Presence Of Hexoses Research Articles

Human SLC2A9a and SLC2A9b isoforms mediate electrogenic transport of urate with different characteristics in the presence of hexoses

Human SLC2A9 (GLUT9) is a novel high-capacity urate transporter belonging to the facilitated glucose transporter family. In the present study, heterologous expression in Xenopus oocytes has allowed us to undertake an in-depth radiotracer flux and electrophysiological study of urate transport mediated by both isoforms of SLC2A9 (a and b). Addition of urate to SLC2A9-producing oocytes generated outward currents, indicating electrogenic transport. Urate transport by SLC2A9 was voltage dependent and independent of the Na(+) transmembrane gradient. Urate-induced outward currents were affected by the extracellular concentration of Cl(-), but there was no evidence for exchange of the two anions. [(14)C]urate flux studies under non-voltage-clamped conditions demonstrated symmetry of influx and efflux, suggesting that SLC2A9 functions in urate efflux driven primarily by the electrochemical gradient of the cell. Urate uptake in the presence of intracellular hexoses showed marked differences between the two isoforms, suggesting functional differences between the two splice variants. Finally, the permeant selectivity of SLC2A9 was examined by testing the ability to transport a panel of radiolabeled purine and pyrimidine nucleobases. SLC2A9 mediated the uptake of adenine in addition to urate, but did not function as a generalized nucleobase transporter. The differential expression pattern of the two isoforms of SLC2A9 in the human kidney's proximal convoluted tubule and its electrogenic transport of urate suggest that these transporters play key roles in the regulation of plasma urate levels and are therefore potentially important participants in hyperuricemia and hypouricemia.

Glucose‐induced production of recombinant proteins in Hansenulapolymorpha mutants deficient in catabolite repression

The most commonly used expression platform for production of recombinant proteins in the methylotrophic yeast Hansenula polymorpha relies on the strong and strictly regulated promoter from the gene encoding peroxisomal enzyme alcohol (or methanol) oxidase (P(MOX)). Expression from P(MOX) is induced by methanol and is partially derepressed in glycerol or xylose medium, whereas in the presence of hexoses, disaccharides or ethanol, it is repressed. The need for methanol for maximal induction of gene expression in large-scale fermentation is a significant drawback, as this compound is toxic, flammable, supports a slow growth rate and requires extensive aeration. We isolated H. polymorpha mutants deficient in glucose repression of P(MOX) due to an impaired HpGCR1 gene, and other yet unidentified secondary mutations. The mutants exhibited pronounced defects in P(MOX) regulation only by hexoses and xylose, but not by disaccharides or ethanol. With one of these mutant strains as hosts, we developed a modified two-carbon source mode expression platform that utilizes convenient sugar substrates for growth (sucrose) and induction of recombinant protein expression (glucose or xylose). We demonstrate efficient regulatable by sugar carbon sources expression of three recombinant proteins: a secreted glucose oxidase from the fungus Aspergillus niger, a secreted mini pro-insulin, and an intracellular hepatitis B virus surface antigen in these mutant hosts. The modified expression platform preserves the favorable regulatable nature of P(MOX) without methanol, making a convenient alternative to the traditional system.

Glucose and galactose regulate intestinal absorption of cholesterol

A dose-dependent increase in cholesterol absorption was induced by glucose addition (0–75 mM) to the apical medium of TC7 cells, a well-characterized clone of Caco-2. The uptake into the cells and the secretion rate to the basolateral space were both enhanced by glucose and galactose. This up-regulation was suppressed by SGLT1 inhibition but not by GLUT2 inhibition. Cholesterol cell uptake was significantly decreased by PMA and increased by chelerythrine, with more pronounced changes in the presence of hexoses. Thus, the involvement of a protein kinase C signalling pathway was evidenced in the regulation processes of intestinal cholesterol absorption. In the presence of antibodies directed to hSR-BI cholesterol absorption was reduced by 40% and glucose or galactose no longer enhanced it. We suggest that glucose or galactose, through an interaction with SGLT1, activates a protein kinase C pathway that regulates the activity of one of the intestinal cholesterol transporters, namely hSR-BI.

Phosphoenolpyruvate carboxylase in developing seeds of Vicia faba L.: gene expression and metabolic regulation.

