Factors influencing the fecundity of Moniliformis moniliformis (Acanthocephala): constant dose and varied diet

The reproductive performance, including survival, growth and mature egg production, of Moniliformis moniliformis was studied experimentally during primary infections in rats given 10 cystacanths each. Four isoenergetic purified diets containing either 1, 3, 6 or 12% fructose (w/w) were used and the amounts of fructose in the intestinal lumen of rats fed on these diets were measured. It was concluded that, while dietary composition had no effect on parasite establishment, there were associations between diet and the survival, growth and fecundity of the parasite. The host diet containing 3% fructose was considered to be more favourable for the worms than the others; 12% fructose was associated with a curtailment of survival time without any compensatory production of eggs. Female worms from rats fed on diets containing 3 or 6% fructose grew larger, and consistently carried more ovaries and produced more eggs than those from rats fed on the 1% fructose diet. It was concluded that these and other findings might be mediated not only through the amount of available fructose in the intestine for worm metabolism, but also by the responses of the host's intestinal physiology to the varying concentrations of fructose in the diet.     

Effect of glucose concentration on the rate of fructose consumption in native strains isolated from the fermentation of Agave duranguensis

Studies on hexose consumption by Saccharomyces cerevisiae show that glucose is consumed faster than fructose when both are present (9:1 fructose to glucose) in the medium during the fermentation of Agave. The objective of this work was to select strains of S. cerevisiae that consume fructose equal to or faster than glucose at high fructose concentrations by analyzing the influence of different glucose concentrations on the fructose consumption rate. The optimal growth conditions were determined by a kinetics assay using high performance liquid chromatography (HPLC) using 50?g of glucose and 50?g of fructose per liter of synthetic medium containing peptone and yeast extract. Using the same substrate concentrations, strain ITD-00185 was shown to have a higher reaction rate for fructose over glucose. At 75?g of fructose and 25?g of glucose per liter, strain ITD-00185 had a productivity of 1.02 gL^?1?h^?1 after 40?h and a fructose rate constant of 0.071?h^?1. It was observed that glucose concentration positively influences fructose consumption when present in a 3:1 ratio of fructose to glucose. Therefore, adapted strains at high fructose concentrations could be used as an alternative to traditional fermentation processes.     

The immediate effects of insulin and fructose on the metabolism of the perfused liver. Changes in lipoprotein secretion, fatty acid oxidation and esterification, lipogenesis and carbohydrate metabolism

1. When livers from fed rats were perfused with blood containing elevated concentrations of rat insulin or blood to which fructose was added, the oxidation of free fatty acids was depressed and their esterification was increased. 2. Raised concentrations of insulin or addition of fructose increased secretion of triglyceride in very-low-density lipoproteins, but only insulin caused more of the free fatty acids taken up by the liver to be incorporated into very-low-density lipoproteins. 3. When insulin and fructose were added together the combined effect on oxidation and esterification of free fatty acids and on secretion of very-low-density lipoproteins was equal to the sum of the effects of either alone. No statistically significant interaction between the effects of fructose and insulin was found for any of the parameters investigated. 4. Bovine insulin had similar effects, in most respects, to comparable studies with raised concentrations of rat insulin. 5. Lipogenesis was increased in the livers treated with fructose plus bovine insulin. 6. A significant proportion of the fatty acids in very-low-density lipoproteins were derived either from the liver triglyceride pool or from lipogenesis. This fraction was increased both by treatment with insulin or fructose, and was augmented further when both insulin and fructose were present together. 7. The uptake of fructose by the perfused liver was similar to that found in vivo. It was unaffected by the presence of insulin. 8. Addition of fructose to the perfused liver caused perfusate lactate concentrations to increase, as a result of diminished hepatic uptake of lactate. 9. The uptake of free fatty acids by the perfused liver was unaffected by the addition of either insulin or fructose. 10. The distribution among the various lipid classes in plasma lipoproteins of label arising from the hepatic uptake of [(14)C]oleate was unaltered by the addition of either fructose or insulin. 11. It is suggested that the effects described are due principally to control of the balance between esterification of fatty acids and lipolysis of the ensuing triglyceride, fructose enhancing esterification and insulin inhibiting lipolysis.     

