Synthesis of 1,3-anhydro-D-glucitol and some derivatives of 1,5-anhydro-D-glucitol

Syntheses of 1,5-anhydro-2,4-di-O-benzyl-D-glucitol and 2,4-di-O-acetyl-1,5-anhydro-D-glucitol are outlined. Some approaches to the synthesis of 1,5-anhydro-2,4-O-benzylidene-D-glucitol, which led to the alternative formation of the corresponding 1,3-anhydrohexitol, are discussed. Relevant properties of the parent 1,3-anhydro-D-glucitol and its derivatives are described, as is the evidence in favour of this novel structural formulation.     

Oxidation of 1,5-anhydro-D-glucitol to 1,5-anhydro-D-fructose catalyzed by an enzyme from bacterial membranes

Bacteria which grow on 1,5-anhydro-D-glucitol (AG) were isolated from soil. One such strain showing the highest AG-assimilating activity was further characterized and identified as a new strain of the Pseudomonas family (named Pseudomonas sp. NK-85001). A subcellular membranous fraction obtained from this strain catalyzed the oxidation of AG to 1,5-anhydro-D-fructose. This oxidation reaction consumed molecular oxygen as the terminal electron acceptor. The AG-oxidizing activity was further purified after solubilization. The AG oxidation catalyzed by this solubilized enzyme utilized molecular oxygen only in the presence of an electron mediator such as 2,6-dichlorophenolindophenol or phenazine methosulfate. Thus, the enzyme was suggested to be a dehydrogenase rather than an oxidase. The solubilized enzyme preparation also showed a strict substrate specificity. The observed specificity indicated that application of the enzyme for AG assay in clinical samples might be possible.     

Transport of 1,5-anhydro-D-glucitol into insulinoma cells by a glucose-sensitive transport system

The uptake of 1,5-anhydro-D-glucitol (1,5-AG) occurs by passive mechanisms in cells or tissues that have passive glucose transporters. It is known that serum 1,5-AG concentrations are reduced in patients with diabetes mellitus. To elucidate the metabolism of this substance and its physiological role in pancreatic beta-cells, we assayed 1,5-AG transport in the insulinoma-derived cell lines, RINr and MIN6. Both cell lines showed an insulin-insensitive, concentration-dependent uptake of 1,5-AG with a saturation time of approximately 120 min, and most of the 1,5-AG in the cytoplasm was in the free form. A biphasic saturation curve was obtained using a wide range of 1,5-AG concentrations, suggesting that accumulation was mediated by a high affinity and a low affinity transporter. The high affinity transporter had a K(m) of 10.4 in RINr cells and 13.0 mM in MIN6 cells, and the low affinity transporter had a K(m)100 times, being much higher than the physiological concentrations of 1,5-AG. These results indicate that the 1,5-AG carrier system in insulinoma cells is distinct from that in either the somatic cells or renal tubular cells. These findings also suggest that a unique 1,5-AG transport system is present in pancreatic beta-cells.     

Transport of 1,5-anhydro-D-glucitol into human polymorphonuclear leukocytes.

1,5-Anhydro-D-glucitol (AG) is one of the main polyols and its structure resembles glucose. It has been proposed that decreased serum AG concentrations in diabetic patients are a novel indicator of diabetic metabolic derangement. However, the pathway of AG metabolism still remains to be clarified. In this study we investigated the transport of AG into human polymorphonuclear leukocytes (PMNLs) isolated from healthy volunteers and found that 0.1 mM 3-O-methy-D-glucose (3OMG) was equilibrated with a half saturation time of 10 s, while the uptake rate of AG was much slower. The concentration dependence of AG uptake revealed that the AG transport velocity reached a plateau, with a Km of about 50 mM and Vmax of about 25 nmol/min/10(7) cells. Transport of 14C-labeled 3OMG was inhibited by unlabeled D-glucose or AG in a dose-dependent manner. The mean inhibition constant (Ki) for D-glucose and for AG were 1.06 and 4.93 mM, respectively. Cytochalasin B (20 microM) inhibited 3OMG transport by 90% but AG transport by only 50%. S/V for 14C-labeled AG transport plotted against the concentration of unlabeled 3OMG showed a non-linear and biphasic pattern. These results suggest that AG influx into PMNLs is mediated not only by the cytochalasin B-sensitive glucose transport system but also via another facilitated transport system.     

