Degradation Of Fructose Research Articles

Effects of honey HMF on enzyme activities and serum biochemical parameters of Wistar rats.

Hydroxymethylfurfural (HMF) is a by-product of thermal degradation of glucose and fructose. In this study, the effects of high HMF content of honey on biochemical parameters of rats were investigated. Experiments were conducted with 40 Wistar albino male rats, each weighing 250-350g and covered a period of 5weeks. The animals were divided into five groups. The first group was served as control group. HMF was injected subcutaneously at a dose of 200mg/kg rat b.w. to the animals in group 2. Group 3 was fed with honey that contains 10mg HMF/kg honey. In group 4 and 5, there were honeys that contain significantly high HMF content due to long storage period (181mg HMF/kg honey) and heat process (140mg HMF/kg honey). At the end of the feeding process, biochemical blood parameters of rats were investigated. It was observed that there were no differences among the glucose, triglyceride, HDL cholesterol, uric acid, Na, GGT, and ALP parameters of the groups. On the other hand, significant differences were observed among the cholesterol, LDL, BUN, creatinine, Ca, P, Mg, K, Cl, total bilirubin, LDH, CPK, AST, ALT, total protein, and pseudocholinesterase values of the rats. The highest adverse effects were obtained from group HMF, and it was followed by groups SH (stored honey) and HH (heated honey). It can be concluded that high HMF content of honey may affect the human health adversely; thus, HMF in honey must be controlled by beekeepers.

Ethanol and 4-methylpyrazole increase DNA adduct formation of furfuryl alcohol in FVB/N wild-type mice and in mice expressing human sulfotransferases 1A1/1A2.

Furfuryl alcohol (FFA) is a carcinogenic food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. The activation by sulfotransferases (SULTs) yields a DNA reactive and mutagenic sulfate ester. The most prominent DNA adduct, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MF-dG), was detected in FFA-treated mice and also in human tissue samples. The dominant pathway of FFA detoxification is the oxidation via alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The activity of these enzymes may be greatly altered in the presence of inhibitors or competitive substrates. Here, we investigated the impact of ethanol and the ADH inhibitor 4-methylpyrazole (4MP) on the DNA adduct formation by FFA in wild-type and in humanized mice that were transgenic for human SULT1A1/1A2 and deficient in the mouse (m) Sult1a1 and Sult1d1 genes (h1A1/1A2/1a1(-)/1d1(-)). The administration of FFA alone led to hepatic adduct levels of 4.5 N(2)-MF-dG/10(8) nucleosides and 33.6 N(2)-MF-dG/10(8) nucleosides in male and female wild-type mice, respectively, and of 19.6 N(2)-MF-dG/10(8) nucleosides and 95.4 N(2)-MF-dG/10(8) nucleosides in male and female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 1.6g ethanol/kg body weight increased N(2)-MF-dG levels by 2.3-fold in male and by 1.7-fold in female wild-type mice and by 2.5-fold in male and by 1.5-fold in female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 100mg 4MP/kg body weight had a similar effect on the adduct levels. These findings indicate that modulators of the oxidative metabolism, e.g. the drug 4MP or consumption of alcoholic beverages, may increase the genotoxic effects of FFA also in humans.

Effects of Increased Night Temperature on Cellulose Synthesis and the Activity of Sucrose Metabolism Enzymes in Cotton Fiber

Temperature is one of the key factors that influence cotton fiber synthesis at the late growth stage of cotton. In this paper, using two early-maturing cotton varieties as experimental materials, night temperature increase was stimulated in the field using far-infrared quartz tubes set in semi-mobile incubators and compared with the normal night temperatures (control) in order to investigate the effects of night temperature on the cotton fiber cellulose synthesis during secondary wall thickening. The results showed that the activity of sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) quickly increased and remained constant during the development of cotton fiber, while the activity of acid invertase (AI) and alkaline invertase (NI) decreased, increased night temperatures prompted the rapid transformation of sugar, and all the available sucrose fully converted into cellulose. With night temperature increasing treatment, an increase in SuSy activity and concentration of sucrose indicate more sucrose converted into UDPG (uridin diphosphate-glucose) during the early and late stages of cotton fiber development. Furthermore, SPS activity and the increased concentration of fructose accelerated fructose degradation and reduced the inhibition of fructose to SuSy; maintaining higher value of allocation proportion of invertase and sucrose during the early development stages of cotton fiber, which was propitious to supply a greater carbon source and energy for cellulose synthesis. Therefore, the minimum temperature in the nightime was a major factor correlated with the activity of sucrose metabolism enzymes in cotton fiber. Consequently, soluble sugar transformation and cellulose accumulation were closely associated with the minimum night temperature.

