Intracellular Sugar Research Articles

Microcolonial fungi on rocks: a life in constant drought?

Black microcolonial fungi (MCF) and black yeasts are among the most stress-resistant eukaryotic organisms known on Earth. They mainly inhabit bare rock surfaces in hot and cold deserts of all regions of the Earth, but some of them have a close phylogenetic relation to human pathogenic black fungi which makes them important model organisms also with respect to clinical mycology. The environment of those fungi is especially characterized by extreme changes from humidity to long periods of desiccation and extreme temperature differences. A key to the understanding of MCF ecology is the question about metabolic activity versus dormancy in the natural environments. In this study, the time lag from the desiccated state to rehydration and full metabolic activity and growth was measured and defined in accordance with simulated environmental conditions. The ability to survive after desiccation and the speed of rehydration as well as changes of the whole cell protein pattern are demonstrated. Whereas both mesophilic strains—Exophiala jeanselmei and Knufia perforans (=Coniosporium perforans)—show a clear reaction toward desiccation by production of small proteins, Cryomyces antarcticus—the extremotolerant MCF—does not show any response to desiccation but seems just to down-regulate its metabolism. Data on intracellular sugar suggest that both trehalose and mannitol might play a cell protective role in those fungi.Electronic supplementary materialThe online version of this article (doi:10.1007/s11046-012-9592-1) contains supplementary material, which is available to authorized users.

Quantification of intracellular nucleotide sugars and formulation of a mathematical model for prediction of their metabolism

Quantification of intracellular nucleotide sugars and formulation of a mathematical model for prediction of their metabolism

Relationship between hexokinase and cytokinin in the regulation of leaf senescence and seed germination

Arabidopsis hexokinase (AtHXK1), an enzyme that catalyses hexose phosphorylation, accelerates leaf senescence, whereas the plant hormone cytokinin inhibits senescence. Previous work in our laboratory has shown that isopentenyl transferase (IPT), a key gene in the biosynthesis of cytokinin, expressed under promoters of the senescence-associated genes SAG12 or SAG13 (P(SAG12)::IPT and P(SAG13)::IPT, respectively), inhibits leaf senescence in tomato plants. To study the relationship between hexokinase and cytokinin in the regulation of leaf senescence, we created and analysed double-transgenic tomato plants expressing both AtHXK1 and either P(SAG12)::IPT or P(SAG13)::IPT. We found that expression of IPT in the double-transgenic plants could not prevent the accelerated senescence induced by over-expression of AtHXK1. Since cytokinin inhibits senescence via an apoplastic invertase that produces extracellular hexoses, whereas AtHXK1 is an intracellular mitochondria-associated hexokinase, our results suggest that intracellular sugar sensing via AtHXK1 is dominant over extracellular sugar sensing with regard to leaf senescence. Interestingly, the heterologous SAG12 and SAG13 promoters are also expressed in germinating tomato seed, around the radicle penetration zone, suggesting that seed germination involves a senescence process that is probably necessary for radicle emergence. Indeed, seed expressing P(SAG12)::IPT and P(SAG13)::IPT exhibited delayed radicle emergence, possibly due to delayed endosperm senescence.

Determinants of Ligand Binding Affinity and Cooperativity at the GLUT1 Endofacial Site

Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport [ Cloherty, E. K., Levine, K. B., and Carruthers, A. ((2001)) The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry 40 ((51)) 15549-15561]. This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, endofacial binding sites for CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogues, asks what structural features of endofacial site ligands determine binding site affinity and cooperativity. Like CB, FSK competitively inhibits exchange 3-O-methylglucose transport (sugar uptake in cells containing intracellular sugar) but noncompetitively inhibits sugar uptake into cells lacking sugar at 4 °C. This refutes the hypothesis that FSK binds at GLUT1 endofacial and exofacial sugar binding sites. Some forskolin derivatives and cytochalasins inhibit equilibrium [(3)H]-CB binding to red cell membranes depleted of peripheral proteins at 4 °C. Others produce a moderate stimulation of [(3)H]-CB binding when introduced at low concentrations but inhibit binding as their concentration is increased. Yet other analogues modestly stimulate [(3)H]-CB binding at all inhibitor concentrations applied. These findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of models for GLUT1-mediated sugar transport and GLUT1 quaternary structure, and we evaluate the major determinants of ligand binding affinity and cooperativity.