Reduced renal reabsorption of 1,5-anhydro-D-glucitol in diabetic rats and mice

A stable amount, approximately 60 micrograms, of 1,5-anhydro-D-glucitol (AG) was detected in the 24-h-urine of normal young rats fed ad libitum. Upon administration of streptozotocin (STZ), this amount was temporarily elevated to as much as 1.1 mg and AG was concomitantly removed from the circulation. The plasma AG level stayed almost null thereafter while the acutely elevated urinary AG excretion declined within 24 h to another stable excretion level that was three times as high as that of the untreated rats. In contrast, glucosuria developed much more slowly in the drug-treated rats. Normal rats and mice retained exogenous [14C]AG to a considerable extent and the radioactivity was distributed all over the body. Only a marginal fraction of the radioactivity was excreted as expired CO2. The radio-activities retained in the body and excreted into the urine were mostly attributed to unmetabolized AG. The observations of AG's metabolic stability and its relatively low level of leakage into urine suggested the concept of effective renal AG reabsorption. On the other hand, the rats with STZ-induced diabetes and NOD-mice with spontaneously developed diabetes retained little of the radioactive AG in their bodies; most of the injected radioactivity was recovered in the urine within 24 h. This observation was interpreted as due to reduced renal AG reabsorption in these animals. The concept of reduction in renal AG reabsorption in diabetes could account for the reduced plasma AG level generally observed in human diabetic cases.     

Transport of 1,5-anhydro-D-glucitol across plasma membranes in rat hepatoma cells

The transport of 1,5-anhydro-D-glucitol (AG) across plasma membranes was investigated in rat hepatoma cells, Reuber H-35. The AG uptake by the cells showed a concentration gradient dependency: the uptake was saturated within 40 s, which was less than one-third of the saturation time for 2-deoxy-D-glucose (DG) uptake. Furthermore, the Km value of the transport system for AG was higher than 100 mM. Though AG has a pyranoid structure resembling that of glucose, AG did not compete for cellular uptake with DG, D-glucose or 3-O-methyl-D-glucose, which are taken into cells through the glucose transporters. Conversely, the DG transport was not inhibited by AG at concentrations up to 50 mM. AG transport was hardly inhibited by 10 microM cytochalasin B, which strongly inhibits glucose transporters. In contrast, the AG transport was inhibited by 100 microM phloretin much more strongly than the DG transport when cells were preincubated with the inhibitor; the inhibition constant was 28.0 microM. The AG transport was not inhibited by 100 microM phloridzin, while the DG uptake was slightly inhibited by phloridzin. On the basis of these observations we propose that the AG uptake into rat hepatoma cells is mediated by a carrier distinct from glucose transporters.     

The daidzein- and estradiol- induced anorectic action in CCK or leptin receptor deficiency rats

We investigated the effect of daidzein feeding and estradiol treatment on food intake in cholecystokinin-1 receptor (CCK1R) deficiency, leptin receptor (ObRb) deficiency rats and their wild-type rats. These rats underwent an ovariectomy or a sham operation. For the 5 week experiment, each rat was divided in three groups: control, daidzein (150 mg/kg diet), and estradiol (4.2 μg/rat/day) groups. In both CCK1R+ and CCK1R? rats, daidzein feeding and estradiol treatment significantly decreased food intake. Daidzein feeding significantly reduced food intake in ovariectomized ObRb? rats, although not in ObRb+ rats. Estradiol treatment significantly lowered food intake in ovariectomized ObRb+ and ObRb? rats. In the ovariectomized rats, estradiol treatment significantly increases uterine weight, while daidzein feeding did not change it, suggesting that daidzein might have no or weak estrogenic effect in our experiment. These results suggest that CCK1R and ObRb signalings were not essential for the daidzein- and estradiol-induced anorectic action.     