To analyze the role of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) during seed development, two cDNA clones encoding two isoforms of PEPCase were isolated from a seed-specific library of Vicia faba. The two sequences (VfPEPCase1 and VfPEPCase2) have a sequence identity of 82 and 89% on the nucleotide and amino acid levels. The VfPEPCase1 mRNA was found to be predominantly expressed in roots and developing cotyledons whereas the VfPEPCase2 mRNA was more abundant in green and maternal tissues. In the cotyledons, PEPCase mRNAs accumulated from early to mid cotyledon stage and decreased thereafter. The PEPCase activity increased continuously during cotyledon development. The enzyme was strongly activated by glucose-6-phosphate, but not by glucose, fructose or sucrose. Asparagine was weakly activating whereas malate, aspartate and glutamate were inhibitory. The inhibitors became less effective with increasing pH. Aspartate was a much stronger inhibitor of cotyledonary PEPCase than glutamate at both pH 7.0 and 7.5. The sensitivity of PEPCase to malate inhibition decreased from early to mid cotyledon stage at a time when storage proteins are synthesized. This indicates activation on the protein level, possibly by protein phosphorylation. Nitrogen starvation in the presence of hexoses but not sucrose decreased mRNA levels of VfPEPCase1 and enzyme activity, indicating control on the mRNA level by both carbon and nitrogen. It is concluded that in developing cotyledons PEPCase is probably important for the synthesis of organic acids to provide carbon skeletons for amino acid synthesis.

Carrier-mediated system for pefloxacin uptake in human monocytes.

Protein transporters present in cellular membranes can influence the intracellular concentration of a variety of drugs. Pefloxacin is a fluoroquinolone which is commonly used in systemic infections. Its transport mechanisms have never been described. In the present study, we demonstrated that pefloxacin uptake is carrier-mediated. Pefloxacin crosses the membrane in zwitterionic form. The transport seems to differ from that of other fluoroquinolones which use amino acid or organic anion transporters. Pefloxacin uptake is not influenced by the presence of hexose in the incubation medium. It does not use a nucleoside transport system to penetrate the cells but adenosine increases the uptake. The carrier is inhibited by verapamil at 60 min and is activated by a Ca2+-dependent system.

Utilization of sucrose by Hymenoscyphus ericae (an ericoid endomycorrhizal fungus) and ectomycorrhizal fungi

Hymenoscyphus ericae utilized sucrose, whereas the ectomycorrhizal fungi either showed poor growth or responded to the presence of sucrose after a lag phase. On sucrose-containing media, fructose and glucose accumulated in the medium at the end of the linear phase of growth of H. ericae and then disappeared during the stationary phase. Dry mass of H. ericae increased with increasing concentration of both sucrose and glucose up to 2·0 g C l −1 , whereas on fructose, it increased up to 4 g C l −1 . Growth of H. ericae was doubled on media containing both glucose and fructose at 1:1 ratio compared with growth on either hexose alone at the same carbon concentration. Sucrose, fructose and glucose were detected in the mycelium of H. ericae , although the main soluble carbohydrates were mannitol and trehalose. Growth of ectomycorrhizal fungi (excluding Cenococcum geophilum ) on sucrose depended solely on the presence of hexoses in the medium and utilization of sucrose was poor. The use of ‘starter glucose’ had an effect on sucrose utilization by Laccaria laccata, Lactarius rufus, Paxillus involutus and Thelephora terrestris but growth was still less than that on equivalent amounts of glucose. These results confirm previous studies that the majority of ectomycorrhizal associations do not utilize sucrose directly and may have to rely on apoplastic host invertase. In contrast, host invertases may not be required in the utilization of sucrose by ericoid mycorrhizal associations.

GLUT2 is the transporter for fructose across the rat intestinal basolateral membrane

Background: The exact roles of disaccharidases and GLUT5 in the brush border membrane and GLUT2 in the basolateral membrane in the absorption of fructose across the intestine have not been fully determined. This paper describes characterization of fructose transport across the jejunal basolateral membrane using isolated membrane vesicles. Methods: Transport of fructose was measured using rapid filtration of vesicles. Luminal perfusion in vivo with glucose and fructose before vesicle preparation was used to assess modulation of GLUT2 activity. Western blotting measured the abundance of GLUT2 in the membrane. Results: The maximal rate of transport for fructose was 1100 pmol/mg protein/s and the Michaelis constant was 16 mmol/L. Fructose and glucose could completely inhibit the transport of each other. Perfusion of the intestinal lumen with fructose or glucose saline for 4 hours produced a fourfold increase in maximal fructose transport. Conclusions: These data indicate that the one transport protein, GLUT2, is responsible for moving both fructose and glucose out of the enterocyte across the basolateral membrane under basal conditions. The activity of this, or a closely related carrier, is rapidly upregulated by the presence of hexoses in the intestinal lumen, explaining the potentiation of fructose absorption by luminal glucose and obviating any need to involve apical disaccharidases.