Trehalose and Sucrose Osmolytes Accumulated by Algae as Potential Raw Material for Bioethanol

Currently, obtaining sustainable fuels, such as biodiesel and bioethanol, from cheap and renewable materials is a challenge. In recent years, a new approach being developed consists of producing, sugars from algae by photosynthesis. Sugar accumulation can be increased under osmotic stress (osmoregulation). The aim of this study is to show the pro-duction of sugars from algae, isolated from natural sources, and the effect of osmotic stress on fermentable sugars ac-cumulation. Strain isolation, production of sugars from each alga and the effect of osmotic stress on growth and sugar production are described. Twelve algal strains were isolated, showing growths between 0.6 and 1.8 g of biomass dry weight /L, all with production of intracellular and extracellular sugars. The strain identified as Chlorella sp. showed an increase in sugar production from 23.64 to 421 mg of sugars/g of biomass dry weight after 24 h of osmotic stress with 0.4 M NaCl. Sucrose and trehalose, both fermentable sugars, were the compatible osmolytes accumulated in response to the osmotic stress. The isolated strains are potential producers of fermentable sugars, using the photosynthetic pathway and osmotic stress.

A Comparative Assessment of the Potential of Polysaccharide Production and Intracellular Sugar Composition within Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (W.Curt.:Fr.)P. Karst. (Aphyllophoromycetideae)

Ganoderma lucidum is a well-known medicinal mushroom species in which polysaccharides are one of the major sources of biological activity. The species was considered as a species-complex due to significant variations in morphological, biochemical, and genetic features among populations with a worldwide distribution. This fact was the basis for setting the aim of this research: to study intraspecific diversity in polysaccharide production and intracellular sugar composition among selected G. lucidum strains. The presence ofintraspecific diversity among 10 G. lucidum strains, from different areas worldwide, was noted. Values of produced mycelia biomass and intracellular polysaccharides were found in wide ranges (3.1 - 28.2 g L(-1) and 20.0 - 53.3 mg g(-1), respectively), while differences in extracellular polysaccharide amounts were minor (0.2 - 1.5 mg mL(-1)). The significant quantitative and qualitative differences in intracellular sugar composition were noted. Glucose was the predominant sugar in almost all strains except one (HAI 447), where sucrose was dominant. The potential of polysaccharide production and intracellular sugar composition could be one more taxonomic criterion for strain characterization within G. lucidum. The differences in intracellular sugar composition and proportions could be reflected in features of produced polysaccharides and also in their biological activities.

Desiccation tolerance in bovine sperm: A study of the effect of intracellular sugars and the supplemental roles of an antioxidant and a chelator

Desiccation preservation holds promise as a simplified alternative to cryopreservation for the long term storage of cells. We report a study on the protective effects of intracellular and extracellular sugars during bovine sperm desiccation and the supplemental effects of the addition of an antioxidant (catalase) or a chelator (desferal). The goal of the study was to preserve mammalian sperm in a partially or completely desiccated state. Sperm loaded intracellularly with two different types of sugars, trehalose or sucrose, were dried with and without catalase and desferal and evaluated for motility and membrane integrity immediately after rehydration. Intracellular sugars were loaded using ATP induced poration. Drying was performed in desiccator boxes maintained at 11% relative humidity (RH). Results indicated that sperm exhibited improved desiccation tolerance if they were loaded with either intracellular trehalose or sucrose. Survival was further enhanced by the addition of 1 mM desferal to the desiccation buffer. Though sperm motility after drying to low dry basis water fractions (DBWF) did not show significant improvement under any of the tested conditions, there was an increase in the sperm membrane integrity that could be retained after partial desiccation through the use of intracellular sugars and desferal.