Metabolic preconditioning with fructose prior to organ recovery attenuates ischemia-reperfusion injury in the isolated perfused rat liver

Objective. Ischemia-reperfusion injury is associated with a high rate of primary organ dysfunction and thereby contributes substantially to morbidity and mortality in the course of liver transplantation. In the present study, the impact of metabolic preconditioning with fructose on ischemia-reperfusion injury in the isolated perfused rat liver model is evaluated. Methods. Fasted rats received a single intravenous fructose injection to induce metabolic preconditioning (fructose group) or a volume equivalent of normal saline (control group) 10 min before liver explantation. After 26 h of cold storage, livers were reperfused for 90 min at 37°C with Krebs-Henseleit buffer. The parameters used to quantify ischemia-reperfusion injury included hepatic oxygen consumption, enzyme release, and cell viability. Results. During reperfusion, livers in the fructose group consumed more oxygen than livers in the control group (p < 0.005), indicating ATP synthesis as a result of glycolytic fructose degradation. Moreover, cell injury was reduced by fructose administration, as reflected by a lower enzyme release during both cold ischemia and reperfusion (p < 0.05). Finally, hepatocyte viability at the end of reperfusion was significantly higher in the fructose group (p < 0.01). However, there was no significant difference between the two experimental groups in reference to the viability of endothelial cells. Conclusion. In clinical use, metabolic preconditioning with fructose prior to organ recovery might contribute to a reduction in the incidence of primary organ dysfunction after liver transplantation.

Kinetics of monosaccharide conversion in the presence of homogeneous Bronsted acids

For the utilization of biomass in the fuel and polymer industry, a wide variety of catalysts have been studied for their activity either on the dehydration reaction in particular or on the monosaccharide degradations in general. Yet, systematic data outlining the effects of acidic features is not available and a common framework for catalytic activity comparison is missing that is needed for rational catalyst design. The current work aimed to provide insight about the effect of the nature of the acid and initial acidity on degradation kinetics of C5 and C6 carbohydrates and thereby built a platform allowing activity comparison. Mineral and organic acids ranging in acidic strength, hydrochloric, sulfuric, phosphoric, maleic, and propane sulfonic acid, were tested for their activity in the degradation of xylose, fructose and glucose at two different pH values; 1.5 and 3.6. In the presence of weak homogeneous acids, glucose undergoes degradation with drastically different activation energies at pH of 1.5 than at pH 3.6. Such a difference does not occur in the presence of strong homogenous acids. On the other hand, xylose and fructose undergo degradation with similar activation energies of approximately 140kJ/mol regardless of the pH or nature of the homogeneous acid. A common framework that compiles the catalytic activities and outlines the differences potentially related to underlying mechanism changes provides the basis required for rational heterogeneous catalyst design.

Microwave-assisted hydrothermal degradation of fructose and glucose in subcritical water

Abstract The microwave-assisted hydrothermal treatment of fructose and glucose is studied under subcritical conditions, in a temperature window between 180 °C and 250 °C. The suitability of microwave heating as well as the impact of the reaction variables temperature and time on the extent of the substrate conversion and on the product composition are elucidated. With rate constants of 3.6*10−3 s−1 (fructose) and 5.2*10−4 s−1 (glucose) at 220 °C the decomposition of fructose took place about eight times faster than that of glucose. 5-HMF was found to be the major decomposition product for both monosaccharides, with maximum yields of 47% 5-HMF (for fructose) and 30% (for glucose) at a maximum selectivity of 65% and 50%, respectively. All other liquid monomeric products like levulinic acid, furfural and trihydroxy benzene are formed at cumulative yields below 16%.

