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.     

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.     

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.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

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.     

Reaction on D-glucal by an inverting phosphorylase to synthesize derivatives of 2-deoxy-beta-D-arabino-hexopyranosyl-(1-->4)-D-glucose (2II-deoxycellobiose)

Four derivatives of 2(II)-deoxycellobiose were synthesized from d-glucal and acceptor sugars (d-glucose, d-xylose, d-mannose, and 2-deoxy-d-arabino-hexose) using a cellobiose phosphorylase from Cellvibrio gilvus. The enzyme was found to be an effective catalyst to synthesize the beta-(1-->4) linkage of 2-deoxy-d-arabino-hexopyranoside. The acceptor specificity for the d-glucal reaction was identical to that for the alpha-d-glucose 1-phosphate reaction, but the activity of d-glucal was approximately 500 times less than that of alpha-d-glucose 1-phosphate, using 10mM substrates.     

Efficient synthesis of 5-thio-D-arabinopyranose and 5-thio-D-xylopyranose from the corresponding D-pentono-1,4-lactones

5-Thio-D-arabinopyranose (5) and 5-thio-D-xylopyranose (10) were synthesized from the corresponding D-pentono-1,4-lactones. After regioselective bromination at C-5, transformation into 5-S-acetyl-5-thio derivatives, reduction into lactols and deprotection afforded the title compounds in 49 and 42% overall yield, respectively.     

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.     

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.     

Molecular characterization of D-amino acid oxidase from common carp Cyprinus carpio and its induction with exogenous free D-alanine

A full-length cDNA encoding D-amino acid oxidase (DAO, EC 1.4.3.3) was cloned and sequenced from the hepatopancreas of carp fed a diet supplemented with D-alanine. This clone contained an open reading frame encoding 347 amino acid residues. The deduced amino acid sequence exhibited about 60 and 19-29% identity to mammalian and microbial DAOs, respectively. The expression of full-length carp DAO cDNA in Escherichia coli resulted in a significant level of protein with DAO activity. In carp fed the diet with D-alanine for 14 days, DAO mRNA was strongly expressed in intestine followed by hepatopancreas and kidney, but not in muscle. During D-alanine administration, DAO gene was expressed quickly in hepatopancreas with the increase of DAO activity. The inducible nature of carp DAO indicates that it plays an important physiological role in metabolizing exogenous D-alanine that is abundant in their prey invertebrates, crustaceans, and mollusks.     

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.     

Crystal structures of the complexes between vancomycin and cell-wall precursor analogs

The crystal structures of three vancomycin complexes with two vancomycin-sensitive cell-wall precursor analogs (diacetyl-Lys-D-Ala-D-Ala and acetyl-D-Ala-D-Ala) and a vancomycin-resistant cell-wall precursor analog (diacetyl-Lys-D-Ala-D-lactate) were determined at atomic resolutions of 1.80 A, 1.07 A, and 0.93 A, respectively. These structures not only reconfirm the "back-to-back" dimerization of vancomycin monomers and the ligand-binding scheme proposed by previous experiments but also show important structural features of strategies for the generation of new glycopeptide antibiotics. These structural features involve a water-mediated antibiotic-ligand interaction and supramolecular structures such as "side-by-side" arranged dimer-to-dimer structures, in addition to ligand-mediated and "face-to-face" arranged dimer-to-dimer structures. In the diacetyl-Lys-D-Ala-D-lactate complex, the interatomic O...O distance between the carbonyl oxygen of the fourth residue of the antibiotic backbone and the ester oxygen of the D-lactate moiety of the ligand is clearly longer than the corresponding N-H...O hydrogen-bonding distance observed in the two other complexes due to electrostatic repulsion. In addition, two neighboring hydrogen bonds are concomitantly lengthened. These observations provide, at least in part, a molecular basis for the reduced antibacterial activity of vancomycin toward vancomycin-resistant bacteria with cell-wall precursors terminating in -D-Ala-D-lactate.     

Copper-induced oxidation of epinephrine: protective effect of D-DAHK,a synthetic analogue of the high affinity copper binding site of human albumin

Epinephrine is known to be rapidly oxidized during sepsis. Ischemia and acidosis, which often accompany sepsis, are associated with the release of weakly bound cupric ions from plasma proteins. We investigated whether copper promotes oxidation of epinephrine at both physiological and acidic pH and whether D-Asp-D-Ala-D-His-D-Lys (D-DAHK), a human albumin (HSA) N-terminus synthetic peptide with a high affinity for cupric ions, attenuates this oxidation. Epinephrine alone [100 microM] or with CuCl(2) [10 microM], and with CuCl(2) [10 microM] and D-DAHK [20 microM] at pH 7.4, 7.0, 6.5, and 6.0 were incubated for 1h at 37 degrees C. Epinephrine oxidation was measured by the spectrophotometric quantification of its oxidation product, adrenochrome. We found that adrenochrome increased, suggesting copper-induced oxidation of epinephrine. At pH 7.4, 7.0, 6.5, and 6.0, adrenochrome increased by 47%, 53%, 24%, and 6% above baseline, respectively. D-DAHK attenuated the copper-induced oxidation of epinephrine to baseline levels. These in vitro results indicate that copper-induced epinephrine oxidation is greatest at the physiological pH 7.4 as well as in severe acidosis, pH 7.0, and that D-DAHK completely inhibits this oxidation.     