Distribution of 1,5-anhydro-D-glucitol in normal, diabetic, and perfused rat bodies.

The concentration of 1,5-anhydro-D-glucitol (AG) was determined in various organs and tissues of normal rats and rats rendered diabetic with streptozocin, using an AG-assay method in which AG was extracted after acid hydrolysis of the whole tissues. The organs and tissues examined included skin, muscle, liver, and kidney. The plasma of control rats contained 3-12 micrograms/ml of AG. In these rats, all the organs examined also contained AG at concentrations not much lower than that in the corresponding plasma, except for adipose tissues and testis, which have relatively small water spaces; the latter two contained AG at relatively low concentrations. In contrast, both the plasma and various organs of the diabetic rats contained only trace amounts of AG. The whole body perfusion of control rats depleted AG from most of the organs, the exception being spleen, the circulation system of which is known to have a structure that is difficult wash by means of perfusion. These observations indicated that AG readily diffused into the inter- and intra-cellular water spaces from the circulation. Accordingly, the plasma membranes of the cells in these organs were suggested to be permeable to AG.     

A novel NAD-dependent dehydrogenase,highly specific for 1,5-anhydro-D-glucitol,from Trichoderma longibrachiatum strain 11-3

A novel NAD-dependent dehydrogenase highly specific for 1,5-anhydro-D-glucitol (1,5-AG) was found in the cell extract of an imperfect fungus, Trichoderma longibrachiatum strain 11-3. This fungus used 1,5-AG as a sole carbon source for growth and transformed 1,5-AG into glucose. 1,5-AG dehydrogenase (AGH) was purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the purified enzyme was estimated to be 36 and 141 kDa by SDS-PAGE and by gel filtration, respectively, suggesting that the enzyme was homotetrameric. The enzyme was highly specific for 1,5-AG and did not exhibit activity with any sugar or sugar alcohol tested in this study other than 1,5-AG. A linear relationship between the initial rate of the enzyme reaction and the concentration of 1,5-AG at the physiological level was observed. The presence of glucose in abundance did not interfere with the relationship. The optimum temperature for the enzyme reaction was 50 degrees C, and the enzyme was stable at temperatures up to 70 degrees C. These results suggested that AGH is a novel enzyme and is useful for specifically diagnosing diabetes mellitus.     

Amylin receptors mediate the anorectic action of salmon calcitonin (sCT)

The teleost salmon calcitonin (sCT), but not mammalian CT, shows similar biologic actions in the skeletal muscle as amylin and calcitonin gene-related peptide (CGRP). The peptides have also been shown to reduce food intake in rams. Because sCT, but not amylin, binds irreversibly to amylin binding sites, the aim of the present study was to compare the anorectic potency of both peptides. To determine whether sCT reduces food intake through interaction with amylin binding sites, we also tested whether appropriate antagonists (CORP 8-37, AC 187) attenuate the anorectic effect of sCT. Finally, we wanted to know whether rat calcitonin (rCT) and sCT reduce food intake to the same extent. Peptides were injected intraperitoneally at dark onset in 24 h food-deprived rats. At doses of 5 or 0.5 microg/kg, the anorectic effect of sCT was more potent and lasted much longer (e.g. 5 microg/kg: sCT > 10 h; amylin approx. 2 h) than that of amylin. Both CORP 8-37 and AC 187 (10 microg/kg) markedly reduced the anorectic action of sCT (0.5 microg/kg). In contrast to sCT, rCT (0.5 microg/kg) had no effect on food intake. It is concluded that sCT s anorectic effect is partly mediated by amylin receptors. Irreversible binding of sCT to amylin receptors may lead to a stronger and prolonged effect in comparison to amylin due to a sustained activation of the binding sites. Similar to other actions of CTs, the anorectic potency of sCT in rats was higher than that of mammalian (rat) CT. This agrees with binding profiles of amylin, sCT, and rCT at amylin binding sites as observed in in vitro studies.     

