Presence Of Fructose Research Articles

Regulation of Fructose Transport and Its Effect on Fructose Toxicity in Anabaena spp

Anabaena variabilis grows heterotrophically using fructose, while the close relative Anabaena sp. strain PCC 7120 does not. Introduction of a cluster of genes encoding a putative ABC transporter, herein named frtRABC, into Anabaena sp. strain PCC 7120 on a replicating plasmid allowed that strain to grow in the dark using fructose, indicating that these genes are necessary and sufficient for heterotrophic growth. FrtR, a putative LacI-like regulatory protein, was essential for heterotrophic growth of both cyanobacterial strains. Transcriptional analysis revealed that the transport system was induced by fructose and that in the absence of FrtR, frtA was very highly expressed, with or without fructose. In the frtR mutant, fructose uptake was immediate, in contrast to that in the wild-type strain, which required about 40 min for induction of transport. In the frtR mutant, high-level expression of the fructose transporter resulted in cells that were extremely sensitive to fructose. Even in the presence of the inducer, fructose, expression of frtA was low in the wild-type strain compared to that in the frtR mutant, indicating that FrtR repressed the transporter genes even in the presence of fructose. FrtR bound to the upstream region of frtA, but binding was not visibly altered by fructose, further supporting the hypothesis that fructose has only a modest effect in relieving repression of frtA by FrtR. A. variabilis grew better with increasing concentrations of fructose up to 50 mM, showing increased cell size and heterocyst frequency. Anabaena sp. strain PCC 7120 did not show any of these changes when it was grown with fructose. Thus, although Anabaena sp. strain PCC 7120 could take up fructose and use it in the dark, fructose did not improve growth in the light.

Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus.

The aim of this research effort was to investigate the role of various sugar substrates in the growth medium upon thermotolerance and upon survival during storage after freeze-drying of Lactobacillus bulgaricus. Addition of the sugars tested to the growth medium, and of these and sorbitol to the drying medium (skim milk) was investigated so as to determine whether a relationship exists between growth and drying media, in terms of protection of freeze-dried cells throughout storage. The lowest decrease in viability of L. bulgaricus cells after freeze-drying was obtained when that organism was grown in the presence of mannose. However, L. bulgaricus clearly survived better during storage when cells had been grown in the presence of fructose, lactose or mannose rather than glucose (the standard sugar in the growth medium). A similar effect could not be observed in terms of thermotolerance; in this case, the growth medium supplemented with lactose was found to yield cells bearing the highest heat resistance. Supplementation of the drying medium with glucose, fructose, lactose, mannose or sorbitol led in most cases to enhancement of protection during storage, to a degree that was growth medium-dependent.

Fructose the odd man out. Why is the genomic control of intestinal GLUT5 expression different?

Fructose the odd man out. Why is the genomic control of intestinal GLUT5 expression different?

A Novel GDP-dependent Pyruvate Kinase Isozyme from Toxoplasma gondii Localizes to Both the Apicoplast and the Mitochondrion

We previously reported a cytosolic pyruvate kinase (EC 2.7.1.40) from Toxoplasma gondii (TgPyKI) that differs from most eukaryotic pyruvate kinases in being regulated by glucose 6-phosphate rather than fructose 1,6-diphosphate. Another putative pyruvate kinase (TgPyKII) was identified from parasite genome, which exhibits 32% amino acid sequence identity to TgPyKI and retains pyruvate kinase signature motifs and amino acids essential for substrate binding and catalysis. Whereas TgPyKI is most closely related to plant/algal enzymes, phylogenetic analysis suggests a proteobacterial origin for TgPyKII. Enzymatic characterization of recombinant TgPyKII shows a high pH optimum at 8.5, and a preference for GDP as a phosphate recipient. Catalytic activity is independent of K+, and no allosteric or regulatory effects were observed in the presence of fructose 1,6-diphosphate, fructose 2,6-diphosphate, glucose 6-phosphate, ribose 5-phosphate, AMP, or ATP. Unlike TgPyKI, native TgPyKII activity was exclusively associated with the membranous fraction of a T. gondii tachyzoite lysate. TgPyKII possesses a long N-terminal extension containing five putative start codons before the conserved region and localizes to both apicoplast and mitochondrion by immunofluorescence assay using native antibody and fluorescent protein fusion to the N-terminal extension. Further deletional and site-directed mutagenesis suggests that a translation product from 1st Met is responsible for the localization to the apicoplast, whereas one from 3rd Met is for the mitochondrion. This is the first study of a potential mitochondrial pyruvate kinase in any system.

