Comparative quantitative analysis of sucrose and related compounds using ion exchange and reverse phase chromatographic methods

Two analytical methods of sugar determination, namely ion exchange chromatography on an anionic resin coupled with electrochemical detection, and reverse phase chromatography on Nucleosil-NH2 resin equipped with a light scattering detector were tested and compared as regards their rapidity, sensitivity and accuracy with sucrose, fructose, glucose, raffinose, maltose, arabinose, fucose, rhamnose and xylose. Excellent resolution and highly reproducible results were obtained in both cases. Greater sensitivity up to the picomolar range was possible however only with ion exchange chromatography. Reverse phase chromatography was successfully applied to the time course of sucrose hydrolysis under chemical (acid) and enzymatic (invertase) conditions. The hydrolysis was monitored by determining sucrose degradation and the corresponding formation of glucose and fructose.     

Changes in T-cell phenotype and adhesion molecules expression in psoriatic lesions after low-dose cyclosporin therapy

The fermentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically, only 12 species ferment it, and only 2 species, Candida molischiana and Candida wickerhamii, also ferment cellodextrins. Candida molischiana produced beta-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of beta-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana beta-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose, maltose, mannitol, or glucitol, C. molischiana preferentially utilized glucose and produced little beta-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose, the yeast preferentially utilized the monosaccharides and produced little beta-glucosidase activity. Candida molischiana produced beta-glucosidase and co-utilized cellobiose and xylose, maltose, or trehalose. Glucose and fructose, mannose, or trehalose were co-utilized; however, no beta-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose, fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol).     

Triosephosphate metabolism by mature boar spermatozoa

Boar sperm rapidly interconverted dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, produced fructose-1,6-bisphosphate, approximately equilibrium concentrations of fructose 6-phosphate and glucose 6-phosphate but not glycerol or glycerol 3-phosphate. In the presence of 3-chloro-1-hydroxypropanone, an inhibitor of stage 2 of the glycolytic pathway, the triosephosphates were metabolized faster, produced less fructose-1,6-bisphosphate, fructose 6-phosphate and glucose 6-phosphate, but not glycerol or glycerol 3-phosphate. This suggests that these cells may have the capacity to convert glycolytic intermediates into a storage metabolite to conserve carbon atoms for the eventual synthesis of lactate.     

Inactivation of glyceraldehyde 3-phosphate dehydrogenase by sugars, prednisolone-21-hemisuccinate, cyanate and other small molecules

Diabetes, diarrhoea, renal failure and glucocorticoid therapy have all been identified as independent risk factors for cataract. Increased post-translational modification of proteins, leading to inactivation of enzymes and induction of conformational changes within proteins could result in lens opacification and cataract. Aspirin has been associated with many beneficial effects, including protection against cataract, in-vivo. alpha-Crystallin has been shown to act as a molecular chaperone in-vitro. This lenticular protein prevented the thermal aggregation of other lens proteins in-vitro and has sequence and functional homology with the small heat shock proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAP-DH) is constitutively expressed in tissues and is susceptible to chemical modification in-vivo. In-vitro incubations of GAP-DH with sugars, cyanate and prednisolone-21-hemisuccinate, all led to significant loss of enzyme activity with time in two buffer systems. Rapid inactivation occurred when GAP-DH was incubated with fructose 6-phosphate or prednisolone-21-hemisuccinate. Slower inactivation was observed when GAP-DH was incubated with fructose, glucose 6-phosphate or potassium cyanate. Glucose did not inactivate GAP-DH under the conditions of our experiments. Aspirin and ibuprofen were shown to inactivate GAP-DH very rapidly in-vitro. Bovine lenticular alpha-crystallin conferred no protection against GAP-DH inactivation. This is the first occasion that alpha-crystallin has been demonstrated to be unable to protect against inactivation in our chemical enzyme inactivation system. This may have implications for the susceptibility of lenticular GAP-DH to post-translational inactivation.     