Structural transitions of chromatin induced by netropsin

Structural changes of chromatin induced by interaction of netropsin (Nt) with DNA has been examined by analysis of CD and electromicroscopic measurements. The results demonstrate the existence of a transition from the condensed globular state of chromatin into nucleosomal fibres generated by extremely low Nt concentration up to 1 mole Nt per 200 nucleotides. A second transition occurs at high Nt ratio per DNA phosphate (v' = 0.3). involving disorganization of nucleosomal particles. The interference of the Nt binding with chromatin proteins maintaining the sub- and superstructure will be discussed.     

Histamine H1 receptors mediate the anorectic action of the pancreatic hormone amylin

We investigated the role of histamine H1 receptors in mediating the anorectic effect of intraperitoneally injected amylin (5 and 20 microg/kg), the amylin agonist salmon calcitonin (sCT; 10 microg/kg), leptin (1.3 mg/kg), and cholecystokinin (CCK; 20 microg/kg). The experiments were performed with mice lacking functional H1 receptors (H1Rko) and wild-type (WT) controls. The mice were also injected with the H3 antagonist thioperamide (20 mg/kg), which reduces feeding by enhancing the release of endogenous histamine through presynaptic H3 receptors. The feeding-suppressive effect of thioperamide was abolished in H1Rko mice. The anorectic effects of amylin and sCT were significantly reduced in 12-h food-deprived H1Rko mice compared with WT mice [1-h food intake: WT-NaCl 0.51 +/- 0.05 g vs. WT-amylin (5 microg/kg) 0.30 +/- 0.06 g (P < 0.01); H1Rko-NaCl 0.45 +/- 0.05 g vs. H1Rko-amylin 0.40 +/- 0.04 g; WT-NaCl 0.40 +/- 0.09 g vs. WT-sCT (10 microg/kg) 0.14 +/- 0.10 g (P < 0.05); H1Rko-NaCl 0.44 +/- 0.08 g vs. H1Rko-sCT 0.50 +/- 0.06 g]. The anorectic effect of leptin was absent in ad libitum-fed H1Rko mice, whereas CCK equally reduced feeding in WT and H1Rko animals. This suggests that the histaminergic system is involved in mediating the anorectic effects of peripheral amylin and sCT via histamine H1 receptors. The same applies to leptin but not to CCK. H1Rko mice showed significantly increased body weight gain compared with WT mice, supporting the role of endogenous histamine in the regulation of feeding and body weight.     

Differential roles of tumor necrosis factor-alpha and interferon-gamma in mouse hypermetabolic and anorectic responses induced by LPS

Lipopolysaccharide (LPS)-induced effects on energy balance are characterized by alterations in energy expenditure (hypermetabolism) and food intake (anorexia). To study the role of tumour necrosis factor alpha (TNF-alpha) on some of these metabolic responses to endotoxin, we have used transgenic mice expressing soluble tumour necrosis factor receptor-1 IgG fusion protein (TNFR1-IgG) as well as TNF-alpha knockout (KO), lymphotoxin-alpha (LT-alpha) KO, and interferon-gamma receptor (IFN-gamma R) KO mice. The results from TNFR1-IgG transgenic mice suggest that the hypermetabolic and anorectic responses induced by LPS are independently regulated since, in the absence of TNF-alpha or LT-alpha, the LPS-induced hypermetabolism is almost prevented but not the anorexia. The anorectic response shows the strongest association with IFN-gamma since both IFN-gamma R KO mice and mice treated with anti-IFN-gamma antibody showed marked reduction in the LPS-induced anorexia compared to other mice. IFN-gamma R KO mice also have an attenuated thermogenic response to endotoxin. Anti-Asialo GM1 antibody treatment attenuated both the hypermetabolic and anorectic responses to LPS, to an extent comparable to that observed in IFN-gamma R KO mice. This finding suggests that natural killer cells (lymphocytic subsets) may be involved in IFN-gamma production and play an important role in the metabolic alterations induced by LPS. We also showed that the hypermetabolic response of control mice is associated with an upregulation of cytokine expression within the brain and an increase in permeability of the blood brain barrier. LPS-induced anorexia appears to involve peripheral cytokine expression.     