C-terminal Loop 13 of Na+/Glucose Cotransporter 1 Contains Both Stereospecific and Non-stereospecific Sugar Interaction Sites

To investigate whether the C-terminal loop 13 of rabbit sodium/glucose cotransporter SGLT1 is involved in the recognition of the substrate d-glucose, isolated loop 13 (amino acids (aa) 541-638) was immobilized to a lipid bilayer. Interactions were investigated by surface plasmon resonance spectroscopy using an antibody directed against the late part of the loop (aa 606-631) or the glucoside transport inhibitor phlorizin. Specific binding of the antibody to the loop could be detected. The number of bound antibodies decreased upon the addition of d-glucose but not upon the addition of l-glucose. Phlorizin also significantly lowered the number of bound antibodies. Binding of phlorizin to the loop could also be demonstrated directly. Binding of phlorizin was, however, reduced to a similar extent upon the addition of either d-glucose or l-glucose, indicating their unspecific competition with the inhibitor's sugar moiety. Thus, the presence of a stereospecific glucose interaction site in the late part of the loop and a second, but non-stereospecific, sugar binding site on the same loop was assumed. To investigate whether the early part of loop 13 contains this non-stereospecific sugar binding site, peptides containing aa 541-598 were expressed in Escherichia coli and purified. Both d-glucose and l-glucose quenched the peptides tryptophan fluorescence and reduced the Trp accessibility to acrylamide to a similar degree. In view of the recently proposed transmembrane orientation of loop 13, the two binding sites may be part of the extracellular (stereospecific) and intracellular (non-stereospecific) sugar interaction sites of SGLT1.

Quantitative analysis of intracellular sugar phosphates and sugar nucleotides in encapsulated streptococci using HPAEC‐PAD

Metabolomics is a powerful tool for the study of biological systems. Besides analytical techniques, cell harvest and extraction are critical steps, especially when studying encapsulated streptococci. We have compared four different harvesting techniques for biomass from liquid culture of the hyaluronic acid (HA)-producing bacterium Streptococcus zooepidemicus. The best method for cell separation was quick (2 min) centrifugation, which allowed efficient medium removal and enabled quantification of the broadest range of sugar metabolites. Unlike observations for other microbes, changes in metabolite pools due to a delay of extraction by the centrifugation were not observed, so metabolite levels accurately reflected the metabolome at the point of cell harvest. A hypothesis is that the capsule itself isolates the cells from the surroundings and still supports it with nutrients during the harvest. Quantification of sugar phosphates and nucleotide sugars was performed using high-performance anion exchange chromatography combined with pulsed amperometric detection, achieving limits of quantification of 2.5 pmol for sugar phosphates and 5 pmol on column for nucleotide sugars. Intracellular pool sizes for intermediates of the HA pathway under production conditions ranged from 0.2 to 0.5 micromol/g cell dry weight.

36. Study of the effect of intracellular sugars and antioxidant/chelator in bovine sperm desiccation

36. Study of the effect of intracellular sugars and antioxidant/chelator in bovine sperm desiccation

Intracellular sucrose communicates metabolic demand to sucrose transporters in developing pea cotyledons.

Mechanistic inter-relationships in sinks between sucrose compartmentation/metabolism and phloem unloading/translocation are poorly understood. Developing grain legume seeds provide tractable experimental systems to explore this question. Metabolic demand by cotyledons is communicated to phloem unloading and ultimately import by sucrose withdrawal from the seed apoplasmic space via a turgor-homeostat mechanism. What is unknown is how metabolic demand is communicated to cotyledon sucrose transporters responsible for withdrawing sucrose from the apoplasmic space. This question was explored here using a pea rugosus mutant (rrRbRb) compromised in starch biosynthesis compared with its wild-type counterpart (RRRbRb). Sucrose influx into cotyledons was found to account for 90% of developmental variations in their absolute growth and hence starch biosynthetic rates. Furthermore, rr and RR cotyledons shared identical response surfaces, indicating that control of transporter activity was likely to be similar for both lines. In this context, sucrose influx was correlated positively with expression of a sucrose/H+ symporter (PsSUT1) and negatively with two sucrose facilitators (PsSUF1 and PsSUF4). Sucrose influx exhibited a negative curvilinear relationship with cotyledon concentrations of sucrose and hexoses. In contrast, the impact of intracellular sugars on transporter expression was transporter dependent, with expression of PsSUT1 inhibited, PsSUF1 unaffected, and PsSUF4 enhanced by sugars. Sugar supply to, and sugar concentrations of, RR cotyledons were manipulated using in vitro pod and cotyledon culture. Collectively the results obtained showed that intracellular sucrose was the physiologically active sugar signal that communicated metabolic demand to sucrose influx and this transport function was primarily determined by PsSUT1 regulated at the transcriptional level.

