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Various glycosyl hydrolase enzymes of the clan GH-H (http:// www.cazy.org/Glycoside-Hydrolases.html) catalyse transglycosylation reactions, mostly by using starch (e.g. , CGTase, family GH13) or sucrose (e.g. , glucansucrase, family GH70) as the donor substrate, thus transferring glucose. Some alternative biocatalysts for glycosyl transfer reactions have also been discovered; these include glycosynthases and glycosyltransferases. In recent years we have introduced the concept of sucrose analogues. Sucrose analogues such as a-d-glycopyranosyl bd-fructofuranosides have been reported as promising tools for the synthesis of functional fructo-oligosaccharides. In recent review articles we have suggested that these analogues can be used as glycopyranosyl donors for artificial transglycosylation reactions, which are not performed in nature. 9] Sucrose analogues are readily available from the cheap and abundant starting material sucrose. They are obtained through a transfructosylation reaction catalysed by levansucrase from Bacillus subtilis or Bacillus megaterium, by transferring fructose from sucrose to mannose, galactose, xylose or fucose. a-d-Allopyranosyl b-d-fructofuranoside (1, All-Fru, Scheme 1) is an allose that contains a sucrose analogue derived from sucrose by dehydrogenisation to 3-ketosucrose and subsequent hydrogenation to All-Fru. This synthesis has been established at an industrial scale. Allose is a rare sugar in nature; only a few plant species, for example, Passiflora edulis, Sideritis grandiflora and Protea rubropilosa, are known to contain glycoconjugates with blinked allose residues. Allose and allosylated glycoconjugates show promising properties for pharmaceutical use in cancer therapy, in immune suppression, for inhibition of neutrophil production and as antioxidants. To the best of our knowledge no a-d-allosyltransferase is known, however, research by Thorson and co-workers indicated a possible b-allosyl transfer performed by the glycosyltransferase OleD enzyme (family GT1). In recent work, we sought to identify enzymes able to transfer d-allose from the sucrose analogue All-Fru as the donor substrate. For this, All-Fru was incubated with various enzymes from the glycosyl hydrolase clan GH-H. As this sucrose analogue is a-linked and enzymes of the GH-H clan generally retain the configuration at the anomeric centre, the formed products should exhibit a-linkage. The sucrose isomerase (SI, GH13) from Protaminobacter rubrum showed no activity towards All-Fru. In contrast, incubation of All-Fru (292 mm) with amylosucrase (NpAs, GH13) from Neisseria polysaccharaea at pH 6.6 and 30 8C showed slow hydrolysis : even after 80 h, consumption of All-Fru was incomplete. Glucansucrases from Lactobacillus reuteri strains 121 (GTFA) and 180 (GTF180), and GTFR from Streptococcus oralis (all GH70) were able to hydrolyse 292 mm All-Fru. As hydrolysis activity on All-Fru was highest for GTFA (complete hydrolysis after 80 h), this enzyme was used in further studies. The glucansucrase GTFA from L. reuteri 121 is an extracellular enzyme that produces the polymer reuteran (mainly a(1!4)-linked glucan, with minor a(1!6) and branches) from sucrose. In contrast, with sucrose as donor, no formation of allose oligoor polysaccharides was observed by TLC. The transferase activity of GTFA-DN-CHis (a N-truncated version was used in all experiments) with All-Fru was observed with several acceptor substrates (note: concentration depended on solubility in the solvent system), thus obtaining a variety of functionalities (Table 1) such as sugars (methyl a-d-glucopyranoside (a-d-Me-Glcp, 2) and methyl 6-O-p-toluenesulfonyl-ad-glucopyranoside (a-d-Me-6-Ts-Glcp, 4)), amino acids (e.g. , N-(tert-butoxycarbonyl)-d-serine methyl ester (N-Boc-d-serineOMe, 6)) and ( )-epicatechin (8). Incubating GTFA with All-Fru (292 mm) and a-d-Me-Glcp (772 mm, 2) as the acceptor resulted in methyl a-d-allopyranosyl-(1!4)-a-d-glucopyranoside (3) as the main product (50% yield). The a-configuration of the glycosidic bond was determined by H NMR (H-1’: d= Scheme 1. The sucrose analogue All-Fru.