Evidence of In Vivo Cross Talk between the Nitrogen-Related and Fructose-Related Branches of the Carbohydrate Phosphotransferase System of Pseudomonas putida

The genome of Pseudomonas putida KT2440 encodes only five recognizable proteins belonging to the phosphoenolpyruvate (PEP)-carbohydrate phosphotransferase system (PTS). Two of these PTS constituents (FruA and FruB) form a complete system for fructose intake. The other three products, encoded by ptsP (EI(Ntr)), ptsO (NPr), and ptsN (EIIA(Ntr)), comprise a branch of the system unrelated to sugar traffic but thought to have an influence on coordination of N and C metabolism. We used a genetic approach to clarify the course of high-energy phosphate through this reduced set of PTS proteins. To this end, we monitored the phosphorylation state in vivo of the EIIA(Ntr) enzyme in various genetic backgrounds and growth conditions. Our results show that the source of phosphate available to the system is PEP and that the primary flow of phosphate through the N/C-sensing PTS proceeds from PEP to EI(Ntr) to NPr to EIIA(Ntr). We also found that in the presence of fructose, unlike in the presence of succinate, EIIA(Ntr) can be phosphorylated in a ptsP strain but not in a ptsP fruB double mutant. This result revealed that the fructose transport system has the ability to cross talk in vivo with the N-related PTS branch. The data reported here thus document an unexpected connection in vivo between the sugar-dependent and sugar-independent PTSs.

Regulation of the expression of phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) genes in Corynebacterium glutamicum R

The phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS) catalyses the transport of carbohydrates by coupling carbohydrate translocation and phosphorylation. In Corynebacterium glutamicum R, the genes ptsH and ptsI encode general components of the PTS, and genes ptsF, ptsS and ptsG each encode fructose-, sucrose- and glucose-specific components of the PTS, respectively. In this study, we examined the mRNA levels of the pts genes in the presence or absence of PTS sugars. Glucose elevated the expression of ptsG, ptsH and ptsI genes, whereas fructose and sucrose induced the expression of all the pts genes examined, i.e. ptsF, -S, -G, -H and -I. We determined the transcriptional start sites of the pts genes and found that these promoters were activated in the presence of fructose. Disruption of fruR, which is a deoxyribonucleoside repressor (DeoR)-type transcriptional regulator co-transcribed with ptsF, resulted in enhanced induction of the fructose-pts operon, ptsI, and ptsH expression in response to fructose, indicating that FruR attenuates the induction of ptsI, ptsH and fructose-pts by fructose.

Lactate favours the dissociation of skeletal muscle 6-phosphofructo-1-kinase tetramers down-regulating the enzyme and muscle glycolysis

For a long period lactate was considered as a dead-end product of glycolysis in many cells and its accumulation correlated with acidosis and cellular and tissue damage. At present, the role of lactate in several physiological processes has been investigated based on its properties as an energy source, a signalling molecule and as essential for tissue repair. It is noteworthy that lactate accumulation alters glycolytic flux independently from medium acidification, thereby this compound can regulate glucose metabolism within cells. PFK (6-phosphofructo-1-kinase) is the key regulatory glycolytic enzyme which is regulated by diverse molecules and signals. PFK activity is directly correlated with cellular glucose consumption. The present study shows the property of lactate to down-regulate PFK activity in a specific manner which is not dependent on acidification of the medium. Lactate reduces the affinity of the enzyme for its substrates, ATP and fructose 6-phosphate, as well as reducing the affinity for ATP at its allosteric inhibitory site at the enzyme. Moreover, we demonstrated that lactate inhibits PFK favouring the dissociation of enzyme active tetramers into less active dimers. This effect can be prevented by tetramer-stabilizing conditions such as the presence of fructose 2,6-bisphosphate, the binding of PFK to f-actin and phosphorylation of the enzyme by protein kinase A. In conclusion, our results support evidence that lactate regulates the glycolytic flux through modulating PFK due to its effects on the enzyme quaternary structure.