Effect of Liposome Charge and Composition on the Delivery of Trehalose into Red Blood Cells

Although the disaccharide trehalose has been shown to protect cells during freezing and desiccation when present intracellularly, a major obstacle to using intracellular sugars for biopreservation applications is the impermeability of plasma membranes. We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of stabilizing sugars into mammalian cells. Previous work has shown that the mechanism of red blood cell (RBC)–liposome interaction includes both liposome fusion with the RBC membranes, as well as tight adsorption of the vesicles onto the RBC surface. However, the fusion efficiency of liposomes was low, with only micromolar concentrations of trehalose delivered to the RBC cytosol. The purpose of this study is to enhance the efficacy of liposomes’ delivery of trehalose into RBCs with minimal detrimental effects on RBC membrane quality, by manipulating liposome physical properties and liposome–RBC incubation conditions. Charged and uncharged un...

Antibiotic Overproduction in Streptomyces coelicolor A3(2) Mediated by Phosphofructokinase Deletion

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by (13)C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria

BackgroundEngineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production.ResultsAn Escherichia coli expression vector compatible with phage λ recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates.ConclusionThe nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating the potential of such sensors as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular metabolite during fermentation.

Investigating Interactions of Trehalose-Containing Liposomes with Human Red Blood Cells

A major obstacle in using intracellular sugars for the cryopreservation of mammalian cells is the inability of cells to synthesize or actively accumulate these sugars. We are investigating the use ...

Inhibitory effects of 7-epiclusianone on glucan synthesis, acidogenicity and biofilm formation by Streptococcus mutans

The aim of this study was to examine the effects of 7-epiclusianone, a new prenylated benzophenone isolated from the plant Rheedia gardneriana, on some of the virulence properties of Streptococcus mutans associated with biofilm development and acidogenicity. The synthesis of glucans by glucosyltransferases B (GTF B) and C (GTF C) was markedly reduced by 7-epiclusianone showing more than 80% inhibition of enzymatic activity at a concentration of 100 microg mL(-1). Double-reciprocal analysis (Lineweaver-Burk plots) revealed that the inhibition of GTF B activity was noncompetitive (mixed) while GTF C was inhibited uncompetitively. The glycolytic pH drop by S. mutans cells was also disrupted by 7-epiclusianone without affecting the bacterial viability, an effect that can be attributed, in part, to inhibition of F-ATPase activity (61.1+/-3.0% inhibition at 100 microg mL(-1)). Furthermore, topical applications (1-min exposure, twice daily) of 7-epiclusianone (at 250 microg mL(-1)) disrupted biofilm formation and physiology. The biomass (dry-weight), extracellular insoluble polysaccharide concentration and acidogenicity of the biofilms were significantly reduced by the test agent (P<0.05). The data show that 7-epiclusianone disrupts the extracellular and intracellular sugar metabolism of S. mutans, and holds promise as a novel, naturally occurring compound to prevent biofilm-related oral diseases.

Effect of sugars on the development of oxidative stress induced by hypothermia in potato plants expressing yeast invertase gene

The influence of sugars on the development of oxidative stress induced by hypothermia was investigated in the leaves of two genotypes of potato (Solanum tuberosum L.) grown in vitro on the Murashige and Skoog medium supplemented with 2% sucrose. We used wild-type plants of potato, cv. Desiree, and potato plants expressing a yeast invertase gene under the control of the B33 class I patatin promoter and carrying a sequence of proteinase inhibitor II leader peptide for the apoplastic enzyme localization. At temperature of 22°C optimal for growth, expression of the yeast invertase gene in the leaves of transformed plants brought about a modification in the carbohydrate metabolism manifested in the activation of acid forms of invertase and accumulation of intracellular sugars (predominantly of sucrose because of its resynthesis). The exposure of plants to light under prolonged hypothermia (5°C, 6 days) activated all the forms of invertase (predominantly of acid invertase) and induced accumulation of sugars. In the leaves of potato expressing the yeast invertase gene, these processes were more intense. Under chilling, superoxide dismutase activity and the rate of lipid peroxidation in the leaves of investigated potato genotypes depended on the level of accumulated intracellular sugars. It was concluded that sugars play an important role as stabilizers of cellular membranes and scavengers of reactive oxygen species decelerating the processes of free radical oxidation of biomolecules upon the development of oxidative stress induced by hypothermia.