Preliminary investigations of an amperometric oligosaccharide dehydrogenase-based electrode for the detection of glucose and some other low molecular weight saccharides

Biosensors for the determination of sugars were constructed using oligosaccharide dehydrogenase (ODH) and soluble phenazine methosulfate (PMS) or an osmium-based three-dimensional redox hydrogel. In the latter case the enzyme and poly(1-vinylimidazole) complexed with osmium (4,4'-dimethylbpy)2Cl were cross-linked with poly(ethylene glycol) diglycidyl ether. Both electrode configurations showed similar sensitivities for glucose in the range between 8 and 21 muAmM-1 cm-2. The responses for 10 mono and oligosaccharides were studied. There was no response for fructose. In the concentration range 0.1-20 mM the relative sensitivities were determined for arabinose (96%), xylose (3%), mannose (50%), galactose (11%), glucose (100%), maltose (24%), lactose (12%), cellobiose (34%) and maltotriose (10%).     

Superoxide dependence of the toxicity of short chain sugars

Erythrose inhibited the growth of a sodA sodB strain of Escherichia coli under aerobiosis; but did not inhibit anaerobic growth of the sodA sodB strain, or the aerobic growth of the superoxide dismutase (SOD)-competent parental strain. A SOD mimic protected the sodA sodB strain against the toxicity of erythrose as did the carbonyl-blocking reagents hydrazine and aminoguanidine. Three carbon sugars, such as glyceraldehyde and dihydroxy acetone, and the two carbon sugar glycolaldehyde, were similarly toxic in an O-2-dependent manner. An unidentified dialyzable component in E. coli extract augmented the oxidation of short chain sugars, and this was partially inhibitable by SOD. The toxicity of the short chain sugars appears to be because of an O-2-dependent oxidation to alpha, beta-dicarbonyl compounds. In keeping with this view was the O-2-independent toxicity of methylglyoxal.     

Kinetics of nonenzymatic glycation of ribonuclease A leading to advanced glycation end products. Paradoxical inhibition by ribose leads to facile isolation of protein intermediate for rapid post-Amadori studies

Nonenzymatic glycation (Maillard reaction) of long-lived proteins is a major contributor to the pathology of diabetes and possibly aging and Alzheimer's disease. We report here kinetic studies of the glycation of the model protein ribonuclease A by glucose and ribose leading to the formation of antigenic advanced glycation end products ("AGEs"), detectable by AGE-specific polyclonal antibodies, and pentosidine, an acid-stable fluorescent AGE. As anticipated, the kinetics of glycation by ribose were considerably faster than by glucose, and the rate of AGE formation initially increased with increasing sugar concentrations. However, ribose above 0.15 M appeared to paradoxically slow the kinetics of AGE formation, suggesting ribose inhibits the conversion of "early" Amadori rearrangement products to "late" AGEs and thus favors the accumulation of reactive Amadori intermediates. The facile isolation of such protein intermediates was achieved by an "interrupted glycation" protocol which free and reversibly bound (Schiff base) ribose was removed following a short (24h) initial incubation of 0.5 M ribose at 37 degrees C. The kinetics of buildup of the Amadori intermediates and the kinetics of their post-Amadori conversion to antigenic AGEs were independently studied. A rapid and reversible inhibition of the post-Amadori kinetics by free ribose was verified by direct re-addition of ribose to the isolated, sugar-free intermediate. The pH dependence of the kinetics of antigenic AGE formation from such intermediates was measured and exhibited an unusual bell-shaped profile over the pH range of 5.0-9.5 with a maximum near pH 8.0. Aminoguanidine, a pharmacological AGE inhibitor, was found to moderately or weakly inhibit antigenic AGE formation in such post- Amadori steps. The isolation of the glycated ribonuclease intermediate thus simplifies kinetic and mechanistic studies of AGE formation, permits AGE studies in the absence of complications arising from free or Schiff base bound sugar, and provides a novel methodology for evaluating the mechanism and efficacy of therapeutic agents that may inhibit AGE formation.     