Implications for cytoplasmic pH, protonmotice force, and amino-acid transport across the plasmalemma of Riccia fluitans

By means of pH-sensitive microelectrodes, cytoplasmic pH has been monitored continuously during amino-acid transport across the plasmalemma of Riccia fluitans rhizoid cells under various experimental conditions. (i) Contrary to the general assumption that import of amino acids (or hexoses) together with protons should lead to cytoplasmic acidification, an alkalinization of 0.1-0.3 pHc units was found for all amino acids tested. Similar alkalinizations were recorded in the presence of hexoses and methylamine. No alkalinization occurred when the substrates were added in the depolarized state or in the presence of cyanide, where the electrogenic H(+)-pump is inhibited. (ii) After acidification of the cytoplasm by means of various concentrations of acetic acid, amino-acid transport is massively altered, although the protonmotive force remained essentially constant. It is suggested that H(+)-cotransport is energetically interconnected with the proton-export pump which is stimulated by the amino-acid-induced depolarization, thus causing proton depletion of the cytoplasm. It is concluded that, in order to investigate H(+)-dependent cotransport processes, the cytoplasmic pH must be measured and be under continuous experimental control; secondly, neither ΔpH nor the protonmotive force across a membrane are reliable quantities for analysing a proton-dependent process.

Hexose-transport-deficient mutants of Chlorella vulgaris

Autotrophically grown cells of Chlorella vulgaris show a strong increase in the uptake rates for hexoses and for seven amino acids when incubated in the presence of hexoses. This increase is due to de-novo synthesis of three transport proteins: one forhexoses and two for amino acids. Mutants deficient in hexose transport were obtained after treatment of wild-type cells with acridine orange, followed by a selection procedure using the toxic hexose analogue, 2-deoxy-D-glucose. Moreover, the two amino-acid-transport systems could not be induced in these mutants by hexoses. The capacity to phosphorylate hexoses was identical in mutants and in the wild-type strain. The loss of transport activities can be correlated with the loss of certain radiolabeled protein bands on fluorograms of sodium dodecylsulfate-polyacrylamide gels. These proteins are assumed to be responsible for the different transport systems in the wild-type strain. With the help of additional mutants defective in one or two of the different aminoacid-transport systems, it has been attempted to assign the different transport activities to individual protein bands on the gel.

Inhibition of rat liver glucokinase by alloxan and ninhydrin.

In a recent paper, we reported that glucokinase in pancreatic islets might be a critical site which mediates the inhibition of glucose-stimulated insulin secretion by alloxan and ninhydrin. The action mechanisms of the two agents on rat liver glucokinase (instead of islet glucokinase) were studied here.Both alloxan and ninhydrin irreversibly inhibited rat liver glucokinase in concentration-dependent manners. The inhibitory effects of alloxan and ninhydrin on glucokinase were blocked by the presence of hexoses (D-glucose and D-mannose) that serve as substrates of the enzyme. The blockade provided by D-glucose showed α-anomeric preference, as was also observed in the phosphorylation of D-glucose by glucokinase. Protection against the inhibition of glucokinase by alloxan or ninhydrin was also afforded by D-mannoheptulose, a competitive inhibitor of the enzyme with respect to D-glucose. These results suggest that the inhibitory sites of alloxan and ninhydrin are at or near the substrate-binding site of glucokinase.

Parallel Inhibition of Islet Glucokinase and Glucose-Stimulated Insulin Secretion by Either Alloxan or Ninhydrin

Treatment of pancreatic islets with alloxan or ninhydrin at concentrations that induced the inhibition of glucokinase activity similarly inhibited glucose-stimulated insulin secretion. Alloxan- or ninhydrin-induced inhibition of glucokinase activity was blocked by the presence of hexoses, which also protected against the inhibitory effect of each drug on insulin secretion. The protective effect of D-glucose against the inhibition of glucokinase activity by alloxan or ninhydrin showed α-anomeric preference, which was similarly observed in the protection by the sugar against the inhibition of insulin secretion. These results suggest that the inhibition of glucose-stimulated insulin secretion by treatment of pancreatic islets with alloxan or ninhydrin is due to the inhibition of glucokinase.

Membrane transport of clindamycin in alveolar macrophages.

