2-Deoxy-D-ribose inhibits hypoxia-induced apoptosis by suppressing the phosphorylation of p38 MAPK

An angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, partially prevented hypoxia-induced apoptosis. 2-Deoxy-d-ribose inhibited hypoxia-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not c-jun NH(2)-terminal kinase/stress-activated protein kinase in human leukemia HL-60 cells. 2-Deoxy-d-ribose also suppressed the levels of Bax attached to mitochondria under hypoxic conditions. SB203580, a specific inhibitor of the p38 MAPK, suppressed the hypoxia-induced apoptosis of HL-60 cells. These findings suggest that one of the molecular bases for resistance to hypoxia-induced apoptosis conferred by 2-deoxy-d-ribose is the inhibition of the p38 signaling pathway. The expression levels of TP are elevated in many malignant solid tumors and thus the 2-deoxy-d-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.     

Efficient synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) from L-arabinose

An efficient and practical route for the large-scale synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) starting from L-arabinose was developed using Barton-type free-radical deoxygenation reaction as a key step. The radical precursor, a phenoxythiocarbonyl ester, was prepared in situ, and the most efficient deoxygenation was achieved by slow addition of tributyltin hydride to the reaction mixture.     

Immunohistochemical localization of D-alanine to beta-cells in rat pancreas

A mouse monoclonal antibody against D-alanine (D-Ala) has been raised and the immunohistochemical localization of this D-amino acids in the rat pancreas is visualized. The obtained anti-D-Ala monoclonal antibody has no significant cross-reactivity to all proteinogenic L-amino acids and their D-enantiomers. Using this antibody, immunohistochemical staining was performed on the pancreas, and specific staining for d-Ala has been observed only in the Langerhans islets. To identify the types of D-Ala-immunopositive cells, double staining was carried out with antibodies against D-Ala and pancreatic hormones. Similar immunostaining patterns have been observed for D-Ala and insulin, while D-Ala is hardly co-localized with other hormones (glucagon, somatostatin, and pancreatic polypeptide). These results indicate for the first time that D-Ala is localized to insulin producing beta-cells in mammalian pancreas, suggesting that this D-amino acid would be involved in the regulation of the blood glucose level.     

Metal-ion interactions with sugars. Crystal structures and FT-IR studies of the LaCl3-ribopyranose and CeCl3-ribopyranose complexes

Single crystals of LaCl3.C5H10O5.5H2O (1) and CeCl3.C5H10O5.5H2O (2) were obtained from ethanol-water solutions and their structures determined by X-ray. The two complexes are isomorphous. Two configurations of complex 1 or complex 2, as a pair of isomers, were found in each single crystal in a disordered state. The ligand of one of the isomer is alpha-D-ribopyranose in the 4C1 conformation, the ligand of the other is beta-D-ribopyranose in the 1C4 conformation. For complex 1, the alpha:beta anomeric ratio is 51:49, and for complex 2, the ratio is 52:48. Both ligands of the two isomers provide three hydroxyl groups in ax-eq-ax orientation for coordination. The Ln3+ (Ln = La or Ce) ion is nine-coordinated with five Ln-O bonds from water molecules, three Ln-O bonds from hydroxyl groups of the D-ribopyranose, and one Ln-Cl bond from chloride ion. The hydroxyl groups, water molecules, and chloride ions form an extensive hydrogen-bond network. The IR spectral C-C, O-H, C-O, and C-O-H vibrations were observed to be shifted in both the two complexes and the IR results are in accord with those of X-ray diffraction.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

Thymidine phosphorylase inhibits the expression of proapoptotic protein BNIP3

An angiogenic factor, thymidine phosphorylase (TP), confers resistance to apoptosis induced by hypoxia. We investigated the molecular basis for the suppressive effect of TP on hypoxia-induced apoptosis using Jurkat cells transfected with TP cDNA, Jurkat/TP, and a mock transfectant, Jurkat/CV. TP and 2-deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, suppressed hypoxia-induced apoptosis. They also inhibited the upregulation of hypoxia-inducible factor (HIF) 1α and the proapoptotic factor, BNIP3, and caspase 3 activation induced by hypoxia. Introduction of siRNA against BNIP3 in Jurkat cells decreased the proportion of apoptotic cells under hypoxic condition. These findings suggest that the suppression of BNIP3 expression by TP prevents, at least in part, hypoxia-induced apoptosis. Expression levels of TP are elevated in many malignant solid tumors and thus 2-deoxy-d-ribose generated by TP in these tumors might play an important role in tumor progression by preventing hypoxia-induced apoptosis.     

Enzymatic synthesis of D-glucosaminic acid from D-glucosamine

D-glucosaminic acid (2-amino-2-deoxy-D-gluconic acid), a component of bacterial lipopolysaccharides and a chiral synthon, is easily prepared on a multigram scale by air oxidation of D-glucosamine (2-amino-2-deoxy-D-glucose) catalysed by glucose oxidase.     

