Characterization of the conformational and orientational dynamics of ganglioside GM1 in a dipalmitoylphosphatidylcholine bilayer by molecular dynamics simulations

The structure and dynamics of a single GM1 (Gal5-β1,3-GalNAc4-β1,4-(NeuAc3-α2,3)-Gal2-β1,4-Glc1-β1,1-Cer) embedded in a DPPC bilayer have been studied by MD simulations. Eleven simulations, each of 10 ns productive run, were performed with different initial conformations of GM1. Simulations of GM1-Os in water and of a DPPC bilayer were also performed to delineate the effects of the bilayer and GM1 on the conformational and orientational dynamics of each other. The conformation of the GM1 headgroup observed in the simulations is in agreement with those reported in literature; but the headgroup is restricted when embedded in the bilayer. NeuAc3 is the outermost saccharide towards the water phase. Glc1 and Gal2 prefer a parallel, and NeuAc3, GalNac4 and Gal5 prefer a perpendicular, orientation with respect to the bilayer normal. The overall characteristics of the bilayer are not affected by the presence of GM1; however, GM1 does influence the DPPC molecules in its immediate vicinity. The implications of these observations on the specific recognition and binding of GM1 embedded in a lipid bilayer by exogenous proteins as well as proteins embedded in lipids have been discussed.     

一种免受乙醇干扰的GOD的酶电极

将酶电极应用于发酵糖的测定时。与糖共存的乙醇常常会影响测糖的准确性,通过对葡萄糖氧化酶(GOD)电极的研究,探讨了乙醇对测糖酶电极测定影响,进而研制出抗干扰的GOD酶电极,若是GOD电极的酶膜通过夹心法制备．在乙醇含量为0．1％(V／V)时·即产生显著的影响,使测定结果偏大4．3%,且乙醇的影响随浓度的升高而增大,若用尼龙网固定GOD膜,GOD电极在测定20mmol／L和5mm01／L左右的葡萄糖溶液时,含量高达9％的乙醇仍未对测定产生显著的影响．表现出良好的不受乙醇干扰的特性,并且,该尼龙网GOD电极具有良好的重复性、稳定的响应活性及较长的保存期．     

An Integrated View of the Kinetics of Glucose and Phosphate Transport, and of Glucose 6-Phosphate Transport and Hydrolysis in Intact Rat Liver Microsomes

The dynamics of the glucose 6-phosphatase system were investigated in intact rat liver microsomes using a fast-sampling, rapid-filtration apparatus. Glucose and phosphate transport followed single exponential kinetics, appeared to be homogeneous, was unaffected by unlabeled substrate concentrations up to 100 mm, proved insensitive to various potential inhibitors, and demonstrated similarly low energies of activation. The extent of tracer accumulation from glucose 6-phosphate depended on which of the glucose or phosphate moieties was the labeled species in the parent molecule. The rates of tracer equilibration reflected those of glucose or phosphate transport but similar initial rates of uptake were observed for the glucose and phosphate products of hydrolysis. However, the latter accounted for only 12–13% of the steady-state rate of total glucose production. It is concluded that tracer uptake cannot represent substrate transport, that labeled glucose 6-phosphate at best represents a tiny fraction of the intramicrosomal glucose or phosphate pools, and that glucose 6-phosphate transport is not an obligatory prerequisite to its hydrolysis. The latter conclusion invalidates a major postulate of the substrate transport-catalytic unit concept but proves compatible with a conformational model whereby glucose 6-phosphate transport and hydrolysis are tightly coupled processes while glucose and phosphate share, along with water and a variety of other organic and inorganic solutes, a common porelike structure for their transport through the microsomal membrane. Received: 26 May 2000/Revised: 16 October 2000     

Differences in fuel utilization between trout and human thrombocytes in physiological media

Cell culture preparations now play a significant and essential role in physiological and biochemical studies of cell biology. However, the fuels offered in cell culture media are only glucose and glutamine, plus whatever might be in the added sera. It is currently difficult to find a rational way forward on this problem, as there are few data on what fuels cells use in vivo or even in an in vitro physiological situation. A recent study on human platelets redressed the situation somewhat by finding that 75% of ATP turnover could be accounted for by aerobic glycolysis, and by the oxidation of glucose, hydroxybutyrate, acetate, glutamine, palmitate and oleate. In the present study we used a similar strategy to investigate fuel choices by trout thymocytes, cells with a similar function but from a different phylogenetic group. When these cells were presented with a physiological medium, we found that aerobic glycolysis accounted for 9% of total ATP turnover, glucose and glutamine oxidation made a combined contribution of 2.3%, oleate and palmitate oxidation accounted for 15%, and 74% was unaccounted for. These patterns of fuel use are very different from that in human platelets. They demonstrate the cell- and animal-specific nature of cellular metabolism and again expose the inadequacy of the fuel component in culture media. Accepted: 21 July 1999     

