 9 d, cell lysis was observed which resulted in the leakage of intracellular substances and a concomitant pH rise. Anthocyanin was never detected in the broth although the broth became darkly pigmented during the lysis period. This suggests that anthocyanin was synthesized only by viable pigmented cells, and degraded rapidly upon cell death and lysis. Based on the results of kinetic analysis, a model was developed by incorporating governing equations for the ratio of pigmented cells into a Bailey and Nicholson's model. This was verified by comparison with the experimental data. The results suggest Bat the model satisfactorily describes the strawberry cell culture process, and may thus be used for process optimization.

Lipid, sugar and liposaccharide based delivery systems

Although there are formidable barriers to the oral delivery of biologically active drugs, considerable progress in the field has been made, using both physical and chemical strategies of absorption enhancement. A possible method to enhance oral absorption is to exploit the phenomenon of lipophilic modification and mono and oligosaccharide conjugation. Depending on the uptake mechanism targeted, different modifications can be employed. To target passive diffusion, lipid modification has been used, whereas the targeting of sugar transport systems has been achieved through drugs conjugated with sugars. These drug delivery units can be specifically tailored to transport a wide variety of poorly absorbed drugs through the skin, and across the barriers that normally inhibit absorption from the gut or into the brain. The delivery system can be conjugated to the drug in such a way as to release the active compound after it has been absorbed (i.e. the drug becomes a prodrug), or to form a biologically stable and active molecule (i.e. the conjugate becomes a new drug moiety). Examples where lipid, sugar and lipid-sugar conjugates have resulted in enhanced drug delivery will be highlighted in this review.

A multitask ATPase serving different ABC-type sugar importers in Bacillus subtilis

Journal of Bacteriology (Out 2010) 5312-5318

A missing sugar prevents glucose entry: a new twist on insulin secretion.

The signaling pathway that regulates glucose-stimulated insulin secretion depends on glucose metabolism, which is itself controlled by glucokinase. In a recent issue of Cell, show that altering N-glycosylation of the GLUT2 glucose transporter prevents its anchoring and retention at the cell surface; this impairs glucose uptake and insulin secretion.

Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants

(Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants). The uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid (2,4-D) were investigated in leaf calluses of Nicotiana tabacum, tuber calluses of Dioscorea opposita and calluses derived from zygotic embryos, leaves and petioles of Dioscorea composita. Striking similarities were evident in the patterns of 2,4-D metabolites and their chemical characteristics in the three callus types of D. composita compared, but significant differences were detected among the patterns of rnetabolites in the three species studied. Preliminary investigations on the stability of various metabolites (separated using TLC) by hydrolysis showed that sugar esters appeared to be the major metabolites in tobacco whilst in yams (D. opposita) glycosides were shown to be the main ones, which indicated a similarity between plants of Gramineae and Dioscoreaceae in terms of 2,4-D metabolism. Release of 2,4-D from tobacco callus cells upon their transfer to 2,4-D-free medium was detected and the implications of this are discussed in relation to the cultural conditions necessary to induce morphogenesis in vitro.