A single membrane-bound enzyme catalyzes the conversion of 2,5-diketo-d-gluconate to 4-keto-d-arabonate in d-glucose oxidative fermentation by Gluconobacter oxydans NBRC 3292

4-Keto-d-arabonate synthase (4KAS), which converts 2,5-diketo-d-gluconate (DKGA) to 4-keto-d-arabonate (4KA) in d-glucose oxidative fermentation by some acetic acid bacteria, was solubilized from the Gluconobacter oxydans NBRC 3292 cytoplasmic membrane, and purified in an electrophoretically homogenous state. A single membrane-bound enzyme was found to catalyze the conversion from DKGA to 4KA. The 92-kDa 4KAS was a homodimeric protein not requiring O₂ or a cofactor for the conversion, but was stimulated by Mn²⁺. N-terminal amino acid sequencing of 4KAS, followed by gene homology search indicated a 1,197-bp open reading frame (ORF), corresponding to the GLS_c04240 locus, GenBank accession No. CP004373, encoding a 398-amino acid protein with a calculated molecular weight of 42,818 Da. An Escherichia coli transformant with the 4kas plasmid exhibited 4KAS activity. Furthermore, overexpressed recombinant 4KAS was purified in an electrophoretically homogenous state and had the same molecular size as the natural enzyme.     

X-ray structure of 1,3,4-tri-O-acetyl-2-deoxy-β-d-erythro-pentopyranose

Peracetylated 2-deoxy-d-erythro-pentose (2-deoxy-d-ribose) was synthesized through the acetylation of 2-deoxy-d-ribose with acetic anhydride in pyridine, and the products (including all four ring forms) exist in form of either a white solid or a syrup. A single crystal of 1,3,4-tri-O-acetyl-2-deoxy-β-d-erythro-pentopyranose was obtained from the syrup and its structure was determined by X-ray diffraction. The crystal adopts the ¹C₄ conformation, presenting an orthorhombic system, space group P2₁2₁2₁ with Z = 4, unit cell dimensions a = 7.2274 (3) Å, b = 8.0938 (5) Å, and c = 22.0517 (11) Å.     

Melting behaviour of D-sucrose, D-glucose and D-fructose

The melting behaviour of d-sucrose, d-glucose and d-fructose was studied. The melting peaks were determined with DSC and the start of decomposition was studied with TG at different rates of heating. In addition, melting points were determined with a melting point apparatus. The samples were identified as d-sucrose, alpha-d-glucopyranose and beta-d-fructopyranose by powder diffraction measurements. There were differences in melting between the different samples of the same sugar and the rate of heating had a remarkable effect on the melting behaviour. For example, T(o), DeltaH(f) and T(i) (initial temperature of decomposition) at a 1 degrees Cmin(-1) rate of heating were 184.5 degrees C, 126.6Jg(-1) and 171.3 degrees C for d-sucrose, 146.5 degrees C, 185.4Jg(-1) and 152.0 degrees C for d-glucose and 112.7 degrees C, 154.1Jg(-1) and 113.9 degrees C for d-fructose. The same parameters at 10 degrees Cmin(-1) rate of heating were 188.9 degrees C, 134.4Jg(-1) and 189.2 degrees C for d-sucrose, 155.2 degrees C, 194.3Jg(-1) and 170.3 degrees C for d-glucose and 125.7 degrees C, 176.7Jg(-1) and 136.8 degrees C d-fructose. At slow rates of heating, there were substantial differences between the different samples of the same sugar. The melting point determination is a sensitive method for the characterization of crystal quality but it cannot be used alone for the identification of sugar samples in all cases. Therefore, the melting point method should be validated for different sugars.     

Chemical synthesis of cholesteryl beta-D-galactofuranoside and -pyranoside

Two isomeric cholesteryl galactosides, cholesteryl beta-D-galactofuranoside and -pyranoside, have been synthesized by the Koenigs-Knorr reaction. Glycosylation of cholesterol with 2,3,5,6-tetra-O-benzoyl-D-galactofuranosyl bromide, followed by Zemplén saponification with sodium methoxide, gave cholesteryl beta-D-galactofuranoside. By using 2,3,4,6-tetra-O-acetyl-D-galactopyranosyl bromide as the glycosyl donor, followed by alkaline hydrolysis, cholesteryl beta-D-galactopyranoside was obtained. The title compounds were characterized by their IR spectra and by their (1)H and (13)C NMR spectra. Structure considerations of the two cholesteryl galactosides correlated with data in the literature, thus confirming that cholesteryl beta-D-galactopyranoside is an antigenic lipid of Lyme disease agent, Borrelia burgdorferi.     

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.     