The use of antibiotics which can penetrate phagocytic cells and kill intracellular organisms is desirable in the treatment of chronic facultative bacterial infections. Recently, we reported that several antibiotics were selectively concentrated by rabbit alveolar macrophages. Clindamycin accumulation was especially marked. In the present study we evaluated the plasma membrane transport (initial uptake) of clindamycin in alveolar macrophages. The transport of clindamycin is an active process, as documented by requirements for cellular viability, elevated environmental temperature, metabolic energy, and establishment of the 40- to 50-fold cellular/extracellular gradient. Energy for membrane transport of the drug depended at least in part upon mitochondrial oxidative respiration and cell membrane Na-K pump activity. Kinetic analysis of active clindamycin transport revealed it to be saturable, with a high binding affinity (Km = 1 mM) and a high velocity of uptake (Vmax = 15.8 nmol/45 s per 10(6) cells). Clindamycin uptake was not influenced by the presence of hexose or amino acids, but was inhibited by nucleosides (adenosine, puromycin). Decreased clindamycin transport in the presence of puromycin was typical of competitive inhibition (increased Km, unchanged Vmax). Conversely, competitive inhibition of adenosine transport by clindamycin was documented. Thus, clindamycin is transported into alveolar macrophages via the nucleoside system. The potential biological consequences of this unique antibiotic transport mechanism are of interest.

Monomeric constituents of the mesogleal polysaccharides of Chrysaora quinquecirrha (scyphozoa: semaeostomeae)

1. 1. Polysaccharides were extracted from the mesoglea of Chrysaora quinquecirrha medusa and recovered by ethanol-NaCl precipitation. 2. 2. After hydrolysis in 6 N HCl, the monomeric constituents of the mesogleal polysaccharides were identified using TLC and quantified using four colorimetric reactions. 3. 3. The presence of hexoses, pentoses, uronic acids and hexosamines indicates that complex carbohydrates are undoubtedly associated with the gelatinous matrix. 4. 4. Of particular interest is the high content of xylose and the almost equal molar amount of fucose in the hydrolysate.

Control of beta-glucosidase synthesis in Mucor racemosus

The beta-glucosidase of Mucor racemosus was shown to be synthesized when the organism was grown in the presence of such diverse carbon sources as glycerol, lactate, xylose, ribose, alpha-methylglucoside, alpha-phenylglucoside, maltose, and cellobiose. Enzyme synthesis was strongly repressed in the presence of hexoses. In addition, exogenous cyclic adenosine 3',5'-monophosphate (cAMP) resulted in enzyme repression. When cAMP was added exogenously after enzyme activity had accumulated, a reversible enzyme inactivation occurred. Growth on disaccharides (maltose or cellobiose) was severely retarded in the presence of cAMP, whereas that on glucose remained unaffected. The results indicate a probable role for cAMP in control of glucosidase synthesis in Mucor.

Saccharomyces mutants with invertase formation resistant to repression by hexoses.

Production of invertase by many strains of yeast is repressed in the presence of hexoses. This phenomenon interferes with studies on the secretion of invertase and with the preparation of large quantities of the enzyme for examination of its chemical and physical characteristics. Saccharomyces strain 303-67, a diploid carrying the single gene SUC-2 for (hexose repressible) invertase production, was subjected to ultraviolet irradiation. No single-step mutations to high level resistance were detected. By a two-step irradiation process mutants were obtained with differing degrees of resistance. The biochemical and genetic characteristics of these mutants are summarized with particular emphasis on FH4C (the most resistant). Although the steady state level of cyclic 3', 5'-adenosine monophosphate (cyclic AMP) was usually slightly higher in cells grown in low- rather than in high-glucose media, the level of cyclic AMP was not correlated with the sensitivity of invertase synthesis to glucose repression. In mutant FH4C, 1 to 2% of the total cell protein is present as invertase; synthesis of alpha-glucosidase is also resistant to repression by hexoses. This mutant does not sporulate and is probably a haploid of a-mating type with low frequency of conjugation and poor viability of conjugants. Mutants 1016 and 1710 are substantially resistant to hexose repression and still sporulate well. They may be useful for genetic analysis of hexose resistance.

Determination of 3 β-hydroxysterols by the cysteine-sulfuric acid reaction

A colorimetric method for the determination of 3β-hydroxysterols in bile and tissue has been described. This is based upon the reaction of the purified sterol digitonides with cysteine and sulfuric acid. Spectrophotometric data has been presented, and evidence that the reaction depends upon the presence of hexoses in digitonin. The accuracy of the proposed method has been assessed and close agreement obtained with results for free cholesterol using the Liebermann-Burchard reaction. The sensitivity of the method is such that 10 μg of cholesterol in bile or tissue extracts can be determined.