Defective Glucose Transport Across Brain Tissue Barriers: A Newly Recognized Neurological Syndrome

Impaired glucose transport across brain tissue barriers causes infantile seizures, developmental delay and acquired microcephaly. Since the first report in 1991 (De Vivo et al, NEJM, 1991) 17 patients have been identified with the glucose transporter protein syndrome (GTPS). The diagnostic feature of the syndrome is an unexplained hypoglycorrhachia in the clinical setting of an infantile epileptic encephalopathy. We review our clinical experience by highlighting one illustrative case: a 6-year old girl who presented at age 2 months with infantile seizures and hypoglycorrhachia. The CSF/blood glucose ratio was 0.33. DNA sequencing identified a missense mutation in exon 7 (C1108T). Erythrocyte GLUT1 immunoreactivity was normal. The time course of 3-0-methyl-glucose (3OMG) uptake by erythrocytes of the patient was 46% that of mother and father. The apparent K_m was similar in all cases (2–4 mmol/L), but the apparent V_max in the patient was only 28% that of the parents (500 versus 1,766 fmol/s/10⁶RBC; p < 0.004). In addition, a 3-month trial of oral thioctic acid also benefited the patient and increased the V_max to 935 fmol/s/10⁶ RBC (p < 3 × 10^–7). Uptake of dehydroascorbic acid by erythrocytes of the patient was impaired to the same degree as that of 3OMG (V_max was 38% of that of the mother's), which supports previous observations of GLUT1 being multifunctional. These studies confirm the molecular basis of the GTPS and the multifunctional role of GLUT1. The need for more effective treatment is compelling.     

Glucose delays seed germination in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Here we report that glucose delays germination of Arabidopsis thaliana (L.) Heynh. seeds at concentrations below those known to inhibit early seedling development. This inhibition acts on embryo growth and is independent of hexokinase (HXK) function. Hormones and hormone inhibitors were applied to the germination media and several hormone biosynthesis and signalling mutants were tested on glucose media to investigate a possible role of abscisic acid (ABA), gibberellin and ethylene in the glucose-induced germination delay. Results indicate that the germination inhibition by glucose cannot be antagonized by ethylene or gibberellin and is independent of the HXK1/ABA/ABI4 signalling cascade. These findings suggest that there is a separate regulatory pathway independent of ABI2/ABI4/ABI5. Thus, in a relatively short time frame sugars utilize different signalling cascades to inhibit germination and post-germination growth, underlining the complexity of sugar responses.Abbreviations ABA Abscisic acid - ABI ABA insensitive - ACC 1-Aminocyclopropane-1-carboxylic acid - BR Brassinosteroid - CAB Chlorophyll a/b-binding protein - FUS3 Fusca3 - GA Gibberellin - GA ₃ Gibberellic acid - HXK Hexokinase - LEC1 Leafy cotyledon1 - RBCS Ribulose-1,5-bisphosphate carboxylase small subunit - WT Wild type     

Temperature affects physiological stress responses to acute confinement in sunshine bass (Morone chrysops x Morone saxatilis)

Sunshine bass (Morone chrysopsxMorone saxatilis) were subjected to a 15-min low-water confinement stressor at temperatures ranging from 5 to 30 degrees C. Physiological responses were evaluated by measuring hematocrit, and plasma chloride, glucose and cortisol. Fish acclimated to 30 degrees C had initial glucose concentrations of 3.13 mM (564 mg/L) which were significantly lower than in fish acclimated to 5 and 10 degrees C (4.32 and 4.82 mM or 779 and 868 mg/l, respectively). Fish survived the conditions imposed at every temperature except 30 degrees C, where 15 out of 42 fish died during the stress and recovery protocol. The general pattern was an initial increase in hematocrit, followed by a delayed decrease in hematocrit and chloride, and an increase in plasma glucose and cortisol. In general, fish stressed at temperatures below 20 degrees C had lower and more delayed changes in plasma glucose and cortisol than fish tested at 20, 25 and 30 degrees C. Initial cortisol concentrations were 65 ng/ml and increased to above 200 ng/ml in fish held at 20 degrees C and above. At the higher temperatures, glucose concentrations were twice the initial concentration after stress and cortisol changes were four to five times the initial concentration after the stress. Quantitative responses for glucose and cortisol were moderate and recovery rapid in fish stressed at 10 and 15 degrees C; therefore, this range of water temperature is recommended when handling sunshine bass.     