A new method to determine the energy saving night temperature for vegetative growth of Phalaenopsis

Knowledge of the energy saving night temperature (i.e. a relatively cool night temperature without affecting photosynthetic activity and physiology) and a better understanding of low night temperature effects on the photosynthetic physiology of Phalaenopsis would improve their production in terms of greenhouse temperature control and energy use. Therefore, Phalaenopsis‘Hercules’ was subjected to day temperatures of 27.5°C and night temperatures of 27.0°C, 24.2°C, 21.2°C, 18.3°C, 15.3°C or 12.3°C in a growth chamber. A new tool for the determination of the energy saving night temperature range was developed based on temperature response curves of leaf net CO2 exchange, chlorophyll fluorescence, organic acid content and carbohydrate concentrations. The newly developed method was validated during a complete vegetative cultivation in a greenhouse environment with eight Phalaenopsis hybrids (i.e. ‘Boston’, ‘Bristol’, ‘Chalk Dust', ‘Fire Fly’, ‘Lennestadt’, ‘Liverpool’, ‘Precious’, ‘Vivaldi’) and day/night temperature set points of 28/28°C, 29/23°C and 29/17°C. Temperature response curves revealed an overall energy saving night temperature range for nocturnal CO2 uptake, carbohydrate metabolism, organic acid accumulation and photosystem II (PSII) photochemistry of 17.1°C to 19.9°C for Phalaenopsis‘Hercules’. At the lower end of this energy saving night temperature range, a high malate-to-citrate ratio switched towards a low ratio and this transition seemed to alleviate effects of night chilling induced photoinhibition. At night temperatures of 24°C or higher, the degradation of starch, glucose and fructose indicated an increased respiratory CO2 production. During the greenhouse validation experiment, the differences between the eight Phalaenopsis hybrids with regard to their response to the warm day/cool night temperature regimes were remarkably large. In general, the day/night temperature of 29/17°C led to a significantly lower biomass accumulation and less leaves which were in addition shorter, narrower and smaller in size as compared to the day/night temperature regimes of 28/28°C and 29/23°C. During week 25 of the cultivation period, plants matured and flower initiation steeply increased for all hybrids and in each day/night temperature regime. Before week 25, early spiking was only sufficiently suppressed in the 29/23°C and 29/17°C temperature regimes for three hybrids (‘Boston’, ‘Bristol’ and ‘Lennestadt’) but not in the other five hybrids. Although a considerable biochemical flexibility was demonstrated for Phalaenopsis‘Hercules’, inhibition of flowering after exposure to a combination of warm days and cool nights appeared to be largely hybrid dependent.

Kinetics of sugars, organic acids and acetaldehyde during simultaneous yeast-bacterial fermentations of white wine at different pH values

Successfully inducing malolactic fermentation in the production of grape wines can be challenging, especially in wines with multiple inhibitors such as low pH values and high ethanol concentrations. In the present study, the kinetics of several chemicals of enological relevance was studied in Chardonnay vinified by traditional, consecutive alcoholic (AF) and malolactic fermentations (MLF), and simultaneous AF/MLF, where bacteria were co-inoculated with yeast. The Chardonnay must was adjusted to four pH values (3.2, 3.35, 3.5 or 3.65), and the concentrations of sugars, organic acids as well as acetaldehyde were followed throughout the fermentations. The degradation of glucose and fructose was slower at the lowest must pH value (3.2) but independent from the time point of bacterial inoculation. In all cases, malolactic conversion was faster after yeast-bacterial co-inoculation and was completed in simultaneous treatments at pH values of 3.35–3.65, and consecutive treatments at pH 3.5 and 3.65. No statistically significant difference was observed among the final acetic acid concentrations among all inoculation and pH treatments, but there was a trend towards higher acetic acid residues in wines produced by co-inoculation, especially at high pH values. Overall, simultaneous AF/MLF allowed for greatly reduced fermentation times, but the must pH remained a strong factor for fermentation success and determined the final concentration of various wine components. The time point of inoculation influenced formation and degradation kinetics of organic acids and acetaldehyde considerably, and these are of relevance for vinification decisions.