Study of the fructose 6-phosphate/fructose 1,6-bi-phosphate cycle in the liver in vivo.

1. The method proposed by Rognstad & Katz [(1976) Arch, Biochem, Biophys, 177, 337-345] for the determination of the fructose 6-phosphate/fructose 1,6-bisphosphate cycle by the randomization of carbon between C-1 and C-6 of glucose glucose formed from [1-14C] galactose was applied to anaesthetized rats and conscious mice. 2. It was checked that the hydrolysis of fructose 6-phosphate by glucose 6-phosphatase is too weak to invalidate the method. The participation of the Cori cycle in the randomization was negligible within the short experimental period used (2-4 min). 3. No detectable randomization of carbon was observed in starved animals, indicating that phosphofructokinase is inactive in this experimental condition. 4. Randomization of carbon was detected as soon as 1 min after administration of [1-14C] galactose to fed animals and was maximal at about 3-4 min. It was calculated that on average 15% of the glucose formed by the liver to fed rats was recycled through the triose phosphates. The extent of cycling was quite variable. Recycling was also observed in starved rats in which glucose had been administered intravenously 10 min previously. In these animals, recycling was completely inhibited by glucagon. 5. The main factors that appear to be responsible for the very large changes in recycling observed in various experimental conditions are the concentrations of fructose 1,6-bisphosphate and of fructose 6-phosphate and also the affinity of phosphofructokinase for fructose 6-phosphate. The concentration of nucleotides does not seem to play a role.     

The Formation of Glucose 1,6-Diphosphate from Fructose 1,6-Diphosphate by Yeast Phosphoglucomutase

It was found that fructose 1,6-diphosphate, the main intermediate of glycolysis, was able to act as a coenzyme of yeast phosphoglucomutase reaction. The mechanism of the coenzymatic activity of fructose 1,6-diphosphate was studied. It was indicated in the fructose 1,6-diphosphate dependent reaction that glucose 1,6-diphosphate was formed by the phosphate-transfer of fructose 1,6-diphosphate to glucose 1-phosphate in the first step, and in the second step the conversion of glucose 1-phosphate to glucose 6-phosphate, the original mutase reaction, occurred in the presence of glucose 1,6-diphosphate. The kinetic constants in the reaction of the first step were determined from the time courses of the fructose 1,6-diphosphate dependent reaction.     

Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6-bisphosphate in yeast

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500-fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP-agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [gamma-32P]ATP-Mg and the catalytic subunit of cyclic-AMP-dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105-kDa peptide and also, but only faintly, into a 162-kDa subunit. A low-Km (0.1 microM) fructose-2,6-bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6-[2-32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300-fold, had an apparent molecular mass of 110 kDa and was made of two 56-kDa subunits. It was inhibited by fructose 6-phosphate (Ki = 5 microM) and stimulated 2-3-fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low-Km fructose-2,6-bisphosphatase clearly separated from each other in all purification procedures used. A high-Km (approximately equal to 100 microM), apparently specific, fructose 2,6-bisphosphatase was separated by anion-exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6-bisphosphate, which it converts to fructose 6-phosphate and Pi, because it is not inhibited by fructose 6-phosphate, glucose 6-phosphate or Pi. Several other phosphatases able to hydrolyze fructose 2,6-bisphosphate into a mixture of fructose 2-phosphate, fructose 6-phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6-bisphosphate (Km greater than 50 microM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.     

The enzymes of fructose metabolism in the liver of chick embryos

The activity of fructose cycle enzymes remains practically constant in chick embryonic liver during ontogenesis. Change in ratio of aldolase A to B activities was detected. It is suggested that fructose enters the cycle via the sorbitol pathway in which aldose reductase and sorbitol dehydrogenase are involved.     