Identification of the facilitative glucose transporter 12 gene Glut12 in mouse preimplantation embryos

In this study we report the cloning and characterisation of the mouse Glut12 gene and examine for the first time its expression pattern in the earliest stages of development. Mouse Glut12 (mGlut12) was cloned from preimplantation embryos by 5'RACE RT-PCR using primers designed from an EST clone corresponding to a human GLUT12 antigenic sequence after positive immunoreactivity was observed in mouse two-cell embryos by western immunoblotting. The mGlut12 gene contains an open reading frame of 1869 base pairs, potentially encoding a polypeptide of 622 amino acids. The predicted mGLUT12 protein bears all the hallmarks of the SLC2A family of hexose transporters and shares an 83% sequence homology to human GLUT12. Consistent with its human homolog mGlut12 mRNA is found highly expressed in skeletal and cardiac muscle and fat. Additionally, it was also found in the uterus and during early embryogenesis. During early development in the mouse, Glut12 expression is clearly apparent in ovulated oocytes and two-cell embryos but declines in day 3 morulae. With the exception of some Glut12 expression apparent in blastocysts, Glut12 mRNA remains at low to undetectable levels until E11.     

Thapsigargin and EGTA inhibit endothelin-1-induced glucose transport

We have previously demonstrated that ET-1 may enhance glucose transport in 3T3-L1 adipocytes, secondarily to its stimulatory effect on GLUT1 gene expression by a mitogen-activated protein kinase (MAPK)-dependent pathway. In the present study, we further tested the involvement of Ca²⁺ in glucose uptake in response to ET-1. Among a variety of Ca²⁺-related agents tested, EGTA and thapsigargin were found to suppress both the glucose uptake and intracellular Ca²⁺ mobilization induced by ET-1, as determined by Fura-2 analysis. However, a phospholipase C inhibitor, U73122, also eliminated the intracellular calcium mobilization induced by ET-1, but had no effect on ET-1-stimulated glucose uptake. The finding that neither EGTA nor thapsigargin had any influence on ET-1-induced MAPK activation implies that some mechanism downstream of MAPK activation is involved. Further investigation showed that both agents exerted global inhibitory effects on protein and RNA syntheses. Since both thapsigargin and EGTA may deplete endoplasmic reticulum (ER) Ca²⁺ stores, our results suggest that (1) ET-1-induced glucose transport is independent of ET-1's effect on Ca²⁺ mobilization and (2) depletion of ER Ca²⁺ stores per se may interfere with ET-1's effect on GLUT1 expression.     

Axin and the Axin/Arrow-binding protein DCAP mediate glucose-glycogen metabolism

Axin was found as a negative regulator of the canonical Wnt pathway. Human LRP5 was originally found as a candidate gene of insulin dependent diabetes mellitus (IDDM), but its Drosophila homolog, Arrow, works as a co-receptor of the canonical Wnt signal. In our previous paper, we found a new Drosophila Axin (Daxin)-binding SH3 protein, DCAP, a homolog of mammalian CAV family protein. Among the subtypes, DCAPL3 shows significant homology with CAP, an essential component of glucose transport in insulin signal. Further binding assay revealed that DCAP binds to not only Axin but also Arrow, and Axin binds to not only GSK3beta but also Arrow. However, overexpression and RNAi experiments of DCAP do not affect the canonical Wnt pathway. As DCAP is expressed predominantly in insulin-target organs, and as RNAi of DCAP disrupts the pattern of endogenous glycogen accumulation in late stage embryos, we suggest that DCAP is also involved in glucose transport. Moreover, early stage embryos lacking maternal Axin show significant delay of initial glycogen decomposition, and RNAi of Axin in S2 cells revealed quite increase of endogenous glycogen level as well as GSK3beta. These results suggest that Axin and DCAP mediate glucose-glycogen metabolism in embryo. In addition, the interaction among Axin, Arrow, and DCAP implies a possible cross-talk between Wnt signal and insulin signal.