Total synthesis and identification of two diastereoisomers of 1-methyl-2-[N-(p-tolylsulfonyl)-2-pyrrolidinyl]ethyl beta-L-fucopyranoside

The reaction of a racemic mixture of (2R,2'S)- and (2S,2'R)-N-(p-tolylsulfonyl)-2-pyrrolidinyl-2-propanol, prepared from (S)-proline, with 2,3,4-tri-O-acetyl-alpha-L-fucopyranosyl trichloroacetimidate led to both diastereoisomers of the title compound after O-deacetylation.     

d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

Comparison between D-[3-3H]- and D-[5-3H]glucose and fructose utilization in pancreatic islets from control and hereditarily diabetic rats

The metabolism of D-glucose and/or D-fructose was investigated in pancreatic islets from control rats and hereditarily diabetic GK rats. In the case of both D-glucose and D-fructose metabolism, a preferential alteration of oxidative events was observed in islets from GK rats. The generation of 3HOH from D-[5-3H]glucose (or D-[5-3H]fructose) exceeded that from D-[3-3H]glucose (or D-[3-3H]fructose) in both control and GK rats. This difference, which is possibly attributable to a partial escape from glycolysis of tritiated dihydroxyacetone phosphate, was accentuated whenever the rate of glycolysis was decreased, e.g., in the absence of extracellular Ca(2+) or presence of exogenous D-glyceraldehyde. D-Mannoheptulose, which inhibited D-glucose metabolism, exerted only limited effects upon D-fructose metabolism. In the presence of both hexoses, the paired ratio between D-[U-14C]fructose oxidation and D-[3-3H]fructose or D-[5-3H]fructose utilization was considerably increased, this being probably attributable, in part at least, to a preferential stimulation by the aldohexose of mitochondrial oxidative events. Moreover, this coincided with the fact that D-mannoheptulose now severely inhibited the catabolism of D-[5-3H]fructose and D-[U-14C]fructose. The latter situation is consistent with both the knowledge that D-glucose augments D-fructose phosphorylation by glucokinase and the findings that D-mannoheptulose, which fails to affect D-fructose phosphorylation by fructokinase, inhibits the phosphorylation of D-fructose by glucokinase.     

Production of l-allose and d-talose from l-psicose and d-tagatose by l-ribose isomerase

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.     

The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc1 complexes

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a_s-type with reduction potentials of + 200, + 400 and + 160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a_s, and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be + 320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane α-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc₁ complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc₁ complexes of bacteria and mitochondria, however with distinct subunits and heme types.     

Simultaneous measurement of D-serine dehydratase and d-amino acid oxidase activities by the detection of 2-oxo-acid formation with reverse-phase high-performance liquid chromatography

N-methyl-D-aspartate receptors (NMDARs) play critical roles in excitatory synaptic transmission in the vertebrate central nervous system. NMDARs need D-serine for their channel activities in various brain regions. In mammalian brains, D-serine is produced from L-serine by serine racemase and degraded by D-amino acid oxidase (DAO) to 3-hydroxypyruvate. In avian organs, such as the kidney, in addition to DAO, D-serine is also degraded to pyruvate by D-serine dehydratase (DSD). To examine the roles of these two enzymes in avian brains, we developed a method to simultaneously measure DAO and DSD activities. First, the keto acids produced from D-serine were derivatized with 3-methyl-2-benzothiazolinone hydrazone to stable azines. Second, the azine derivatives were quantified by means of reverse-phase high-performance liquid chromatography using 2-oxoglutarate as an internal standard. This method allowed the simultaneous detection of DAO and DSD activities as low as 100 pmol/min/mg protein. Chicken brain showed only DSD activities (0.4+/-0.2 nmol/min/mg protein) whereas rat brain exhibited only DAO activities (0.7+/-0.1 nmol/min/mg protein). This result strongly suggests that DSD plays the same role in avian brains, as DAO plays in mammalian brains. The present method is applicable to other keto acids producing enzymes with minor modifications.     

Enzymatic synthesis of tyrosol and hydroxytyrosol β-d-fructofuranosides

Tyrosol β-d-fructofuranoside and hydroxytyrosol β-d-fructofuranoside have been synthesized as new compounds in 27.6 and 19.5% respective yields through transfructosylation of tyrosol and hydroxytyrosol. Yeast β-galactosidase Lactozym 3000?L comprising invertase activity was used as catalyst. Besides the main monofructosides, an equimolar mixture of tyrosol β-d-fructofuranosyl-((2→1)-β-d-fructofuranoside and tyrosol β-d-fructofuranosyl-(2→6)-β-d-fructofuranoside was isolated as additional product fraction in 14.3% yield.     

Photoinduced electron-transfer alpha-deoxygenation of aldonolactones. Efficient synthesis of 2-deoxy-D-arabino-hexono-1,4-lactone

A photoinduced electron-transfer (PET) reaction was used for the deoxygenation at C-2 of aldonolactones derivatized as 2-O-[3-(trifluoromethyl)benzoyl] or benzoyl esters. By irradiation of different D-galactono- and D-glucono-1,4-derivatives, with a 450W lamp, using 9-methylcarbazole as photosensitizer, the corresponding 2-deoxy-D-lyxo- and 2-deoxy-D-arabino-hexono-1,4-lactones were efficiently obtained.