application/pdf, p‐Aminobenzoic ethyl ester (ABEE)‐converted four D‐aldohexoses including rare sugar D‐allose (the C‐3 epimer of glucose) were analyzed by high‐performance liquid chromatography (HPLC) on two reverse‐phase silica columns with a 0.2 M potassium borate buffer (pH 8.9) and acetonitrile (93:7) as the eluent using a detector of fluorescence (excitation of 305 nm and emission of 360 nm). On Cosmosil AR column, the four aldohexoses, which have identical molecular weight, were eluted in the order galactose < mannose < allose < glucose, although mannose, allose, and glucose were insufficiently separated. By contrast, on XBridge column containing polyethoxysilane, the four sugars were completely separated with the sharp peak. Each sugar could be sufficiently detected up to 0.5 nmol (90 ng) with a UV (305 nm) detector and 1 pmol (0.18 ng) with a fluorescence detector. Furthermore, HPLC analysis on XBridge column revealed that rare sugar D‐altrose (C‐2 and C‐3 epimer of glucose) labeled with ABEE was eluted in the position between galactose and mannose. The HPLC analysis described here is simple, reliable, and will be applicable to the identification and quantification of traces of D‐allose and D‐altrose in biomaterials such as animal blood and plant cells., 希少糖であるD‐アロース（グルコースのC‐3エピマー）を含む4種のアルドヘキソースをp‐アミノ安息香酸メチルエステル（ABEE）化し、2種の逆相シリカカラムを用いて0.2Mホウ酸カリウム緩衝液（pH8.9）とアセトニトリル（93:7）を溶出液とした高速液体クロマトグラフィー（HPLC）で蛍光検出器（励起波長305nm、測定波長360nm）を用いて分析した。Cosmosil AR カラムでは、分子量が同一な4種のアルドヘキソースはガラクトース、マンノース、アロース、グルコースの順に溶出したが、マンノース、アロースとグルコースの相互の分離は不完全であった。それに対してポリエトキシシランを含むXBridgeカラムを用いると、4種の糖は相互にシャープなピークとして完全に分離した。各々の糖はUV検出器（305nm）では0.5nmol（90ng）以上、蛍光検出器では1pmol（0.18ng）以上が十分検出できた。さらに、XBridgeカラムを用いたHPLC分析でABEE標識した希少糖であるD‐アルトロース（グルコースのC‐2とC‐3エピマー）はガラクトースとマンノースの間の位置に溶出した。ここで示したHPLC分析は簡単で信頼性があるので、動物血清や植物細胞のような生体試料中での微量なD‐アロースやD‐アルトロースの同定や定量に応用可能である。

Streptococcus agalactiae est la première cause d’infections néonatales, et est aussi un pathogène émergent chez l’adulte immunodéprimé. L’objectif de ce travail de thèse a été de caractériser l’opéron métabolique fru2 de S. agalactiae (i) en étudiant sa phylogénie, (ii) en identifiant ses inducteurs, et (iii) en élaborant son schéma de régulation. Cet opéron est composé de 7 gènes qui codent un activateur transcriptionnel de la famille DeoR-like (Fru2R), un transporteur PTS (PTSFru2), et trois enzymes qui sont potentiellement impliquées dans la voie non oxydative des pentoses phosphates. Nous avons mis en évidence que cet opéron avait été acquis au cours de l’évolution, et n’était présent que chez les souches de complexes clonaux responsables d’infections chez l’adulte immunodéprimé et la personne âgée. Nous avons ensuite montré que certains milieux complexes, sources de carbone, et liquides biologiques humains permettaient l’activation de cet opéron. Le rôle et fonctionnement de la protéine Fru2R ont été caractérisés (i) en montrant son rôle d’activateur transcriptionnel, (ii) en identifiant les acides aminés essentiels à son activité, et (iii) en démontrant sa capacité à se fixer au niveau de la région promotrice de fru2. Nous avons ensuite décrit le rôle des protéines du PTSFru2 dans la régulation de l’opéron fru2 (i) en caractérisant l’impact d’une délétion du PTSFru2 sur la régulation de l’opéron fru2, (ii) en identifiant les acides aminés essentiels à l’activité des protéines EIIAFru2 et EIIBFru2, et (iii) en révélant l’interaction physique de EIIBFru2 avec Fru2R. Enfin, nous avons montré l’implication de la répression catabolique, plus particulièrement de CcpA, dans la régulation de cet opéron. Les données obtenues ont permis d’élaborer un schéma de régulation de l’opéron fru2 en fonction de la source de carbone disponible.

