Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B

This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations.     

Determination of endotoxins in sugar with the Limulus test

The Limulus amebocyte lysate test has been used for determination of pyrogens in sugar of different qualities. All the samples of domestic white sugar and beet raw sugar produced in Sweden during 1976 had a very low content of endotoxins, less than 10 ng/g of sugar. Imported cane raw sugar was, however, highly contaminated. The highest value obtained corresponds to about 100 mg of Escherichia coli endotoxin per g of raw sugar. Such crude sugar cannot, even after refining, be used for medical purposes. Instead, Swedish beet sugar is used as the raw material for production of invert sugar solutions for parenteral administration. The amount of endotoxin in this sugar is less than 1 ng/g.     

Determination of endotoxins in sugar with the Limulus test.

The Limulus amebocyte lysate test has been used for determination of pyrogens in sugar of different qualities. All the samples of domestic white sugar and beet raw sugar produced in Sweden during 1976 had a very low content of endotoxins, less than 10 ng/g of sugar. Imported cane raw sugar was, however, highly contaminated. The highest value obtained corresponds to about 100 mg of Escherichia coli endotoxin per g of raw sugar. Such crude sugar cannot, even after refining, be used for medical purposes. Instead, Swedish beet sugar is used as the raw material for production of invert sugar solutions for parenteral administration. The amount of endotoxin in this sugar is less than 1 ng/g.     

Remodeling of sugar chain structures of human interferon-gamma

Natural human interferon (IFN)-gamma has mainly biantennary complex-type sugar chains and scarcely has multiantennary structures. We attempted to remodel the sugar chain structures using IFN-gamma as a model glycoprotein. To obtain the branching glycoforms of IFN-gamma, we introduced the genes for GnT-IV (UDP-N-acetylglucosamine:alpha-1,3-D-mannoside beta-1, 4-N-acetylglucosaminyltransferase) and/or GnT-V (UDP-N-acetylglucosamine:alpha-1,6-D-mannoside beta-1, 6-N-acetylglucosaminyltransferase) into Chinese hamster ovary (CHO) cells producing human IFN-gamma. The parental CHO cells produced IFN-gamma with biantennary sugar chains mainly. When the GnT-IV activity was increased, triantennary sugar chains with a branch produced by GnT-IV increased up to 66.9% of the total sugar chains. When the GnT-V activity was increased, triantennary sugar chains with a corresponding branch increased up to 55.7% of the total sugar chains. When the GnT-IV and -V activities were increased at a time, tetraantennary sugar chains increased up to 56.2% of the total sugar chains. The proportion of these multiantennary sugar chains corresponded to the intracellular activities of GnT-IV and -V. What is more, lectin blot and flow cytometric analysis indicated that the multi-branch structure of the sugar chains was increased not only on IFN-gamma, one of the secretory glycoproteins, but also on almost CHO cellular proteins by introducing either or both of the GnT genes. The results suggest that the branching structure of sugar chains of glycoproteins could be controlled by cellular GnT-IV and GnT-V activities. This technology can produce glycoforms out of natural occurrence, which should enlarge the potency of glycoprotein therapeutics.     

Sugar uptake analysis of suspension Arabidopsis, tobacco, and rice cells in various media using an FT-IR/ATR method

The kinetic behavior of the sugar uptake phenomena of a suspension of Arabidopsis cells was investigated by mid-infrared spectroscopy using Fourier transform infrared spectrometers and attenuated total reflection techniques. The kinetic behavior of the cell growth was also studied and the growth and the sugar uptake behaviors were discussed for three typical plant cells (Arabidopsis, TBY-2, and rice cells). The cell growth rate and the lag period were influenced by not only the types of the plant cells, but also the sugar species used as the carbon source. The characteristics of the sugar uptake behavior were clarified based on the difference in the three types of plant cells. The cell growth and the sugar uptake progressed at approximately the same time in the TBY-2 cells. In the rice cells, the sugar uptake rate was relatively lower than that of the others. On the other hand, the sugar uptake of the Arabidopsis cells started before the cell growth. Furthermore, glucose as the carbon source of the Arabidopsis cell cultivation seems to significantly influence the sugar metabolism. Glucose had a significant influence on the sugar metabolism of the other sugar under the conditions for the mixture of glucose and the other sugar. The characteristics of the sugar uptake phenomena based on the cell growth stage was typical for each plant cell except for some sugars, such as galactose and trehalose, and the behavior of the total sugar uptake had not changed. These results suggested that the cell growth and the sugar uptake in the plant cell cultivation processes may be controlled by the combined supply of the sugar species as the carbon source. The detailed data for plant cell cultivation using each sugar obtained in this study would be useful for bioscience research and for cultivation process control using various sugars, for example, purified or sugar mixtures formed from biomass materials.     