Synthesis of 4-cyano and 4-nitrophenyl 1,6-dithio-D-manno-, L-ido- and D-glucoseptanosides possessing antithrombotic activity

1,6-Anhydro-3,4-O-isopropylidene-1-thio-D-mannitol was converted into its sulfoxide which after hydrolysis, acetylation and subsequent Pummerer rearrangement gave the penta-O-acetyl-1-thio-D-mannoseptanose anomers in excellent yield. This anomeric mixture was used as donor for the glycosylation of 4-nitro- and 4-cyanobenzenethiol in the presence of boron trifluoride etherate and trimethylsilyl triflate, respectively, to yield the corresponding thioseptanosides in high yield. The same strategy was applied for the synthesis of the corresponding L-idothioseptanosides using 1,6-anhydro-3,4-O-isopropylidene-1-thio-L-iditol as starting material. The penta-O-acetyl-D-glucothioseptanose donors could not be synthesised the same way, as the Pummerer reaction of the corresponding tetra-O-acetyl-1,6-thioanhydro-1-thio-D-glucitol sulfoxides led to an inseparable mixture of the corresponding L-gulo- and D-glucothioseptanose anomers. Therefore, D-glucose diethyl dithioacetal was converted via its 2,3,4,5-tetra-O-acetyl-6-S-acetyl derivative into an anomeric mixture of its 6-thio-septanose and -furanose peracetates which could be separated by column chromatography. Condensation of the 6-thio-glucoseptanose peracetates with 4-cyano- and 4-nitrobenezenethiol in the presence of boron trifluoride etherate afforded anomeric mixtures of the corresponding thioseptanosides. The D-manno-, L-ido- and D-glucothioseptanosides obtained after Zemplén deacetylation of these mixtures were tested for their oral antithrombotic activity.     

A specific enzymatic high-performance liquid chromatography method to determine N-methyl-D-aspartic acid in biological tissues

Recently we demonstrated that N-methyl-D-aspartic acid (NMDA) is present as an endogenous compound in the nervous tissues and endocrine glands of the rat where it plays a role in the regulation of the luteinizing hormone, growth hormone, and prolactin (FASEB J. 14 (2000) 699; Endocrinology 141 (2000) 3861). Based on the prediction that NMDA could have future importance in neuroendocrinology, we have devised an improved method for the specific and routine determination of NMDA in biological tissue. This method is based on the detection by HPLC of methylamine (CH(3)NH(2)) which comes from the oxidation of NMDA by D-aspartate oxidase, an enzyme which specifically oxidizes NMDA, yielding CH(3)NH(2) as one of the oxidative products of the reaction. The sensitivity of the method permits the accurate determination of NMDA in the supernatant of a tissue homogenate at levels of about 5-10 picomol/assay. However, for those tissues in which the concentration of NMDA is less than 1nmol/g, the sample must be further purified by treatment with o-phthaldialdehyde in order to separate the NMDA from the other amino acids and amino compounds and then concentrated and analyzed by HPLC. Using this method we have conducted a comparative study in order to measure the amount of NMDA in neuroendocrine and other tissues of various animal phyla from mollusks to mammals.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

Enzymatic synthesis of D-glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) and NMR analysis of its isomeric forms

D-Glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) was prepared from D-glucosone (D-arabino-hexos-2-ulose) by enzymatic conversion with hexokinase. The isomeric composition of D-glucosone 6-phosphate in aqueous solution was quantitatively determined by NMR spectroscopy and compared to D-glucosone. The main isomers are the alpha-anomer (58%) and the beta-anomer (28%) of the hydrated pyranose form, and the beta-D-fructofuranose form (14%).     

An improved synthesis of 5-thio-D-ribose from D-ribono-1,4-lactone

5-Thio-D-ribopyranose was synthesized from D-ribono-1,4-lactone (1) by two approaches: (i) 5-bromo-5-deoxy-D-ribono-1,4-lactone (2) was successively transformed into 5-bromo-5-deoxy, 5-S-acetyl-5-thio or 5-thiocyanato-D-ribofuranose derivatives; appropriate treatment then lead to 5-thio-D-ribopyranose (7) in 46-48% overall yield and; (ii) 2 was transformed into the 5-S-acetyl-5-thio-D-ribono-1,4-lactone derivative (11). Reduction and deprotection of 11 afforded 5-thio-D-ribopyranose (7) in 57% overall yield.     

Synthesis of 4-O-glycosylated 1,5-anhydro-d-fructose and of 1,5-anhydro-d-tagatose from a common intermediate 2,3-O-isopropylidene-d-fructose

Four novel disaccharides of glycosylated 1,5-anhydro-d-ketoses have been prepared: 1,5-anhydro-4-O-β-d-glucopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-galactopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-glucopyranosyl-d-tagatose, and 1,5-anhydro-4-O-β-d-galactopyranosyl-d-tagatose. The common intermediate, 1,5-anhydro-2,3-O-isopropylidene-β-d-fructopyranose, was prepared from d-fructose and was converted into the d-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The anhydroketoses thus prepared were glycosylated and deprotected to give the disaccharides.