Mutation of Clostridium thermocellum in the presence of certain carbon sources

Abstract When a cellobiose-grown inoculum of Clostridium thermocellum was transferred to either glucose or fructose as the sole carbon sourcem growth occurred only after a long lag of 180–200 h. We established that sugar uptake and phosphorylation were not limiting growth nor was the lag period the time take for a physiological adaptation process or for the growth of a mutant carried over in the cellibiose-grown incoculum. It became apparent that a mutation was occuring during the lag period in response to the selection pressure exerted by the presence of glucose or fructose as the sole carbon source. Once growth occurred on glucose and fructose, the cells could be transferred to cellobiose and back to glucose or fructose without exhibiting the long lag period. The change was stable over several transfers in the respective sugars.     

Studies on the in vitro phosphorylation of 6-phosphofructo-1-kinase from rat liver

Rat hepatic 6-phosphofructo-1-kinase (ATP:d-fructose-6-phosphate 1-phosphotransferase) was purified to homogeneity and its phosphorylation by the catalytic subunit of the cyclic AMP-dependent protein kinase examined. Up to 4 mol of phosphate could be incorporated per mole of tetrameric enzyme, and the phosphate was incorporated into seryl residues. Phosphorylation did not alter the affinity of the enzyme for fructose 6-phosphate or fructose 2,6-bisphosphate. The rate of phosphorylation was enhanced by allosteric activators of 6-phosphofructo-1-kinase such as AMP and fructose 2,6-bisphosphate, and it was decreased by the allosteric inhibitors ATP and H⁺. The phosphopeptide region of the enzyme subunit was susceptible to limited proteolysis by trypsin. Removal of the phosphopeptide did not affect the subunit molecular weight nor the maximum activity of the enzyme, but it enhanced the apparent affinity of the enzyme for both fructose 6-phosphate and fructose 2,6-bisphosphate. It is concluded that the phosphopeptide region of the enzyme subunit is an important determinant of the affinity of the enzyme for its substrate as well as for the allosteric activator fructose 2,6-bisphosphate.     

Study of the microbial aggregation in Mycobacterium using image analysis and electron microscopy

Cellular aggregation, which occurs in both prokaryotes and eukaryotes, is controlled by the hydrophobicity as well as the electrokinetic potential of the cell surface and substratum. It is known that the Mycobacterium genus form aggregates, but the influence of sugar on the cellular aggregation has not been reported for this genus. The mutant strain Mycobacterium sp. MB-3683 that transforms sterol to androstenedione (AD), a steroidal precursor used by the pharmaceutical industries, was employed in this study. This strain was cultivated in a synthetic medium on three sugars (glycerol, glucose and fructose) at different concentrations, and at 144 h microbial growth, cellular aggregation, hydrophobicity, lipid content, fatty acid composition, and width of cellular walls were measured. It was observed that at different sugar concentrations, similar growth and pH were obtained. However, in fructose, the aggregation level was significantly high, followed by glycerol and glucose (fructose < glycerol < glucose). These results were confirmed using electron microscopy and the aggregate area quantified by image analysis. Hydrophobicity was the highest in fructose and the lowest in glucose. The total lipids, in contrast to cellular hydrophobicity, were higher in glucose than glycerol. Although, the hydrophilic-lipophilic balance (HLB) of principal fatty acids isolated was similar regardless of sugar used. In glycerol and fructose, the paraffins were observed, which are responsible for the high cellular hydrophobicity detected above. The width of cell wall of the organisms grown on glucose and fructose was similar, but in glycerol the walls were very thin. There is a correspondence between cell wall width and lipid content.     