Modulation of L-type Ca channel activity by P2-purinergic agonist in cardiac cells

Plant cell wall polysaccharides are primarily composed of hexose or hexose derivatives, but a significant fraction is hemicellulose which contains pentose sugars. Prevotella ruminicola B14, a predominant ruminal bacterium, simultaneously metabolized pentoses and glucose or maltose, but the organism preferentially fermented pentoses over cellobiose and preferred xylose to sucrose. Xylose and arabinose transport at either low (2 microM) or high (1 mM) substrate concentrations were observed only in the presence of sodium and if oxygen was excluded during the harvest and assay procedures. An artificial electrical potential (delta psi) or chemical gradient of sodium (delta pNa) drove transport in anaerobically prepared membrane vesicles. Because (i) transport was electrogenic, (ii) a delta pNa drove uptake, and (iii) the number of sodium binding sites was approximately 1, it appeared that P. ruminicola possessed pentose/sodium support mechanisms for the transport of arabinose and xylose at low substrate concentrations. Pentose uptake exhibited a low affinity for xylose or arabinose (> 300 microM), and transport of xylose exhibited bi-phasic kinetics which suggested that a second sodium-dependent xylose transport system was present. Little study has been made on solute transport by Prevotella (Bacteroides) species and this work represents the first use of isolated membrane vesicles from these organisms.     

Urinary sugar phosphates and related organic acids in fructose-1,6-diphosphatase deficiency

Two sisters with fructose-1,6-diphosphatase deficiency are reported. They presented with ketonuria, elevated plasma transaminase activity and severe metabolic acidosis during hypoglycaemic crises, which resembled Reye syndrome. Intravenous fructose tolerance tests provoked severe hypoglycaemia and metabolic acidosis. Fructose-1,6-diphosphatase activities in both peripheral leukocytes and cultured lymphocytes were below the limit of detection. Urinary organic acid analysis during crises revealed markedly increased excretion of lactate, ketone bodies, glycerol and glycerol-3-phosphate. We newly identified other glycolytic intermediates, glyceraldehyde, 3-phosphoglycerate and fructose-1,6-diphosphate, in the urine during hypoglycaemic attacks or after fructose tolerance tests. Identification of such compounds may be useful in the early diagnosis of this disease.     

Potentiation of glucose-induced insulin secretion by fagomine, a pseudo-sugar isolated from mulberry leaves

The mechanisms of potentiation by fagomine, an N-containing pseudo-sugar derived from mulberry leaves, of insulin secretion from isolated rat pancreatic islets in response to glucose was studied. Fagomine at more than 1 mmol/L significantly potentiated insulin secretion induced by 10 mmol/L glucose. The pseudo-sugar, however, did not affect the basal insulin secretion assessed at a glucose concentration of 3.5 mmol/L. The effects of fagomine on 10 mmol/L and 20 mmol/L glucose-induced insulin secretion were not significantly different. Fagomine (4 mmol/L) also potentiated glyceraldehyde-induced insulin secretion, but not the leucine-induced type. Glycolysis assessed by lactate production from glucose was significantly enhanced. The amounts of all intermediates (from glucose 6-phosphate to glyceraldehyde 3-phosphate) of the upper part of the glycolytic pathway in islets incubated with 20 mmol/L glucose were not affected by 4 mmol/L fagomine. The rise in the ATP/ADP ratio through both the glycolytic pathway and the citric acid cycle is believed to be pivotal in glucose- and glyceraldehyde-induced insulin secretion; whereas the ATP/ADP ratio rise through the citric acid cycle via the formation of acetyl-CoA is involved in leucine-induced insulin secretion. Our findings, together with these considerations, suggest that fagomine potentiates glucose-induced insulin secretion through acceleration of some step(s) after the formation of glyceraldehyde 3-phosphate in the glycolytic pathway.     

Power Doppler findings in nasopharingeal angiofibroma

Three fractions of polysaccharides that have high biological activity were extracted from Chinese bamboo leaves (Indocalamus Tesselatus) with 0.9% sodium chloride, 1% ammonium oxalate and 85% ethanol. Their component sugars were investigated by paper chromatography developed with n-butanol-pyridine-water (6:4:3 by vol), and by gas chromatography with crown ester stationary-phase-fused silica capillary column. Their monosaccharide compositions are different from each other with glucuronic acid, galactose, arabinose, mannose, glucose, and xylose (52, 18, 9, 7 and 6%, respectively) in one fraction, only glucuronic acid in another and glucuronic acid, fucose, arabinose and xylose (36, 31, 24 and 9%, respectively) in the third fraction. The protection effect of two polysaccharides against experimental hepatitis was studied in mice.     