Multifactorial analysis of acetaldehyde kinetics during alcoholic fermentation by Saccharomyces cerevisiae

Acetaldehyde is the terminal electron acceptor in the alcoholic fermentation by Saccharomyces cerevisiae. Quantitatively the most important carbonyl by-product, it has relevance for ethanol production yields as well as product stabilization and toxicology. The aim of this study was to investigate the effect of various enological parameters on acetaldehyde kinetics during alcoholic fermentations. Two commercial yeast strains were tested in two grape musts and the pH, temperature, SO2 and nutrient addition were varied. All incubations had uniform kinetics where acetaldehyde reached an initial peak value followed by partial reutilization. Peak acetaldehyde concentrations and residual concentrations after 15days of fermentations ranged from 62 to 119mgl−1 and 22 to 49mgl−1, respectively. A positive linear relationship was found between peak and final acetaldehyde levels in Gewürztraminer, but not Sauvignon Blanc fermentations, where sluggish fermentations were observed. Several factors had a significant effect on peak and/or final acetaldehyde levels. SO2 addition, grape cultivar and fermentation nutrition were important regulators of peak acetaldehyde production, while final acetaldehyde concentrations were correlated with SO2 addition, grape cultivar and temperature. The results allowed to estimate the acetaldehyde increase caused by SO2 addition to 366μg of acetaldehyde per mg of SO2 added to the must. The course of the final fermentation phase was shown to determine acetaldehyde residues. Comparison of acetaldehyde and hexose kinetics revealed a possible relationship between the time of occurrence of peak acetaldehyde concentrations and the divergence of glucose and fructose degradation rates.

Sporosalibacterium faouarense gen. nov., sp. nov., a moderately halophilic bacterium isolated from oil-contaminated soil

A novel strictly anaerobic, moderately halophilic and mesophilic bacterium, designated strain SOL3f37(T), was isolated from a hydrocarbon-polluted soil surrounding a deep petroleum environment located in south Tunisia. Cells of strain SOL3f37(T) stained Gram-positive and were motile, straight and spore-forming. Strain SOL3f37(T) had a typical Gram-positive-type cell-wall structure, unlike the thick, multilayered cell wall of its closest relative Clostridiisalibacter paucivorans. The major fatty acids were iso-C(15 : 0) (41 %), iso-C(14 : 0) 3-OH and/or iso-C(15 : 0) dimethyl acetal (21.6 %), iso-C(13 : 0) (4.4 %), anteiso-C(15 : 0) (3.9 %) and iso-C(15 : 1) (2.8 %). Strain SOL3f37(T) grew between 20 and 48 °C (optimum 40 °C) and at pH 6.2-8.1 (optimum pH 6.9). Strain SOL3f37(T) required at least 0.5 NaCl l(-1) and grew in the presence of NaCl concentrations up to 150 g l(-1) (optimum 40 g l(-1)). Yeast extract (2 g l(-1)) was required for degradation of pyruvate, fumarate, fructose, glucose and mannitol. Also, strain SOL3f37(T) grew heterotrophically on yeast extract, peptone and bio-Trypticase, but was unable to grow on Casamino acids. Sulfate, thiosulfate, sulfite, elemental sulfur, fumarate, nitrate and nitrite were not reduced. The DNA G+C content was 30.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SOL3f37(T) was a member of the family Clostridiaceae in the order Clostridiales; strain SOL3f37(T) was related to members of various genera of the family Clostridiaceae. It exhibited highest 16S rRNA gene sequence similarity (93.4 %) with Clostridiisalibacter paucivorans 37HS60(T), 91.8 % with Thermohalobacter berrensis CTT3(T) and 91.7 % with Caloranaerobacter azorensis MV1087(T). On the basis of genotypic, phenotypic and phylogenetic data, it is suggested that strain SOL3f37(T) represents a novel species in a new genus. The name Sporosalibacterium faouarense gen. nov., sp. nov. is proposed, with SOL3f37(T) (=DSM 21485(T) =JCM 15487(T)) as the type strain of Sporosalibacterium faouarense.