Enhanced production of human monoclonal antibodies by the use of fructose in serum-free hybridoma culture media

It was found that the production of human monoclonal antibodies (MoAbs) by human-human hybridomas can be significantly enhanced by replacing glucose with fructose in the dish culture medium. Optimization of initial concentrations of fructose and glutamine, another influencing factor for MoAb production, enabled an enhanced production of human MoAb 2.1 times higher than that obtained using the conventional culture media employing glucose. It was shown by kinetic analysis that enhanced MoAb production at the optimum fructose concentration can be attributed to the retention of high specific antibody production rates and diminished time lag during the course of culture.These dish culture results with fructose-containing medium were successfully applied to the continuous perfusion culture with a slight modification, where 2.9- and 1.9-fold enhancements in specific antibody production rate and MoAb concentration, respectively, were attained as compared with the conventional glucose-containing medium.An inverse relationship was observed between the secreted concentrations of lactic acid and MoAb when the hybridoma was cultured in the media containing varying concentrations of fructose, i.e., the lower the lactic acid concentration, the higher the MoAb production andvice versa, suggesting that fructose at appropriate concentrations in the medium can serve as an alternative sugar for the efficient production of human MoAbs, with reduced pH shifts, for the serum-free culture of human-human hybridomas.     

A protein from rat liver confers to glucokinase the property of being antagonistically regulated by fructose 6-phosphate and fructose 1-phosphate

At a concentration of 1 mM, fructose 1-phosphate stimulated about twofold, and glucose 6-phosphate inhibited by about 30%, the phosphorylation of 5 mM glucose in high-speed supernatants prepared from rat liver or from isolated hepatocytes, but did not affect, or barely so, the activity of a partially purified preparation of glucokinase. Anion-exchange chromatography of liver extracts separated glucokinase from a fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive inhibitor of that enzyme. This inhibitor could be further purified by chromatography on phospho-Ultrogel. It was destroyed by trypsin and was heat-labile. It inhibited glucokinase competitively with respect to glucose and its inhibitory effect was greatly reinforced by fructose 6-phosphate although not by glucose 6-phosphate. Fructose 1-phosphate relieved the enzyme of the inhibitory effect of the regulator and antagonised the effect of fructose 6-phosphate in a competitive manner. It is concluded that the regulator plays a role in the physiological control of the activity of glucokinase, particularly with respect to the stimulatory effect of fructose in isolated hepatocytes (see preceding paper in this journal).     

Binding of hexose bisphosphates to muscle phosphofructokinase

On the basis of kinetic activation assays, the apparent affinity of muscle phosphofructokinase for fructose 2,6-bisphosphate was about 9-fold greater than that for fructose 1,6-bisphosphate, which in turn was about 10 times higher than that for glucose 1,6-bisphosphate. Equilibrium binding experiments showed that both fructose bisphosphates bind to phosphofructokinase with negative cooperativity; the affinity for fructose 2,6-bisphosphate was about 1 order of magnitude greater than the affinity for fructose 1,6-bisphosphate. Binding of fructose 2,6-bisphosphate to phosphofructokinase was antagonized by fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and vice versa. Both fructose bisphosphates promoted aggregation of the enzyme to higher polymers as indicated by sucrose density gradient centrifugation. Other indicators of phosphofructokinase conformation such as thiol reactivity and maximum activation of in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase gave identical results in the presence of fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, or glucose 1,6-bisphosphate, indicating a common conformation is produced by all three ligands. It is concluded that the sugar bisphosphates bind to a single site on the enzyme.     

Fructose 2,6-bisphosphate. A new activator of phosphofructokinase

A new activator of rat liver phosphofructokinase was partially purified from rat hepatocyte extracts by DEAE-Sephadex chromatography. The activator, which eluted in the sugar diphosphate region, was sensitive to acid treatment but resistant to heating in alkali. Mild acid hydrolysis resulted in the appearance of a sugar monophosphate which was identified as fructose 6-phosphate by gas chromatography/mass spectroscopy. These observations suggest that the activator is fructose 2,6-bisphosphate. This compound was synthesized by first reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide and then treating the cyclic intermediate with alkali. The structure of the synthetic compound was definitively identified as fructose 2,6-bisphosphate by 13C NMR spectroscopy. Fructose 2,6-bisphosphate had properties identical with those of the activator purified from hepatocyte extracts. It activated both the rat liver and rabbit skeletal muscle enzyme in the 0.1 microM range and was several orders of magnitude more effective than fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate was not a substrate for aldolase or fructose 1,6-bisphosphatase. It is likely that this new activator is an important physiologic factor of phosphofructokinase in vivo.     