Growth inhibition by hexoses of a temperature-sensitive thiazoleless mutant of Salmonella typhimurium.

A Salmonella typhimurium mutant showing impairment in the utilization of hexoses was isolated after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. At 30 C, it grew with hexoses (glucose, fructose, galactose, mannitol), glycerol, succinate, or acid-hydrolyzed casein. At 37 C, it failed to grow with any of the hexoses. Enzymatic determinations demonstrated, however, that the enzymes of the glycolytic pathway (up to the formation of triose phosphates) were present and active at 25 and 32 C. At 42 C, the mutant did not grow with any of the carbon sources used. At both 37 and 42 C, the mutant grew perfectly well with hexoses if yeast extract was present. The metabolite required for growth was thiamine or, specifically, its thiazole moiety. If glucose was added to a culture growing in glycerol, at 37 C, growth was inhibited. This inhibition was relieved by the addition of thiamine or thiazole. Thus, at 37 C and only in the presence of hexoses, the mutant manifests a requirement for thiazole. This auxotrophy is absolute at 42 C. These data indicate that, in this mutant, some derivative of hexoses inhibits the synthesis of thiazole, and that this inhibition is also dependent on the temperature of incubation. The position in the bacterial chromosome of the genetic locus of this lesion (thz(-)) was determined by conjugation and found to coincide with the only thiamine (thi) locus so far reported.

Gastric pepsin, mucus and clinical secretory studies. II. The role of the mucous barrier in the defense of the stomach vs. peptic ulceration. Carbohydrate histochemistry of gastric epithelial secretions in dog.

SummaryHistochemical methods specific for carbohydrate constituents distinguish a number of widely different entities in several types of gastric epithelial cells in the dog.The surface gastric epithelium reveals basophilia, indicating mucosubstances rich in both carboxyl groups (presumably sialic acid) and sulfate esters. The carboxyl‐type basophilia predominates in the superficial convolutions, and that attributed to sulfate is found in the foveolar walls. The strong reactivity with both the periodic acid‐Schiff (PAS) and the periodic acid‐para diamine (PAD) methods for vicinal‐hydroxyls indicates presence of hexose‐ or deoxyhexose‐rich mucosubstances in both of these areas. Susceptibility of basophilia to blockage by initial periodate‐meta diamine treatment points to presence of vicinal‐glycols in proximity of the acid groups in surface epithelium mucin. The surface epithelium in the pylorus differs slightly in that its secretion is less strongly basophilic.The mucous neck cells secrete an apparently neutral mucosubstance which differs from the surface epithelium in its lack of both PAD reactivity and basophilia. The parietal cells also form a diastase‐resistant, neutral, carbohydrate‐rich substance. The zymogen cells are abundant, deep in the glands of the cardiac portion and the body of the stomach, but less numerous in the intermediate zone. They contain apical granular secretion apparently rich in sulfated mucosubstances. Lack of reactivity toward vic‐glycol methods, resistance of alcianophilia or azurophilia to a previous periodate‐meta diamine sequence, and resistance of mixed diamine staining to previous periodate oxidation indicate absence of residues with unsubstituted adjacent hydroxyls (e.g., hexose or deoxyhexose), in this acid mucosubstance.The pyloric glands resemble mucous neck cells in their strong PAS staining and lack of PAD reactivity, but differ in that they show a weak staining indicative of sulfomucin.

COLOR REACTION OF PENTOSE WITH ANTHRONE. 3. FLUORESCENCE FORMED BY THE REACTION OF PENTOSE WITH ANTHRONE.

Fluorescence was observed in the reaction of pentose with anthrone in strong sulfuric acid. The range of its fluorescence spectrum was from 470 to 600 mμ and the maximum was at about 510 mμ. The anthrone reagent itself had a flat fluorescence spectrum from 420 to 600 mμ for it contains anthranol, but the shape of its spectrum was quite different from pentose fluorescence, and, furthermore, the intensity was far weaker than pentose fluorescence. In the case of glucose the fluorescence spectrum resembled that of the blank. This reaction was specific for pentose in the presence of hexose, but excess glucose decreased the pentose fluorescence.

Paper electrophoresis of polyhydric alcohols.

THE polyhydric alcohols or polyols are a group of carbohydrates important both biologically and industrially. Apart from laborious column techniques1, there is no satisfactory micro-method available for the resolution of mixtures of hexitols (sugar alcohols) by themselves or, in particular, in the presence of hexoses.