Unravelling the kinetics of the formation of acrylamide in the Maillard reaction of fructose and asparagine by multiresponse modelling

A kinetic model for the formation of acrylamide in a fructose–asparagine reaction system at initial pH 5.5 is proposed, based on an approach called multiresponse kinetic modelling. The formation of acetic acid and formic acid from the degradation of fructose and its isomer glucose was included in the proposed kinetic model. The kinetic model suggests that the effect of temperature on acrylamide formation with fructose is more due to the preceding steps with the formation of the Schiff base. The use of fructose and lower pH resulted in a higher yield of acrylamide (3%), suggesting that both can play an important role in acrylamide mitigation. Furthermore, these models have shown that, at high temperatures (120–200 °C), the Maillard reaction rapidly goes into the advanced stages, forming high amounts of organic acids and high molecular weight melanoidins. Overall, these mechanistic models provide more insight of the formation of acrylamide in a quantitative way.

Hydrolytic Methods for the Quantification of Fructose Equivalents in Herbaceous Biomass

A low, but significant, fraction of the carbohydrate portion of herbaceous biomass may be composed of fructose/fructosyl-containing components ("fructose equivalents"); such carbohydrates include sucrose, fructooligosaccharides, and fructans. Standard methods used for the quantification of structural-carbohydrate-derived neutral monosaccharide equivalents in biomass are not particularly well suited for the quantification of fructose equivalents due to the inherent instability of fructose in conditions commonly used for hemicellulose/cellulose hydrolysis (>80% degradation of fructose standards treated at 4% sulfuric acid, 121 degrees C, 1 h). Alternative time, temperature, and acid concentration combinations for fructan hydrolysis were considered using model fructans (inulin, beta-2,1, and levan, beta-2,6) and a grass seed straw (tall fescue, Festuca arundinacea) as representative feedstocks. The instability of fructose, relative to glucose and xylose, at higher acid/temperature combinations is demonstrated, all rates of fructose degradation being acid and temperature dependent. Fructans are shown to be completely hydrolyzed at acid concentrations well below that used for the structural carbohydrates, as low as 0.2%, at 121 degrees C for 1 h. Lower temperatures are also shown to be effective, with corresponding adjustments in acid concentration and time. Thus, fructans can be effectively hydrolyzed under conditions where fructose degradation is maintained below 10%. Hydrolysis of the beta-2,1 fructans at temperatures > or =50 degrees C, at all conditions consistent with complete hydrolysis, appears to generate difructose dianhydrides. These same compounds were not detected upon hydrolysis of levan, sucrose, or straw components. It is suggested that fructan hydrolysis conditions be chosen such that hydrolysis goes to completion; fructose degradation is minimized, and difructose dianhydride production is accounted for.

A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P(2) (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P(2) in regulating glucose metabolism and insulin secretion in pancreatic beta-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P(2), in regulating beta-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P(2) were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P(2) content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P(2) in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P(2), in potentiating glucose-induced insulin secretion. Whereas down-regulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P(2) is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

Characterization of a new liver- and kidney-specific pfkfb3 isozyme that is downregulated by cell proliferation and dedifferentiation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P 2), a signalling molecule that controls the balance between glycolysis and gluconeogenesis in several cell types. Four genes, designated Pfkfb1–4, code several PFK-2 isozymes that differ in their kinetic properties, molecular masses, and regulation by protein kinases. In rat tissues, Pfkfb3 gene accounts for eight splice variants and two of them, ubiquitous and inducible PFK-2 isozymes, have been extensively studied and related to cell proliferation and tumour metabolism. Here, we characterize a new kidney- and liver-specific Pfkfb3 isozyme, a product of the RB2K3 splice variant, and demonstrate that its expression, in primary cultured hepatocytes, depends on hepatic cell proliferation and dedifferentiation. In parallel, our results provide further evidence that ubiquitous PFK-2 is a crucial isozyme in supporting growing and proliferant cell metabolism.