Protective role of fructose in the metabolism of astroglial C6 cells exposed to hydrogen peroxide

Astroglial cells represent the main line of defence against oxidative damage related to neurodegeneration. Therefore, protection of astroglia from an excess of reactive oxygen species could represent an important target of the treatment of such conditions. The aim of our study was to compare the abilities of glucose and fructose, the two monosaccharides used in diet and infusion, to protect C6 cells from hydrogen peroxide (H₂O₂)-mediated oxidative stress. It was observed using confocal microscopy with fluorescent labels and the MTT test that fructose prevents changes of oxidative status of the cells exposed to H₂O₂ and preserves their viability. Even more pronounced protective effects were observed for fructose 1,6-bis(phosphate). We propose that fructose and its intracellular forms prevent H₂O₂ from participating in the Fenton reaction via iron sequestration. As fructose and fructose 1,6-bis(phosphate) are able to pass the blood–brain barrier, they could provide antioxidative protection of nervous tissue in vivo. So, in contrast to the well-known negative effects of frequent consumption of fructose under physiological conditions, acute infusion or ingestion of fructose or fructose 1,6-bis(phosphate) could be of benefit in the cytoprotective therapy of neurodegenerative disorders related to oxidative stress.     

Carbon catabolite repression of invertase during batch cultivations of Saccharomyces cerevisiae: the role of glucose, fructose, and mannose

When Saccharomyces cerevisiae are grown on a mixture of glucose and another fermentable sugar such as sucrose, maltose or galactose, the metabolism is diauxic, i.e. glucose is metabolized first, whereas the other sugars are metabolized when glucose is exhausted. This phenomenon is a consequence of glucose repression, or more generally, catabolite repression. Besides glucose, the hexoses fructose and mannose are generally also believed to trigger catabolite repression. In this study, batch fermentations of S. cerevisiae in mixtures of sucrose and either glucose, fructose or mannose were performed. It was found that the utilization of sucrose is inhibited by concentrations of either glucose or fructose higher than 5 g/l, and thus that glucose and fructose are equally capable of exerting catabolite repression. However, sucrose was found to be hydrolyzed to glucose and fructose, even when the mannose concentration was as high as 17 g/l, indicating, that mannose is not a repressing sugar. It is suggested that the capability to trigger catabolite repression is connected to hexokinase PII, which is involved in the in vivo phosphorylation of glucose and fructose. Received: 5 May 1998 / Received revision: 3 August 1998 / Accepted: 8 August 1998     

Kinetics of the selective fermentation of glucose from glucose/fructose mixtures using Saccharomyces cerevisiae ATCC 36859

The kinetics of batch fermentation during the growth of S. cerevisiae ATCC 36859 was studied in various glucose/fructose mixtures. It was found that the growth is inhibited equally by glucose and fructose even though fructose is not consumed to any large extent by the yeast under the conditions tested here. The inhibition of growth by the substrate and ethanol is represented by linear equations. These equations were combined with the MONOD expression in order to formulate equations for the biomass growth, glucose and fructose consumption and ethanol production. Parameter estimates were obtained by fitting these equations to batch fermentation data and so developing models which indicate that the growth is completely inhibited when 62 g/l ethanol is produced by the yeast, while glucose consumption and ethanol production continue up to an ethanol concentration of 152 g/l. Products containing a high concentration of fructose are best produced by using a high initial biomass concentration.     