Thiol oxidation induced by oxidative action of adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H₂O₂ (HRP-H₂O₂) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H₂O₂ was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H₂O₂. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H₂O₂ in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.     

Structural changes in bacteriorhodopsin induced by electric impulses

Electric impulses of high field intensity (2 × 10⁵ to 3 × 10⁶ Vm^?1, 1 to 20 μs duration) cause transient changes in the optical absorbance of suspended purple membranes of Halobacterium halobium. The electric dichroism at 1 mm NaCL, pH ≈ 6 and at 293K is dependent on field strength, pulse duration and wavelength of the monitoring, plane-polarized light in the range 400 to 650 nm. The optically detected processes are, however, independent of bacteriorhodopsin concentration, of ionic strenght and of the intensity of the monitoring light. These data together with the analysis of time course ands steady state of the reduced dichroism, suggest electric field-sensitive, intramemembraneous structural changes which lead to restricted orientation changes of the chromophore. A thoretical analysis of restricted orientation is developed and applied to the electro-optic data. As a result, it is found that the electric dichroism of purple membrane is associated with a large polarizability anisotropy of 2.4 × 10^?30 Fm² (2.2 × 10^?14 cm³); the electric permanent dipole moment which is involved amounts to 4.7 × 10^?28 Cm(140 Debye). The kinetic data suggest a cyclic reaction scheme with at least five different conformations. The high polarizability is probably due to displaceable ionic groups within the cooperative lattice of bacteriorhodopsin molecules in purple membranes.     

Structural changes induced by glycine on Streptomyces antibioticus

Abstract Germination and vegetative growth of Streptomyces antibioticus in liquid medium with different concentrations of glycine was examined. Both processes proved to be sensitive to the amino acid, being inhibited by 5 and 2.5% glycine, respectively. At concentrations of 5% or more, lysis of the vegetative mycelium occurred. Subinhibitory concentrations of glycine induced structural changes on germinating spores. These included an increase in the number of germ tubes produced by spore, in relation to the control. Moreover, soon after outgrowth the tubes bifurcate, giving rise to germinated spores with a characteristic aspect, and anomalous formation of cross-walls that appear both within the spores and in the newly formed germinative tubes, at or close to the region of outgrowth. The branching effect of glycine was also observed during vegetative growth of S. antibioticus .     

Structural rearrangements in membranes induced by anesthetics]

Studies were carried out on the effects of anesthetics on the stability of membranes to sodium dodecylsulfate and trypsin. The initial rate of membrane desintegration by detergent evaluated by light scattering with stopped flow method was increased in the presence of ethanol, propanol, benzyl alcohol, procain-amide (erthrocyte membrane) and chlorpromazine (rat brain synaptosomes). Concentrations of anesthetics which correspond to half of the maximum effect were very close to that of blocking the action potential and inducing 50%--antihemolysis. Alcohols and procaine at anesthetic concentrations inhibited proteolysis of isolated erythrocyte membranes but were without effect on their ultrasonic fragments. Increase of membrane desintegtation rate is interpreted as structural rearrangements of membranes with the weakening of detergent sensitive intermolecular interactions. Proteolysis inhibition of ghosts but not the gragments indicates against the participation of the basic func of proteins in the structural membrane rearrangements.