Activities of plasma membrane-bound enzymes isolated from roots of spruce (Picea abies) grown in the presence of aluminium

In batch cultures of Petunia hybrida cv. Rosy Morn Fertile. one respiratory peak is usually observed shortly after subculturing. However, two types of peak respiration could be distinguished, one connected with the dilution process and one with sugar addition at low biomass concentrations. The dilution peak was observed when cells were diluted in medium without sugar, in the presence or absence of mannitol. The sugar peak occurred only after previous dilution of the cells and not when sugar is added at high biomass concentrations Apparently the existence of a dilute suspension is a prerequisite for the induction of the peak. The presence of sugar is not a prerequisite for the increased respiratory activity but it is necessary lor growth: however, growth is possible without the increase in respiration, as was shown by the addition of sugar to a culture with a high biomass concentration. The peak caused by dilution either in the presence or absence of sugar showed no significant differences in height. The height of the peak caused by sugar addition to a previously diluted cell suspension was correlated with the sugar concentration. The respiratory peak disappeared long before the end of the growth period; this decline of the respiratory rates was not connected to sugar or oxygen limitation. In a continuous culture of Petunia hybrida growing at low biomass concentration, the respiration was always at the high level as observed during the peak of batch culture. Growing at lower biomass concentrations might be more expensive for plant cell suspensions.     

D-阿拉伯糖结晶影响因素分析

D-阿拉伯糖是多种功能性糖合成的中间体,其纯度高低决定了功能性糖转化率的高低,所以得到高纯度的D-阿拉伯糖尤为重要。通过对D-阿拉伯糖结晶温度、搅拌速度、结晶液中离子含量等因素进行试验,确定了采用梯度降温形式、搅拌速度控制在5 r/min,离子含量控制在100 μs/cm以下能够得到纯度达99.8%的D-阿拉伯糖晶体,实验结果为后续功能性糖的高效转化奠定了基础。     

Effects of culture temperature shift and light-dark time cycle on the cellular sugar accumulation of Chlorella pyrenoidosa

We investigated the effect of culture temperature on the maximum specific growth rate and the cellular sugar accumulation, and the effect of a temperature shift on the sugar accumulation of Chlorella pyrenoidosa cells in a batch culture system. Increase in temperature below 30?°C appeared to correlate with increase in the maximum specific growth rate, on the contrary the cellular sugar content showed a reverse tendency against temperature. We attempted to utilize this tendency for improving sugar productivity in Chlorella. First, we cultured Chlorella at 28?°C during the logarithmic growth phase to obtain a high specific growth rate. The culture temperature was then shifted from 28?°C to 22?°C at the late logarithmic growth phase in order to reduce the specific growth rate and enhance the cellular sugar accumulation. As a result, we obtained a 15% increase in sugar production over that obtained by cultivation at 28?°C throughout the culture. We also investigated the effect of light-dark time cycle on the sugar productivity and found that this operating variable did not affect the cellular sugar content but influenced the final cell concentration. Among the examined light-dark time cycles, maximum sugar productivity was obtained in the case of 12?h light and 12?h dark period.     

Effect of some alkaline phosphatase inhibitors on intestinal calcium transfer

1. There is a good correlation between the capacity of sugars to stimulate calcium transfer and their capacity to be phosphorylated by the intestinal alkaline phosphate with a part of the phosphate liberated from an ester phosphate. 2. On the sugar dependent and sugar independent calcium transfer, inhibitors of this enzyme act differently. 3. Phosphate, a competitive inhibitor suppresses both transfers. 4. Only the dependent sugar transfer was suppressed with phloridzin acting competitively at the sugar site, or with EDTA which could react close to the active site. 5. L-phenylalanine and phenobarbital, not competitive inhibitors does not act on either type of calcium transfer, the sugar dependent or the sugar independent.     

Ab initio molecular dynamics simulations of beta-D-glucose and beta-D-xylose degradation mechanisms in acidic aqueous solution

Ab initio molecular dynamics simulations were employed to investigate, with explicit solvent water molecules, beta-D-glucose and beta-D-xylose degradation mechanisms in acidic media. The rate-limiting step in sugar degradation was found to be protonation of the hydroxyl groups on the sugar ring. We found that the structure of water molecules plays a significant role in the acidic sugar degradation pathways. Firstly, a water molecule competes with the hydroxyl group on the sugar ring for protons. Secondly, water forms hydrogen bonds with the hydroxyl groups on the sugar rings, thus weakening the C-C and C-O bonds (each to a different degree). Note that the reaction pathways could be altered due to the change of relative stability of the C-C and C-O bonds. Thirdly, water molecules that are hydrogen-bonded to sugar hydroxyls could easily extract a proton from the reaction intermediate, terminating the reaction. Indeed, the sugar degradation pathway is complex due to multiple protonation probabilities and the surrounding water structure. Our experimental data support multiple sugar acidic degradation pathways.     