Direct heterogeneous electron transfer reactions and molecular orientation of fructose dehydrogenase adsorbed onto pyrolytic graphite electrodes

Abstract Direct heterogeneous electron transfer reactions and the molecular orientation of d -fructose dehydrogenase (FDH, from Gluconobacter sp.) adsorbed onto electrodes were investigated. Catalytic oxidation currents based on the direct electron transfer reactions of FDH adsorbed onto basal-plane, highly oriented pyrolytic graphite (basal-plane HOPG), plastic formed carbon plate and tin-doped indium oxide electrodes were observed from a potential around −0.1 V (vs. Ag/AgCl/saturated KCl) in phosphate solution (pH 5.0) in the presence of fructose as a substrate for FDH. The catalytic current for the FDH adsorbed onto basal-plane HOPG electrodes indicated a pH dependence. The catalytic oxidation currents were only observed in acidic solutions (pH ⩽ 6), and were not observed in neutral and alkaline solutions. The reason why the catalytic current was not obtained in neutral and alkaline solutions was because the FDH complex decomposed in neutral and alkaline solutions based on the AFM measurements. The differential pulse voltammetric investigations for FDH adsorbed onto basal-plane HOPG electrode together with the AFM and electrophoresis results indicated that the flavin-containing subunit in FDH accepts electrons from d -fructose, and transfers these electrons to the heme c -containing subunit, and then the direct electron transfer reaction of FDH occurred at the heme c site.

Role of Transglycosylation Products in the Expression of Multiple Xylanases in Myceliophthora sp. IMI 387099

This study reports the regulation of multiple xylanases produced by Myceliophthora sp. IMI 387099. Fructose was found to positively regulate the expression of multiple xylanase when used as sole carbon source. The xylanases (EX(1 )and EX(2)) of acidic pI were expressed in the presence of simple sugars (glucose, arabinose, and xylose), whereas xylanase of both acidic as well as basic pI (EX(1,) EX(2,) EX(3), and EX(5)) were expressed in the presence of fructose, xylan, and combination of xylan and alcohol. The combination of fructose and xylan also led to expression of an additional xylanase (EX(4)). The positional isomer (iso-X4) was found to be the key transglycosylation product when cultures were grown in the presence of fructose and xylan. In the presence of alcohols, the higher expression of xylanase was ascribed to the synergistic effect of alkyl glycoside and other transglycosylation products present in the culture extracts.

Another 'soberade' on the market: does Outox keep its promise?

Several products are being widely promoted for reduction of the concentration of alcohol in the human body. One of these preparations, the fructose soft drink Outox, claims to noticeably increase the alcohol elimination rate (beta 60). Theories to explain this 'fructose effect' are based on the assumption that NAD+, the coenzyme for alcohol dehydrogenase, is regenerated faster in the presence of fructose. A randomized double-blind, placebo-controlled cross-over study was performed with 30 volunteers in two drinking sessions each. Under strictly identical conditions, the same amount of alcohol was consumed, followed by the consumption of either 250 ml Outox or 250 ml placebo. Periodical measurements of blood (BAC), breath (BrAC) and urine alcohol concentration (UAC) were performed. Analyses revealed a significant difference (P<0.0001) between the mean alcohol levels of the Outox and the placebo drinking sessions. The overall mean BAC difference was 0.077 g/l (BAC 0.748 g/l without vs 0.671 g/l with Outox), equivalent to 10.3%. The mean BrAC difference was 0.045 mg/l (BrAC 0.314 mg/l without vs 0.269 mg/l with Outox), equivalent to 14.3%. Differences were lower for women than for men. A significant difference between the alcohol elimination rates (beta 60) was not found. The results show that the soft drink Outox may decrease the alcohol concentration by about 10%. However, BAC and BrAC differences are rather a consequence of slower gastric absorption of alcohol, because Outox does not increase the alcohol elimination rate. Our study demonstrates that the claim of Outox or other fructose drinks to work as a 'soberade' cannot be proven from a scientific point of view. It should be the task of physicians to warn potential consumers, especially in connection with drinking and driving.