Purpose The anti-tumor activity of glucose analogs 2-deoxy-glucose (2-DG) and D-allose was investigated alone or in combination with p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 or platinum analogs as a strategy to pharmacologically target glycolytic tumor phenotypes. Methods Hypoxia inducible factor-1 alpha (HIF-1α) protein accumulation in pancreatic cell lines treated with SB202190 alone and in combination with glucose analogs was analyzed by Western blot. HIF-1α transcriptional activity was measured in MIA PaCa-2 cells stably transfected with a hypoxia response element luciferase reporter following treatment with glucose analogs alone, and in combination with SB202190. Induction of cleaved poly(ADP-ribose) polymerase (PARP) was measured by Western blot in the MIA PaCa-2 cells. In vitro anti-proliferative activity of 2-DG and D-allose alone, or in combination with oxaliplatin (pancreatic cell lines), cisplatin (ovarian cell lines), or with SB202190 were investigated using the MTT assay. Results SB202190 decreased HIF-1α protein accumulation and transcriptional activity. 2-DG demonstrated greater anti-proliferative activity than D-allose. Pre-treatment with SB202190 enhanced activity of both 2-DG and D-allose in MIA PaCa-2, BxPC-3, ASPC-1, and SK-OV-3 cells. The combination of D-allose and platinum agents was additive to moderately synergistic in all but the OVCAR-3 and HEY cells. SB202190 pre-treatment further enhanced activity of D-allose and 2-DG with platinum agents in most cell lines investigated. Conclusions SB202190 induced sensitization of tumor cells to 2-DG and D-allose may be partially mediated by inhibition of HIF-1α activity. Combining glucose analogs and p38 MAPK inhibitors with chemotherapy may be an effective approach to target glycolytic tumor phenotypes.

application/pdf, D-Allose is one of the rare sugars present in small quantities in nature. We studied the effect of D-allose on glycemic responses after oral carbohydrate tolerance test in rats. Male Wistar rats (13 weeks old) were administrated (2g/kg body weight) glucose, sucrose, maltose, or soluble starch together with (0, 0.2 or 0.5g/kg body weight) D-allose. D-Allose has no inhibitory effect of the increment of plasma glucose concentration in all of cases. These results suggest that D-allose is an ineffective monosaccharide on glycemic response after carbohydrate ingestion.

Only D-allose, among various rare monosaccharides tested, induced resistance to Xanthomonas oryzae pv. oryzae in susceptible rice leaves with defence responses: reactive oxygen species, lesion mimic formation, and PR-protein gene expression. These responses were suppressed by ascorbic acid or diphenylene iodonium. Transgenic rice plants overexpressing OsrbohC, encoding NADPH oxidase, were enhanced in sensitivity to D-allose. D-Allose-mediated defence responses were suppressed by the presence of a hexokinase inhibitor. 6-Deoxy-D-allose, a structural derivative of D-allose unable to be phosphorylated, did not confer resistance. Transgenic rice plants expressing Escherichia coli AlsK encoding D-allose kinase to increase D-allose 6-phosphate synthesis were more sensitive to D-allose, but E. coli AlsI encoding D-allose 6-phosphate isomerase expression to decrease D-allose 6-phosphate reduced sensitivity. A D-glucose 6-phosphate dehydrogenase-defective mutant was also less sensitive, and OsG6PDH1 complementation restored full sensitivity. These results reveal that a monosaccharide, D-allose, induces rice resistance to X. oryzae pv. oryzae by activating NADPH oxidase through the activity of D-glucose 6-phosphate dehydrogenase, initiated by hexokinase-mediated conversion of D-allose to D-allose 6-phosphate, and treatment with D-allose might prove to be useful for reducing disease development in rice.

BackgroundPretransplant restoration of islets damaged during isolation remains to be solved. In this study, we examined the effect of D‐allose on islets isolated from rat pancreata prior to islet transplantation., MethodsRat islets isolated from fresh pancreata were cultured overnight in Roswell Park Memorial Institute 1640 solution in the absence (group 1) or presence (group 2) of D‐allose. Then we assessed stimulation index of insulin, and cure rate after islet transplantation to diabetic nude mice. We also measured malondialdehyde level and caspase 3 activity of islets after the overnight culture for assessment of the oxidative stress and the apoptosis., ResultsD‐allose significantly improved insulin secretion of islets. The stimulation index in group 2 was significantly higher than in group 1. Cure rate after transplantation in group 2 was higher than in group 1 especially in the first week. The malondialdehyde level in group 2 was significantly lower than in group 1. But the caspase 3 activities in both groups did not differ., ConclusionsD‐allose treatment of isolated islet culture prior to transplantation restored islet function and increased successful transplant rate. The results of this study suggested that D‐allose improved function of damaged islets through its anti‐oxidative activity.