Teratogenicity of 3-deoxyglucosone and diabetic embryopathy

The increased rate of embryonic dysmorphogenesis in diabetic pregnancy is correlated with the severity and duration of the concurrent hyperglycemia during early gestation. Whole embryo culture was used to investigate a possible association of hyperglycemia-induced disturbances of embryo development with tissue levels of the three alpha-oxoaldehydes: glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). Rat embryos exposed to high glucose levels in vitro showed severe dysmorphogenesis and a 17-fold increased concentration of 3-DG compared with control embryos cultured in a low glucose concentration. Exogenous 3-DG (100 micromol/l) added to the medium of control cultures yielded an increased embryonic malformation rate and a 3-DG concentration similar to that of embryos cultured in high glucose. Addition of superoxide dismutase (SOD) to the culture medium decreased the malformation rates of embryos exposed to either high glucose or high 3-DG levels, but it did not decrease the high embryonic 3-DG concentrations caused by either agent. Our results implicate the potent glycating agent 3-DG as a teratogenic factor in diabetic embryopathy. In addition, the anti-teratogenic effect of SOD administration appears to occur downstream of 3-DG formation, suggesting that 3-DG accumulation leads to superoxide-mediated embryopathy.     

Characterization of Pseudomonas paucimobilis FP2001 which forms flagella depending upon the presence of rhamnose in liquid medium

A bacterial strain FP2001 isolated from the exudate of land reclaimed for municipal waste was identified as Pseudomonas paucimobilis. Cells of strain FP2001 were mobile by means of polar monotrichous flagellum, only when rhamnose was added as a carbon source in the liquid medium. The replacement of rhamnose by arabinose, galactose, glucose or xylose did not lead to the formation of flagella.     

Genetic analysis of the central untranslated genome region and the proximal coding part of the F gene of wild-type and vaccine canine distemper morbilliviruses

Formaldehyde and glycolaldehyde (substrates of the formose autocatalytic cycle) were shown to react with ammonia yielding alanine and homoserine under mild aqueous conditions in the presence of thiol catalysts. Since similar reactions carried out without ammonia yielded alpha-hydroxy acid thioesters (Weber, 1984a, b), the thiol-dependent synthesis of alanine and homoserine is presumed to occur via amino acid thioesters--intermediates capable of forming peptides (Weber and Orgel 1979). A pH 5.2 solution of 20 mM formaldehyde, 20 mM glycolaldehyde, 20 mM ammonium chloride, 23 mM 3-mercaptopropionic acid, and 23 mM acetic acid that reacted for 35 days at 40 degrees C yielded (based on initial formaldehyde) 1.8% alanine and 0.08% homoserine. In the absence of thiol catalyst, the synthesis of alanine and homoserine was negligible. Alanine synthesis required both formaldehyde and glycolaldehyde, but homoserine synthesis required only glycolaldehyde. At 25 days the efficiency of alanine synthesis calculated from the ratio of alanine synthesized to formaldehyde reacted was 2.1%, and the yield (based on initial formaldehyde) of triose and tetrose intermediates involved in alanine and homoserine synthesis was 0.3 and 2.1%, respectively. Alanine synthesis was also seen in similar reactions containing only 10 mM each of aldehyde substrates, ammonia, and thiol. The prebiotic significance of these reactions that use the formose reaction to generate sugar intermediates that are converted to reactive amino acid thioesters is discussed.     

Separation of 1-phenyl-3-methyl-5-pyrazolone derivatives of monosaccharides by capillary electrochromatography

1-Phenyl-3-methyl-5-pyrazolone (PMP) derivatives of component monosaccharides in glycoproteins (fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine) and epimeric aldopentoses (arabinose, lyxose, ribose and xylose) were well separated from each other by capillary electrochromatography on a Hypersil ODS column with a mixture of 50 mM N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) buffer, pH 6.0 to approximately 6.3, and acetonitrile (2.2:1 v/v) as eluent. The elution of these compounds showed relatively strong dependence on the pH and concentration of the buffer salts contained in the eluent, as compared to the elution by pressure-driven high-performance liquid chromatography (HPLC) on the same stationary phase, but separation of PMP-monosaccharides was better than that by HPLC. Retention times of PMP-monosaccharides were highly reproducible with a relative standard deviation (RSD) of approximately 0.6%, and quantification with an RSD less than 5% could be achieved using 3-O-methylglucose as an internal standard.     

NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax. The first identified archaeal member of the aldehyde dehydrogenase superfamily is a glycolytic enzyme with unusual regulatory properties

The hyperthermophilic archaeum Thermoproteus tenax possesses two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity and phosphate dependence of the catalyzed reaction. NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase catalyzes the phosphate-independent irreversible oxidation of D-glyceraldehyde 3-phosphate to 3-phosphoglycerate. The coding gene was cloned, sequenced, and expressed in Escherichia coli. Sequence comparisons showed no similarity to phosphorylating glyceraldehyde-3-phosphate dehydrogenases but revealed a relationship to aldehyde dehydrogenases, with the highest similarity to the subgroup of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenases. The activity of the enzyme is affected by a series of metabolites. All effectors tested influence the affinity of the enzyme for its cosubstrate NAD+. Whereas NADP(H), NADH, and ATP reduce the affinity for the cosubstrate, AMP, ADP, glucose 1-phosphate, and fructose 6-phosphate increase the affinity for NAD+. Additionally, most of the effectors investigated induce cooperativity of NAD+ binding. The irreversible catabolic oxidation of glyceraldehyde 3-phosphate, the control of the enzyme by energy charge of the cell, and the regulation by intermediates of glycolysis and glucan degradation identify the NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase as an integral constituent of glycolysis in T. tenax. Its regulatory properties substitute for those lacking in the reversible nonregulated pyrophosphate-dependent phosphofructokinase in this variant of the Embden-Meyerhof-Parnas pathway.     

Selective fermentation of xylose by a mutant of Tetragenococcus halophila defective in phosphoenolpyruvate:mannose phosphotransferase, phosphofructokinase, and glucokinase

Tetragenococcus halophila is a Gram-positive halophilic lactic acid bacterium used for soy sauce fermentation. We isolated a mutant, T. halophila 3E4, triply defective in phosphoenolpyruvate:mannose phosphotransferase, phosphofructokinase, and glucokinase. 3E4 selectively metabolized pentoses such as xylose and arabinose in the presence of hexoses such as glucose and galactose. We present here an example of the metabolic engineering of catabolite control.     

Increased intestinal glucose absorption and postprandial hyperglycaemia at the early step of glucose intolerance in Otsuka Long-Evans Tokushima Fatty rats

Otsuka Long-Evans Tokushima Fatty (OLETF) rats are reported to be obese Type II (non-insulin-dependent) diabetic rats with insulin resistance and impaired insulin secretion. To investigate the contribution of intestinal glucose absorption to postprandial hyperglycaemia, we determined the plasma xylose concentrations after an 0.8 g/kg oral xylose load which was used as a test of small intestinal glucose absorption in 6-week-old OLETF rats and weight-matched Long-Evans Tokushima Otsuka (LETO) rats. An oral glucose tolerance test showed that OLETF rats developed hyperglycaemia at 60 and 90 min after the glucose load, though the fasting plasma glucose concentration, insulin concentration and insulin-induced in vivo glucose utilization rate were similar. Consistently, in an oral D-xylose loading test, the peak concentration of plasma xylose in OLETF rats was increased by 58.7% compared with that of LETO rats (p < 0.005). The disappearance rate of plasma xylose concentrations after intravenous xylose loading did not differ between the two strains. Co-treatment with 0.4 g/kg phlorizin, a specific inhibitor of sodium-dependent glucose transporter 1 (SGLT1), abolished both plasma glucose and xylose concentrations after the loads. Morphological studies showed that both the small intestinal wet weight and surface area were 30% larger in the OLETF rats than in the LETO rats. Furthermore, the SGLT1 mRNA content of OLETF rats also increased compared with LETO rats. These results suggest that an increased SGLT1 expression concomitant with intestinal hypertrophy in OLETF rats is partly associated with postprandial hyperglycaemia before the onset of insulin resistance and hyperinsulinaemia.