Reactivity and oxidative potential of fructose and glucose in enkephalin-sugar model systems

The reactions of Leu- and Met-enkephalin (Tyr-Gly-Gly-Phe-Leu/Met) with fructose resulted in the parallel formation of Heyns compounds (N-peptidyl-D-mannosamine and -D-glucosamine) and sugar-peptide generated imidazolidinone diastereomers. Glucose showed higher level of reactivity than fructose with respect to the extent of glycated product formation. The presence of fructose in the incubation mixtures makes Met residue more susceptible to oxidation than glucose.

Fructose metabolism in hepatocytes: primary cells or cell lines?

Aims: The beneficial action of D-fructose in parenteral nutrition was attributed to the fact that this hexose is metabolized more rapidly than glucose, and that its metabolization is independent of insulin (1). Since it became known that a high intravenous dose of fructose reduces the intracellular ATP levels in human and murine liver, use of fructose in parenteral nutrition was discontinued. Recently, we reported that effective anti-neoplastic therapy with melphalan requires sufficient intracellular ATP levels (2,3). It still remains open, whether or not fructose-mediated ATP depletion has any significance on cell death or viability of healthy liver parenchymal cells. Aims: We compared the activity of fructose metabolizing enzymes in primary hepatocytes and commonly used cell lines to determine whether cell lines can model hepatocyte fructose metabolism. Results: The specific activity of fructokinase and aldolase b was investigated in the cell lines HepG2, Huh7 and in primary hepatocytes. The specific activity of fructokinase was similar in all three cell types. We detected a lower activity of aldolase b in the two cell lines in comparison with the primary hepatocytes. Additionally, the activity of hexokinases, which catalyze the first step of the fructose metabolism in muscle and other tissue, was determined. Compared to the primary cells, the specific activity of hexokinases was greater in HepG2, Huh7 and also in the murine cell lines Hepa 1–6 and AML–12. Western blot analysis revealed a stronger expression level of Hexokinase II (HK II) in all cell lines. These results indicated that the high activity of HK II might result in a shift from liver-type to muscle-type fructose metabolism in the cell-lines. Therefore, the HK II was inhibited with 3-bromopyruvate in HepG2 cells to study the resulting fructose metabolism. Under HK II inhibition, fructose resulted in a decrease of the ATP-levels in HepG2-cells comparable to that in primary hepatocytes in the absence of 3-bromopyruvate. Conclusions: Our data provide evidence that commonly used cell lines are not suitable to study fructose metabolism in hepatocytes, due to specific changes in enzyme activity in the cell lines. This appears to result in a shift from liver-type to muscle-type fructose metabolism in HepG2-cells. These findings suggest that in the presence of fructose, primary hepatocytes could be less susceptible to chemotherapeutics such as melphalan than neoplastically transformed cells.

Fructose as a reducing agent for in situ generation of Cu(I) species via an electron-transfer reaction in copper-catalyzed living/controlled radical polymerization of styrene

Copper-catalyzed living radical polymerization (LRP) of styrene was carried out in the presence of fructose as a reducing agent for Cu(II) by using either 1-chloro-1-phenyl ethane (1-PECl), ethyl-2-bromoisobutyrate (EiBr) or methyl-2-bromopropionate (MBP) as an initiator and N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) as ligand at 110°C. Kinetic experiments were carried out in order to reveal the effect of fructose concentration on copper-catalyzed LRP of St. This technique was successfully applied for the preparation of homo-polymers (linear and star-type polystyrene (PS)) and corresponding block co-polymers (PS-b-PMMA). The polymers thus obtained were characterized using GPC, 1H-NMR and MALDI-TOF measurements.