Kinetic analyses of the sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose enzyme II complex of the bacterial phosphotransferase system.

The sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose Enzyme II complex of the phosphotransferase system has been analyzed kinetically. Initial rates of phosphoryl transfer from glucose-6-P to methyl alpha-glucopyranoside were determined with butanol/urea-extracted membranes from Salmonella typhimurium strains. The kinetic mechanism was shown to be Bi-Bi Sequential, indicating that the Enzyme II possesses nonoverlapping binding sites for sugar and sugar phosphate. Binding of the two substrates appears to occur in a positively cooperative fashion. A mutant with a defective glucose Enzyme II was isolated which transported methyl alpha-glucoside and glucose with reduced maximal velocities and higher Km values. In vitro kinetic studies of the transphosphorylation reaction catalyzed by the mutant enzyme showed a decrease in maximal velocity and increases in the Km values for both the sugar and sugar phosphate substrates. These results are consistent with the conclusion that a single Enzyme II complex catalyzes both transport and transphosphorylation of its sugar substrates.     

Neutral oligosaccharide structures linked to asparagines of porcine zona pellucida glycoproteins

N-Linked sugar chains were liberated by hydrazinolysis from porcine zona pellucida glycoproteins obtained from ovarian follicular oocytes. Neutral sugar chains were separated from acidic ones by paper electrophoresis and fractionated with a serial lectin column chromatography and Bio-Gel P-4 column chromatography. Their structural analysis by sequential glycosidase digestion in combination with methylation analysis revealed that the neutral sugar chains are of bi-, tri-, and tetraantennary complex type with a fucosylated trimannosyl core. Twenty-six percent of the sugar chains contain N-acetyllactosamine repeating structures in their outer chain moieties. Only linear N-acetyllactosamine repeats, the maximum size of which is hexasaccharide, are detected. A characteristic feature is that 39% of the sugar chains contain N-acetylglucosamine residues at their nonreducing termini in spite of the absence of bisected sugar chains. This study provided, for the first time, the substantial information about the sugar chain structures of mammalian zona pellucida glycoproteins.     

慈竹构件和分株水平的可溶性糖含量研究

采用蒽酮比色法,测定了克隆植物慈竹构件和分株水平的可溶性糖含量．以探讨其对环境的生理适应性．结果表明,在构件水平,可溶性糖含量以慈竹叶最大,枝条次之,秆最小．不同构件的可溶性糖含量对分株年龄和分株部位的反应格局不同,1龄分株枝和秆的可溶性糖含量较高,而分株年龄对叶的可溶性糖含量影响不显著;分株下层叶和秆的可溶性糖含量大于上中层．高光条件下1龄分株叶的可溶性糖含量显著高于弱光条件下的1龄分株．叶可溶性糖含量受季节影响显著,1月最低,4月最高．但2龄和3龄分株间差异不显著．母分株和子分株上、中两层叶可溶性糖含量呈显著正相关,而下层相关性不显著．在分株水平,慈竹5个龄级间的可溶性糖含量无显著差异．总之,慈竹可溶性糖含量受生物和非生物因子的影响显著,且分株和构件层次存在差异．     

Partitioning of Sugar between Growth and Nitrate Reduction in Cotton Roots

The level of endogenous sugars was inversely related to nitrate availability in young cotton (Gossypium hirsutum L.) plants, with high nitrate causing a greater decline in sugar content of roots than of shoots. High nitrate (low sugar) plants also displayed relatively more shoot growth and less root growth than low nitrate (high sugar) plants. These data are consistent with the theory that roots are poor competitors for sugar, and that sugar supply is a major factor limiting root growth in vivo.

The effects of endogenous sugar level on root growth and on nitrate reductase activity in the root were different. When root sugar level was experimentally controlled by varying nitrate concentration in the nutrient solution, root growth was less sensitive than nitrate reductase activity to sugar deficiency. Also, in sterile root tips cultured on media containing a wide range of sucrose concentrations, growth rate was considerably less sensitive to endogenous sugar deficiency than was nitrate assimilation rate. Similarly, in plants which were detopped or girdled, nitrate reductase activity in the roots declined more rapidly than did root sugars, especially glucose and fructose. These results suggest that when sugar is deficient, cotton roots preferentially use it for growth at the expense of nitrate reduction.