D-Allose is classified as a 'rare sugar,' i.e., part of the group of monosaccharides that are present in low quantities in the natural world. D-Allose has been demonstrated to exert many physiological functions. The effects of the rare sugars on immune responses are largely unexplored. Here, we investigated the physiological effects of D-allose on murine dendritic cells' cytokine production. When plasmacytoid dendritic cells (pDCs) were stimulated with a Toll-like receptor 7 (TLR7) ligand, a single-stranded RNA (ssRNA), or a TLR9 ligand, CpG DNA, in the medium containing D-allose, the productions of both interferon-alpha (IFN-α) and interleukin (IL)-12p40 were severely decreased. In contrast, a normal production of these cytokines was observed when pDCs were stimulated with other TLR7 ligands, an imidazoquinoline, or a guanosine analog. In contrast to the pDCs, conventional dendritic cells (cDCs) produced IL-12p40 and tumor necrosis factor-alpha (TNF-α) in response to an imidazoquinoline or CpG DNA even though D-allose was present in the medium. D-Allose did not induce pDC death, and not inhibit the endocytic uptake of fluorophore-labeled CpG DNA into pDCs. These results suggested that D-allose exerts its inhibitory effects after CpG DNA is internalized. We analyzed the TLR7/9 signal-induced activation of downstream signaling molecules in pDCs and observed that when pDCs were stimulated with a ssRNA or CpG DNA, the phosphorylation status of the MAPK family, which includes Erk1/2, JNK/SAPK, and p38 MAPK, was attenuated in the presence of D-allose compared to D-glucose controls. The stimulation of pDCs with an imidazoquinoline induced a strong phosphorylation of these MAPK family members even in the presence of D-allose. These findings reveal that D-allose can inhibit the cytokine production by pDCs stimulated with ssRNA or CpG DNA via an attenuation of the phosphorylation of MAPK family members.

D-allose is a rare sugar that has been reported to up-regulate thioredoxin-interacting protein (TXNIP) expression and affect the production of intracellular reactive oxygen species (ROS). However, the antitumor effect of D-allose is unknown. This study aimed to determine whether orally administered D-allose could be a candidate drug against bladder cancer (BC). To this end, BC cell lines were treated with varying concentrations of D-allose (10, 25, and 50 mM). Cell viability and intracellular ROS levels were assessed using cell viability assay and flow cytometry. TXNIP expression was evaluated using Western blotting. The antitumor effect of orally administered D-allose was assessed using a xenograft mouse model. D-allose reduced cell viability and induced intracellular ROS production in BC cells. Moreover, D-allose stimulated TXNIP expression in a dose-dependent manner. Co-treatment of D-allose and the antioxidant L-glutathione canceled the D-allose-induced reduction in cell viability and intracellular ROS elevation. Furthermore, oral administration of D-allose inhibited tumor growth without adverse effects (p < 0.05). Histopathological findings in tumor tissues showed that D-allose decreased the nuclear fission rate from 4.1 to 1.1% (p = 0.004). Oral administration of D-allose suppressed BC growth in a preclinical mouse model, possibly through up-regulation of TXNIP expression followed by an increase in intracellular ROS. Therefore, D-allose is a potential therapeutic compound for the treatment of BC.

BackgroundThe present study investigates the effects of d-allose, a rare sugar, on the inflammatory response after transient forebrain ischemia in the gerbil and whether it reduces oxidative stress (8-hydroxyl-2′-deoxyguanosine levels) and behavioral deficits., MethodsTransient forebrain ischemia was induced by occlusion of the bilateral common carotid arteries for 5 minutes. d-Allose was intraperitoneally injected immediately after ischemia (400 mg/kg). Inflammatory cytokines and oxidative damage in the hippocampus and behavioral deficits were examined 3 days after ischemia., Resultsd-Allose administration reduced ischemia-induced cytokine production, oxidative stress, and behavioral deficits (motor and memory related)., ConclusionsThe present results suggest that d-allose reduces brain injury after transient global ischemia by suppressing inflammation as well as by inhibiting oxidative stress.

Glucose is a major energy source for mammalian cells and is transported into cells via cell-specific expression of various glucose transporters (GLUTs). Especially, cancer cells require massive amounts of glucose as an energy source for their dysregulated growth and thus over-express GLUTs. d-allose, a C-3 epimer of d-glucose, is one of rare sugars that exist in small quantities in nature. We have shown that d-allose induces the tumor suppressor gene coding for thioredoxin interacting protein (TXNIP) and inhibits cancer cell growth by G1 cell cycle arrest. It has also been reported that GLUTs including GLUT1 are over-expressed in many cancer cell lines, which may contribute to larger glucose utilization. Since d-allose suppresses the growth of cancer cells through the upregulation of TXNIP expression, our present study focused on whether d-allose down-regulates GLUT1 expression via TXNIP expression and thus suppresses cancer cell growth. Western blot and real-time PCR analyses revealed that d-allose significantly induced TXNIP expression and inhibited GLUT1 expression in a dose-dependent manner in three human cancer cell lines: hepatocellular carcinoma (HuH-7), Caucasian breast adenocarcinoma (MDA-MB-231), and neuroblastoma (SH-SY5Y). In these cell lines, d-allose treatment inhibited cell growth. Importantly, d-allose treatment decreased glucose uptake, as measured by the uptake of 2-deoxy d-glucose. Moreover, the reporter assays showed that d-allose decreased the expression of luciferase through the hypoxia response element present in the tested promoter region. These results suggest that d-allose may cause the inhibition of cancer growth by reducing both GLUT1 expression and glucose uptake.