Enhanced poly-?-hydroxybutyrate accumulation in a unicellular cyanobacterium, Synechocystis sp. PCC 6803

To stimulate poly-beta-hydroxybutyrate (PHB) accumulation in Synechocystis sp. PCC 6803 by manipulating culture conditions. Stationary phase cultures of Synechocystis sp. PCC 6803 were subjected to N- and P-deficiency, chemoheterotrophy and limitations of gas-exchange. Enhanced PHB accumulation was observed under all the above conditions. However, interaction of P-deficiency with gas-exchange limitation (GEL) in the presence of exogenous carbon boosted PHB accumulation maximally. Combined effects of P-deficiency and GEL boosted PHB accumulation up to 38% (w/w) of dry cell weight (dcw) in Synechocystis sp. PCC 6803 in the presence of fructose and acetate. This value is about eightfold higher as compared with the accumulation under photoautotrophic growth condition. These results showed a good potential of Synechocystis sp. PCC 6803 in accumulating poly-beta-hydroxybutyrate, an appropriate raw material for biodegradable and biocompatible plastic. Poly-beta-hydroxybutyrate could be an important material for plastic and pharmaceutical industries.

The Production of Bacillus cereus Enterotoxins Is Influenced by Carbohydrate and Growth Rate

Enterotoxin production is a key factor in Bacillus cereus food poisoning. Herein, the effect of the growth rate (mu) on B. cereus toxin production when grown on sucrose was studied and the Hemolytic BL enterotoxin (HBL) and nonhemolytic enterotoxin (Nhe) production by B. cereus was compared according to carbohydrate at mu = 0.2 h(-1). The anaerobic growth was carried out on continuous cultures in synthetic medium supplemented with glucose, fructose, sucrose, or an equimolar mixture of glucose and fructose. Concerning the HBL and Nhe enterotoxin production: (1) the highest enterotoxin production has occurred at mu = 0.2 h(-1) when growing on sucrose; (2) HBL production was repressed when glucose was consumed and the presence of fructose (alone or in mixture) cancelled glucose catabolite repression; (3) the consumption of sucrose increased Nhe production, which was not affected by the catabolite repression. Furthermore, analysis of the fermentative metabolism showed that whatever the mu or the carbon source, B. cereus used the mixed acid fermentation to ferment the different carbohydrates. The enterotoxin productions by this strain at mu = 0.2 h(-1) are highly influenced by the carbohydrates that do not involve any fermentative metabolism changes.

Identification of Nostoc punctiforme akinete‐expressed genes using differential display

Akinetes are spore-like resting cells formed by certain filamentous cyanobacteria that have increased resistance to environmental stress. They can be found at low frequencies in dense cultures experiencing low light or phosphate limitation, but also form at high frequencies in a zwf mutant strain of Nostoc punctiforme following dark incubation in the presence of fructose. The wild-type strain is capable of facultative heterotrophic growth under these conditions and does not form akinetes. To identify genes associated with akinete development, differential display was used to amplify and compare cDNA from a wild-type and zwf mutant strain of N. punctiforme following a switch to dark heterotrophic conditions. Screening of candidate genes by reverse transcriptase real-time quantitative PCR and subsequent testing for akinete-specific expression using GFP transcriptional reporter plasmids lead to the identification of three novel akinete-expressed genes. The genes identified from the screening encoded for proteins homologous to an aminopeptidase (aapN), a zinc protease (hap) and an ATP-binding cassette (ABC)-type transporter (aet). Expression of hap was also increased in developing hormogonia, a transient type of differentiated filament capable of gliding motility. Transcriptional start sites for akinete-expressed genes were determined using random amplification of cDNA ends (RACE), and promoter regions were compared with orthologues in other filamentous cyanobacteria to identify putative regulatory sequences.

Induction of 8-Hydroxydeoxyguanosine Formation in DNA by Fructose and Glucose in the Presence of Copper

The effects of glucose and fructose in the presence or absence of iron or copper on the formation of 8-hydroxydeoxyguanosine (8-OHdG) in calf-thymus DNA were examined. Glucose (5 and 10 mM) and fructose (10–200 μM) increased 8-OHdG formation 1.9–2.8 times and 3.0–8.3 times compared to the control, respectively. In the presence of fructose (200 μM), copper (50 μM) was found to increase 8-OHdG formation 46.2 times compared to the control, and 3.8 times compared to the iron (50 μM)-treated value. The stimulatory potency of 8-OHdG formation by fructose (200 μM) in the presence of copper (50 μM) was much stronger than that by glucose (10 mM) in the presence of iron (50 μM) or of copper (50 μM). Sorbitol, 3-deoxyglucosone and methylglyoxal in the presence or absence of copper had no or weaker stimulating effects on 8-OHdG formation than fructose in the presence or absence of copper. These results suggest that fructose in the presence of copper has the potential to dramatically induce oxidative DNA damage.