     

Development of probiotic diets for the olive fly: evaluation of their effects on fly longevity and fecundity

One possible control strategy against the olive fly, Bactrocera oleae, the most serious olive crop pest, is the Sterile Insect Technique (SIT) application. However, a number of problems associated with this method remain that decrease the effectiveness of SIT, including the quality of reared insects. Taking the importance of the relationship between the olive fly and bacteria into consideration, the effects of probiotic diets enriched with Pseudomonas putida on B. oleae longevity and fecundity were evaluated. First, we found that the probiotic bacterium, P. putida, is conveyed from diets to the oesophageal bulb as well as to the fly midgut after feeding on the probiotic diet. Subsequently, B. oleae adults fed on either: (a) a standard full protein and sugar diet; (b) a sugar only diet; (c) a probiotic standard full protein and sugar diet; or (d) a probiotic sugar diet. Flies fed on probiotic diets were supplied with an inoculated gel containing P. putida; non‐inoculated gel was provided to the flies fed on non‐probiotic diets. B. oleae males and females that fed on sugar diets did not survive as long as those that fed on protein diets. A comparison of the longevity of adults fed on full diet and sugar with their respective probiotic diets revealed no significant difference. Males fed on the sugar only diet survived longer than males fed on probiotic sugar diet, and females fed on the full protein and sugar diet survived much longer than females fed on the full probiotic diet. As regarding fecundity, both full diets resulted in a higher number of eggs laid per female. Females fed on the probiotic sugar diet laid a higher number of eggs than females that fed on sugar only. The inoculated gel of the probiotic sugar diet contained a significantly higher quantity of leucine, isoleucine and proline than the non‐inoculated gel of the sugar only diet. The possible role of dietary bacteria in relation to functional aspects of olive fly physiology is discussed.     

Reprogramming of sugar transport pathways in Escherichia coli using a permeabilized SecY protein-translocation channel

In the initial step of sugar metabolism, sugar-specific transporters play a decisive role in the passage of sugars through plasma membranes into cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. The present work shows that permeabilized SecY channels can be used as nonspecific sugar transporters in Escherichia coli. SecY with the plug domain deleted allowed the passage of glucose, fructose, mannose, xylose, and arabinose, and, with additional pore-ring mutations, facilitated lactose transport, indicating that sugar passage via permeabilized SecY was independent of sugar stereospecificity. The engineered E. coli showed rapid growth on a wide spectrum of monosaccharides and benefited from the elimination of transport saturation, improvement in sugar tolerance, reduction in competitive inhibition, and prevention of carbon catabolite repression, which are usually encountered with native sugar uptake systems. The SecY channel is widespread in prokaryotes, so other bacteria may also be engineered to utilize this system for sugar uptake. The SecY channel thus provides a unique sugar passageway for future development of robust cell factories for biotechnological applications.     

Pollination ecology of Christmas Bells (Blandfordia nobilis): Patterns of standing crop of nectar

Standing crop of nectar (both volume and sugar concentrations) was measured from flowers in a population of Blandfordia nobilis. Nectar in this honeyeater-pollinated species was extremely viscous during one flowering season and averaged approximately 95% sugar, while it averaged only 25% sugar two seasons later. Nectar volume was significantly and negatively correlated with sugar concentration on nine often sampling dates. There was no significant relationship between either nectar volume or sugar concentration and a number of measures of plant size (inflorescence height, numbers of open flowers, buds, spent flowers and total flowers). Similarly, no consistent relationship was found between measures of flower size and reward level. There were significant positive correlations between the reward (both volume and sugar concentration) offered by blossoms on the same plant. The nectar volume of flowers from nearest neighbouring inflorescences were significantly and positively correlated with one another on three of six occasions while the sugar concentrations of those blossoms were significantly correlated on just two of six sampling dates. There was no significant difference in reward (both volume and sugar concentration) between open flowers and those bagged to exclude pollinators. There was significant heterogeneity in the nectar volumes offered by plants to pollinators during a single flowering season.     

Wie der Mensch zum Zucker kam

How sugar came to man Sucrose is a disaccharide, which is normally not stored in plants with a few exceptions, namely sugar beet and sugar cane. Both are economically significant plants which supply approximately 180 million tons of sugar per year. 70 per cent of the world's sugar production comes from sugar cane, which grows only in tropical and subtropical regions, and has a history of about 3,000 years. The concentrated, dried and pulverized cane sap (raw sugar) contains the most minerals, but has a strong flavour and is not universally applicable.     

烤烟糖含量的空间变异特征

以中国烤烟(Nicotiana tabacum)主产省份2005年B2F(上部二级桔黄色烤烟)、C3F(中部三级桔黄色烤烟)和X2F(下部二级桔黄色烤烟)3个等级烤烟为研究对象,利用地统计学与GIS技术结合的方法研究了中国烤烟主产省份烤烟水溶性总糖、还原糖含量和糖碱比的空间分布特征。结果表明:各等级烤烟水溶性总糖、还原糖含量和糖碱比空间相关性表现为近似相关,东西方向上,均呈现由西向东先降低后升高的趋势,南北方向上,水溶性总糖含量、还原糖含量趋势不明显,糖碱比各等级趋势各异。