Six New Methyl Apiofuranosides from the Bark of Phellodendron chinense Schneid and Their Inhibitory Effects on Nitric Oxide Production.

A chemical investigation on 70% EtOH extract from the bark of Phellodendron chinense Schneid (Rutaceae) led to six new methyl apiofuranosides (1-6), and ten known compounds (7-16). All these compounds were characterized by the basic analysis of the spectroscopic data including extensive 1D-, 2D-NMR (HSQC, HMBC), and high-resolution mass spectrometry, and the absolute configurations were determined by both empirical approaches and NOESY. Inhibitory effects of compounds 1-9 and 11-16 on nitric oxide production were investigated in lipopolysaccharide (LPS)-mediated RAW 264.7 cells, as a result, most of these isolates inhibited nitric oxide (NO) release, and among them 9, 11, and 12 displayed the strongest inhibition on NO release at the concentration of 12.5 µM.

Synthesis and Antiviral Activity Evaluation of 2',5',5'-Trifluoro-Apiosyl Nucleoside Phosphonic Acid Analogs.

Racemic synthesis of novel 2',5',5'-trifluoro-apiose nucleoside phosphonic acid analogs were performed as potent antiviral agents. Phosphonation was performed by direct displacement of triflate intermediate with diethyl (lithiodifluoromethyl) phosphonate to give the corresponding (α,α-difluoroalkyl) phosphonate. Condensation successfully proceeded from a glycosyl donor with persilylated bases to yield the nucleoside phosphonate analogs. Deprotection of diethyl phosphonates provided the target nucleoside analogs. An antiviral evaluation of the synthesized compounds against various viruses such as HIV, HSV-1, HSV-2, and HCMV revealed that the pyrimidine analogues have significant anti-HCMV activity.

Selenofuranoside Ameliorates Memory Loss in Alzheimer-Like Sporadic Dementia: AChE Activity, Oxidative Stress, and Inflammation Involvement.

Alzheimer's disease (AD) is becoming more common due to the increase in life expectancy. This study evaluated the effect of selenofuranoside (Se) in an Alzheimer-like sporadic dementia animal model. Male mice were divided into 4 groups: control, Aß, Se, and Aß + Se. Single administration of Aß peptide (fragments 25-35; 3 nmol/3 µL) or distilled water was administered via intracerebroventricular (i.c.v.) injection. Selenofuranoside (5 mg/kg) or vehicle (canola oil) was administered orally 30 min before Aß and for 7 subsequent days. Memory was tested through the Morris water maze (MWM) and step-down passive-avoidance (SDPA) tests. Antioxidant defenses along with reactive species (RS) were assessed. Inflammatory cytokines levels and AChE activity were measured. SOD activity was inhibited in the Aß group whereas RS were increased. AChE activity, GSH, and IL-6 levels were increased in the Aß group. These changes were reflected in impaired cognition and memory loss, observed in both behavioral tests. Se compound was able to protect against memory loss in mice in both behavioral tests. SOD and AChE activities as well as RS and IL-6 levels were also protected by Se administration. Therefore, Se is promising for further studies.

Cadmium inhibits the ovary δ-aminolevulinate dehydratase activity in vitro and ex vivo: protective role of seleno-furanoside.

Cadmium (Cd) toxicity is a concern to the tobacco-smoking sub-population which includes millions of people worldwide. Although this metal may cause severe damage to embryos and the reproductive organs, the precise mechanisms underlying its toxicity remain unclear. In the present study, the Cd effect on ovary δ-aminolevulinate dehydratase (δ-ALA-D) activity was investigated in vitro and ex vivo. We observed that low concentrations of Cd inhibited cow ovary δ-ALA-D activity in vitro and the IC50 value obtained was 19.17 µM. Furthermore, the protective effect of a novel organic selenium compound (seleno-furanoside) in restoring enzyme activity was evaluated. Seleno-furanoside (10, 50, 100, 200, 400 and 1000 µM) did not reverse the Cd toxicity in bovine ovarian tissue in vitro. According to the in vitro reults, acute Cd exposure (2.5 and 5 mg kg(-1)) caused a significant inhibition in ovary δ-ALA-D activity in mice (around 27% and 34%, respectively). Therapy with seleno-furanoside (100 µmol kg(-1)) was able to restore enzyme activity. Thus, we demonstrated for the first time that δ-ALA-D activity from ovary is inhibited by Cd both in vitro and ex vivo. Additionally, seleno-furanoside therapy was effective in restoring ovarian enzyme activity inhibited by Cd exposure in mice, but it did not reverse the in vitro metal effect. This study detected a new toxicity marker of Cd toxicity on ovarian tissue as well as the beneficial effect of a new compound to manage the metal effect after acute exposure.