Identification and Determination of α-Dicarbonyl Compounds Formed in the Degradation of Sugars

The alpha-dicarbonyl compounds formed in the degradation of glucose and fructose were analyzed by HPLC using 2,3-diaminonaphthalene as derivatizing reagent, and identified as glucosone (GLUCO), 3-deoxyglucosone (3DG), 3-deoxyxylosone (3DX), tetrosone (TSO), triosone (TRIO), 3-deoxytetrosone (3DT), glyoxal (GO), and methylglyoxal (MGO). The results suggest that alpha-dicarbonyl compounds were formed from glucose via non-oxidative 3-deoxyglucosone formation and oxidative glucosone formation in glucose degradation. In addition, TRIO, GO, and MGO were also formed from glyceraldehyde as intermediate. The alpha-dicarbonyl compounds might be formed from glucose via these pathways in diabetes.

Glucose controls multiple processes inSaccharomyces cerevisiaethrough diverse combinations of signaling pathways

We have studied how the lack of glucose sensors in the plasma membrane, or of the enzymes Hxk1, Hxk2, Glk1, which catalyze the first intracellular step in glucose metabolism, affect the different responses of Saccharomyces cerevisiae to glucose. Lack of the G-protein-coupled receptor Gpr1 or of Snf3/Rgt2 did not affect glucose repression of different genes or activation by glucose of plasma membrane ATPase, whereas lack of Gpr1 decreased, in an additive manner with lack of Mth1, the degradation of fructose 1,6-bisphosphatase that takes place in the presence of glucose. In an hxk1 hxk2 glk1 strain, unable to phosphorylate glucose, all of these responses to the sugar were suppressed or strongly reduced. In the absence of Hxk2 (or Hxk1 and Hxk2), glucose repression of SUC2, GAL1 and GDH2 was relieved, but that of FBP1 and ICL1 was maintained. Hxk1 or Hxk2 were needed for activation of plasma membrane ATPase but not for degradation of FbPase.

Multivariate analysis of microarray data by principal component discriminant analysis: prioritizing relevant transcripts linked to the degradation of different carbohydrates in Pseudomonas putida S12

The value of the multivariate data analysis tools principal component analysis (PCA) and principal component discriminant analysis (PCDA) for prioritizing leads generated by microarrays was evaluated. To this end, Pseudomonas putida S12 was grown in independent triplicate fermentations on four different carbon sources, i.e. fructose, glucose, gluconate and succinate. RNA isolated from these samples was analysed in duplicate on an anonymous clone-based array to avoid bias during data analysis. The relevant transcripts were identified by analysing the loadings of the principal components (PC) and discriminants (D) in PCA and PCDA, respectively. Even more specifically, the relevant transcripts for a specific phenotype could also be ranked from the loadings under an angle (biplot) obtained after PCDA analysis. The leads identified in this way were compared with those identified using the commonly applied fold-difference and hierarchical clustering approaches. The different data analysis methods gave different results. The methods used were complementary and together resulted in a comprehensive picture of the processes important for the different carbon sources studied. For the more subtle, regulatory processes in a cell, the PCDA approach seemed to be the most effective. Except for glucose and gluconate dehydrogenase, all genes involved in the degradation of glucose, gluconate and fructose were identified. Moreover, the transcriptomics approach resulted in potential new insights into the physiology of the degradation of these carbon sources. Indications of iron limitation were observed with cells grown on glucose, gluconate or succinate but not with fructose-grown cells. Moreover, several cytochrome- or quinone-associated genes seemed to be specifically up- or downregulated, indicating that the composition of the electron-transport chain in P. putida S12 might change significantly in fructose-grown cells compared to glucose-, gluconate- or succinate-grown cells.