Role of sugars in regulating transfer cell development in cotyledons of developing Vicia faba seeds

Transfer cell formation in cotyledons of developing faba bean (Vicia faba L.) seeds coincides with an abrupt change in seed apoplasm composition from one dominated by hexoses to one in which sucrose is the principal sugar. On the basis of these observations, we tested the hypothesis that sugars induce and/or sustain transfer cell development. To avoid confounding effects of in planta developmental programs, we exploited the finding that adaxial epidermal cells of cotyledons, which do not become transfer cells in planta, can be induced to form functional transfer cells when cotyledons are cultured on an agar medium. Growth rates of cotyledons cultured on hexose or sucrose media were used to inform choice of sugar concentrations. The same proportion of adaxial epidermal cells of excised cotyledons were induced to form wall ingrowths independent of sugar species and concentration supplied. In all cases, induction of wall ingrowths coincided with a marked increase in the intracellular sucrose-to-hexose ratio. In contrast, further progression of wall ingrowth deposition was correlated positively with intracellular sucrose concentrations that varied depending upon external sugar species and supply. Sucrose symporter induction and subsequent maintenance behaved identically to wall ingrowth formation in response to an external supply of hexoses or sucrose. However, in contrast to wall ingrowth formation, induction of sucrose symporter activity was delayed. We discuss the possibility of intracellular sugars functioning both as signals and substrates that induce and control subsequent development of transfer cells.

Crosstalk between Auxin, Cytokinins, and Sugars in the Plant Cell Cycle

Plant meristems are utilization sinks, in which cell division activity governs sink strength. However, the molecular mechanisms by which cell division activity and sink strength are adjusted to a plant's developmental program in its environmental setting are not well understood. Mitogenic hormonal as well as metabolic signals drive and modulate the cell cycle, but a coherent idea of how this is accomplished, is still missing. Auxin and cytokinins are known as endogenous mitogens whose concentrations and timing, however, can be externally affected. Although the sites and mechanisms of signal interaction in cell cycle control have not yet been unravelled, crosstalk of sugar and phytohormone signals could be localized to several biochemical levels. At the expression level of cell cycle control genes, like cyclins, Cdks, and others, synergistic but also antagonistic interactions could be demonstrated. Another level of crosstalk is that of signal generation or modulation. Cytokinins affect the activity of extracellular invertases and hexose-uptake carriers and thus impinge on an intracellular sugar signal. With tobacco BY-2 cells, a coordinated control of cell cycle activity at both regulatory levels could be shown. Comparison of the results obtained with the root cell-representing BY-2 cells with literature data from shoot tissues or green cell cultures of Arabidopsis and Chenopodium suggests opposed and tissue-specific regulatory patterns of mitogenic signals and signal crosstalk in root and shoot meristems.

Biosynthesis of radiolabeled cellodextrins by the Clostridium thermocellum cellobiose and cellodextrin phosphorylases for measurement of intracellular sugars

The Clostridium thermocellum cellobiose and cellodextrin phosphorylases (glucosyl transferases) in the cell extract were used to synthesize radiolabeled cellodextrins with a degree of polymerization (DP=2-6) from nonradioactive glucose-1-phosphate and radioactive glucose. Chain lengths of synthesized cellodextrin were controlled by the absence or presence of dithiothreitol and by reaction conditions. All cellodextrins have the sole radioactive glucose unit located at the reducing ends. Mixed cellodextrins (G2-G6) were separated efficiently by size-exclusion chromatography or less efficiently by thin-layer chromatography. A new rapid sampling device was developed using disposable syringes containing an ultracold methanol-quenching buffer. It was simple, less costly, and especially convenient for anaerobic fermentation. After an impulse feed of radiolabeled cellobiose, the intracellular sugar levels were measured after a series of operations-sampling, extracting, concentrating, separating, and reading. Results showed that the largest amount of radioactivity was cellobiose with lesser amounts of glucose, cellotriose, and cellotetraose, and an average DP of intracellular cellodextrins was ca. 2.