Hormonal regulation of fructose 2,6-bisphosphate levels in epididymal adipose tissue of rat

The participation of fructose 2,6-bisphosphate on glycolysis stimulated by insulin and adrenaline in incubated white adipose tissue of rat was investigated. Adrenaline addition to incubated fat-pads strongly decreased the intracellular levels of fructose 2,6-bisphosphate. When the tissue was preincubated with glucose, the presence of insulin in the incubation medium increased fructose 2,6-bisphosphate levels 2-fold. These variations were related to changes in the substrates, ATP and fructose 6-phosphate. It therefore appears that fructose 2,6-bisphosphate may be involved in the control of insulin-induced glycolysis, but it does not seem to play a role in the stimulation of glucolysis by adrenaline.     

Fructose 2,6-bisphosphate and the climacteric in bananas

This work was done to test the view that there is a marked rise in the content of fructose 2,6-bisphosphate during the climacteric of the fruit of banana (Musa cavendishii Lamb ex. Paxton). Bananas were ripened in the dark in a continuous stream of air in the absence of exogenous ethylene. CO2 production and the contents of fructose 2,6-bisphosphate and sucrose were monitored over a 15-day period. A range of extraction procedures for fructose 2,6-bisphosphate were compared. Recovery of fructose 2,6-bisphosphate added to samples of unripe fruit varied from poor to unmeasurable. Recoveries from samples of ripe fruit were high. It is argued that this differential recovery of fructose 2,6-bisphosphate undermines claims that the amount of this compound increases at the climacteric. When recoveries are taken into account, our data suggest that there is no major change in fructose 2,6-bisphosphate content during the onset of the climacteric in bananas.     

Effects of various metabolic conditions and of the trivalent arsenical melarsen oxide on the intracellular levels of fructose 2,6-bisphosphate and of glycolytic intermediates in Trypanosoma brucei

Upon differential centrifugation of cell-free extracts of Trypanosoma brucei, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase behaved as cytosolic enzymes. The two activities could be separated from each other by chromatography on both blue Sepharose and anion exchangers. 6-phosphofructo-2-kinase had a Km for both its substrates in the millimolar range. Its activity was dependent on the presence of inorganic phosphate and was inhibited by phosphoenolpyruvate but not by citrate or glycerol 3-phosphate. The Km of fructose-2,6-bisphosphatase was 7 microM; this enzyme was inhibited by fructose 1,6-bisphosphate (Ki = 10 microM) and, less potently, by fructose 6-phosphate, phosphoenolpyruvate and glycerol 3-phosphate. Melarsen oxide inhibited 6-phosphofructo-2-kinase (Ki less than 1 microM) and fructose-2,6-bisphosphatase (Ki = 2 microM) much more potently than pyruvate kinase (Ki greater than 100 microM). The intracellular concentrations of fructose 2,6-bisphosphate and hexose 6-phosphate were highest with glucose, intermediate with fructose and lowest with glycerol and dihydroxyacetone as glycolytic substrates. When added with glucose, salicylhydroxamic acid caused a decrease in the concentration of fructose 2,6-bisphosphate, ATP, hexose 6-phosphate and fructose 1,6-bisphosphate. These studies indicate that the concentration of fructose 2,6-bisphosphate is mainly controlled by the concentration of the substrates of 6-phosphofructo-2-kinase. The changes in the concentration of phosphoenolpyruvate were in agreement with the stimulatory effect of fructose 2,6-bisphosphate on pyruvate kinase. At micromolar concentrations, melarsen oxide blocked almost completely the formation of fructose 2,6-bisphosphate induced by glucose, without changing the intracellular concentrations of ATP and of hexose 6-phosphates. At higher concentrations (3-10 microM), this drug caused cell lysis, a proportional decrease in the glycolytic flux, as well as an increase in the phosphoenolypyruvate concentrations which was restricted to the extracellular compartment. Similar changes were induced by digitonin. It is concluded that the lytic effect of melarsen oxide on the bloodstream form of T. brucei is not the result of an inhibition of pyruvate kinase.