Expression of Inducible 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase/PFKFB3 Isoforms in Adipocytes and Their Potential Role in Glycolytic Regulation

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (F2,6BP), which is a powerful activator of 6-phosphofructo-1-kinase, the rate-limiting enzyme of glycolysis. Four genes encode PFK-2/FBPase (PFKFB1-4), and an inducible isoform (iPFK-2/PFKFB3) has been found to mediate F2,6BP production in proliferating cells. We have investigated the role of iPFK-2/PFKFB3 and related isoforms in the regulation of glycolysis in adipocytes. Human visceral fat cells express PFKFB3 mRNA, and three alternatively spliced isoforms of iPFK-2/PFKFB3 are expressed in the epididymal fat pad of the mouse. Forced expression of the iPFK-2/PFKFB3 in COS-7 cells resulted in increased glucose uptake and cellular F2,6BP content. Prolonged insulin treatment of 3T3-L1 adipocytes led to reduced PFKFB3 mRNA expression, and epididymal fat pads from db/db mice also showed decreased expression of PFKFB3 mRNA. Finally, anti-phospho-iPFK-2(Ser461) Western blotting revealed strong reactivity in insulin-treated 3T3-L1 adipocyte, suggesting that insulin induces the phosphorylation of PFKFB3 protein. These data expand the role of these structurally unique iPFK-2/PFKFB3 isoforms in the metabolic regulation of adipocytes.

MODELING AND OPTIMIZATION OF LACTIC ACID SYNTHESIS BY THE ALKALINE DEGRADATION OF FRUCTOSE IN A BATCH REACTOR

ABSTRACT The present work deals with the determination of the optimal operating conditions of lactic acid synthesis by the alkaline degradation of fructose. It is a complex transformation for which detailed knowledge is not available. It is carried out in a batch or semi-batch reactor. The “Tendency Modeling” approach, which consists of the development of an approximate stoichiometric and kinetic model, has been used. An experimental planning method has been utilized as the database for model development. The application of the experimental planning methodology allows comparison between the experimental and model response. The model is then used in an optimization procedure to compute the optimal process. The optimal control problem is converted into a nonlinear programming problem solved using the sequencial quadratic programming procedure coupled with the golden search method. The strategy developed allows simultaneously optimizing the different variables, which may be constrained. The validity of the methodology is illustrated by the determination of the optimal operating conditions of lactic acid production.

Different glycolytic pathways for glucose and fructose in the halophilic archaeon Halococcus saccharolyticus

The glucose and fructose degradation pathways were analyzed in the halophilic archaeon Halococcus saccharolyticus by 13C-NMR labeling studies in growing cultures, comparative enzyme measurements and cell suspension experiments. H. saccharolyticus grown on complex media containing glucose or fructose specifically 13C-labeled at C1 and C3, formed acetate and small amounts of lactate. The 13C-labeling patterns, analyzed by 1H- and 13C-NMR, indicated that glucose was degraded via an Entner-Doudoroff (ED) type pathway (100%), whereas fructose was degraded almost completely via an Embden-Meyerhof (EM) type pathway (96%) and only to a small extent (4%) via an ED pathway. Glucose-grown and fructose-grown cells contained all the enzyme activities of the modified versions of the ED and EM pathways recently proposed for halophilic archaea. Glucose-grown cells showed increased activities of the ED enzymes gluconate dehydratase and 2-keto-3-deoxy-gluconate kinase, whereas fructose-grown cells contained higher activities of the key enzymes of a modified EM pathway, ketohexokinase and fructose-1-phosphate kinase. During growth of H. saccharolyticus on media containing both glucose and fructose, diauxic growth kinetics were observed. After complete consumption of glucose, fructose was degraded after a lag phase, in which fructose-1-phosphate kinase activity increased. Suspensions of glucose-grown cells consumed initially only glucose rather than fructose, those of fructose-grown cells degraded fructose rather than glucose. Upon longer incubation times, glucose- and fructose-grown cells also metabolized the alternate hexoses. The data indicate that, in the archaeon H. saccharolyticus, the isomeric hexoses glucose and fructose are degraded via inducible, functionally separated glycolytic pathways: glucose via a modified ED pathway, and fructose via a modified EM pathway.