Production of Bacteriocin ST33LD, Produced by Leuconostoc mesenteroides subsp. mesenteroides, as Recorded in the Presence of Different Medium Components

Bacteriocin ST33LD, produced by Leuconostoc mesenteroides subsp. mesenteroides, is approximately 2.7 kDa in size and inhibits Enterococcus faecalis, Escherichia coli, Lactobacillus casei and Pseudomonas aeruginosa. Good growth was recorded in the presence of 10% (w/v) soy milk or 10% (w/v) molasses, but there was no bacteriocin production. Growth in MRS broth adjusted to pH 4.5 yielded low bacteriocin levels (800 AU/ml). However, the same medium adjusted to pH 5.0, 5.5 and 6.5, respectively, yielded 3200 AU/ml. Tween 80 decreased bacteriocin production by more than 50%. Growth in the presence of tryptone yielded maximal activity (12,800 AU/ml), whereas different combinations of tryptone, meat extract and yeast extract produced activity levels of 1600 AU/ml and less. Growth in the presence of 2.0% (w/v) sucrose, or maltose, yielded much higher levels of bacteriocin activity (12,800 AU/ml) compared to growth in the presence of 2.0% (w/v) glucose or lactose (6400 AU/ml). Lower yields were also recorded in the presence of fructose and mannose. KH2PO4 at 10.0% (w/v) stimulated bacteriocin production. Glycerol concentrations of 0.5% (w/v) and higher (up to 5.0%, w/v) repressed bacteriocin production by 50%. The addition of cyanocobalamin, thiamine and L-ascorbic acid to MRS broth (1.0 ppm) yielded 12,800 AU/ml bacteriocin, whereas the addition of DL-6,8-thioctic acid yielded only 6 400 AU/ml.

The Influence of Glucose and Fructose on the Degradation of 2-Chlorophenol by Pseudomonas putida CP1

Pseudomonas putida CP1 grew on 2-chlorophenol when supplied as the sole source of carbon. Chlorophenol degradation was stimulated in the presence of low concentrations of glucose (0.05–1%, w/v). Substrate removal was inhibited and there was a significant fall in pH with concentrations of glucose greater than 1.0% (w/v). When the pH was controlled at pH 7.0 inhibition of substrate removal was alleviated. The rate of removal of 2-chlorophenol was greater in the presence of fructose than in the presence of glucose. P. putida CP1 formed clumps of cells when grown on 2-chlorophenol and fructose but not on glucose. When the organism was grown on a combination of 2-chlorophenol and an additional carbon source clumping was present but to a lesser degree.

The high-affinity phosphate-binding protein PstS is accumulated under high fructose concentrations and mutation of the corresponding gene affects differentiation in Streptomyces lividans

The secreted protein pattern of Streptomyces lividans depends on the carbon source present in the culture media. One protein that shows the most dramatic change is the high-affinity phosphate-binding protein PstS, which is strongly accumulated in the supernatant of liquid cultures containing high concentrations (>3 %) of certain sugars, such as fructose, galactose and mannose. The promoter region of this gene and that of its Streptomyces coelicolor homologue were used to drive the expression of a xylanase in S. lividans that was accumulated in the culture supernatant when grown in the presence of fructose. PstS accumulation was dramatically increased in a S. lividans polyphosphate kinase null mutant (Deltappk) and was impaired in a deletion mutant lacking phoP, the transcriptional regulator gene of the two-component phoR-phoP system that controls the Pho regulon. Deletion of the pstS genes in S. lividans and S. coelicolor impaired phosphate transport and accelerated differentiation and sporulation on solid media. Complementation with a single copy in a S. lividans pstS null mutant returned phosphate transport and sporulation to levels similar to those of the wild-type strain. The present work demonstrates that carbon and phosphate metabolism are linked in the regulation of genes and that this can trigger the genetic switch towards morphogenesis.