Alcohol-, diol-, and carbohydrate-substituted indenoisoquinolines as topoisomerase I inhibitors: investigating the relationships involving stereochemistry, hydrogen bonding, and biological activity.

The DNA-relaxing enzyme topoisomerase I (Top1) can be inhibited by heterocyclic compounds such as indolocarbazoles and indenoisoquinolines. Carbohydrate and hydroxyl-containing side chains are essential for the biological activity of indolocarbazoles. The current study investigated how similar functionalities could be "translated" to the indenoisoquinoline system and how stereochemistry and hydrogen bonding affect biological activity. Herein is described the preparation and assay of indenoisoquinolines substituted with short-chain alcohols, diols, and carbohydrates. Several compounds (including those derived from sugars) display potent Top1 poisoning and antiproliferative activities. The Top1 poisoning activity of diol-substituted indenoisoquinolines is dependent upon stereochemistry. Although the effect is striking, molecular modeling and docking studies do not indicate any reason for the difference in activity due to similar calculated interactions between the ligand and Top1-DNA complex and ambiguity about the binding mode. A stereochemical dependence was also observed for carbohydrate-derived indenoisoquinolines. Although similar trends were observed in other classes of Top1 inhibitors, the exact nature of this effect has yet to be elucidated.

Production of surfactin using pentose carbohydrate by Bacillus subtilis.

Interest in microbial surfactants has been steadily increasing in recent years due to their diversity, mass production possibility, selectivity, performance under extreme conditions and potential applications in environmental protection. In this study two pentose sugars (xylose and arabinose) were investigated for the submerged fermentation (SmF) of Bacillus subtilis in surfactant production medium for bio-surfactant surfactin production. An excellent vegetative growth of B. subtilis (× 10(10) CFU/mL) was observed for xylose and arabinose containing medium which were comparable to glucose supplemented medium. Low growth (× 10(8) CFU/mL) was found when medium was not supplemented with any of the sugars. Surfactin production in xylose, arabinose and glucose containing medium was 2700, 2600 and 2000 mg/L, respectively, whereas, medium without any sugar showed low surfactin (700 mg/L) production. These results clearly indicate the effect of pentose sugars on production of surfactin. Gradual depletion of the xylose and arabinose were confirmed by HPLC analysis during the growth phase of the strain that ultimately produced the surfactin.

Synthetic UDP-furanoses inhibit the growth of the parasite Leishmania.

The chemical synthesis of UDP-6-NHAc-6-deoxy-Galf was performed and it led to the isolation of both pure anomers. They were then evaluated together with the previously prepared UDP-furanoses for their anti-parasitic properties against Leishmania donovani promastigotes, one of the agents responsible for visceral leishmaniasis. Amongst them, the unnatural 1,2-trans UDP-6-NHAc-Galf demonstrated a high potency in inhibiting the growth of the parasite.

D-xylose metabolism in Hypocrea jecorina: loss of the xylitol dehydrogenase step can be partially compensated for by lad1-encoded L-arabinitol-4-dehydrogenase.

With the goal of the genetic characterization of the D-xylose pathway in Hypocrea jecorina (anamorph: Trichoderma reesei), we cloned the xdh1 gene, encoding NAD-xylitol dehydrogenase, which catalyzes the second step of fungal D-xylose catabolism. This gene encodes a 363-amino-acid protein which has a mass of 38 kDa, belongs to the zinc-containing alcohol dehydrogenase family, exhibits high sequence identity to the published sequences of xylitol dehydrogenases from yeast origins, but contains a second, additional binding site for Zn2+. The enzyme catalyzed the NAD-dependent oxidation of xylitol and D-sorbitol and the NADH-dependent reduction of D-xylulose and D-fructose. No activity was observed with NADP, L-arabinose, or L-arabinitol. A single 1.4-kb transcript was formed during growth on xylan, D-xylose, L-arabinose, L-arabinitol and, at a lower abundance, xylitol, D-galactose, galactitol, and lactose but not on D-glucose and glycerol. xdh1 deletion mutants exhibited 50% reduced growth rates on D-xylose, whereas growth rates on xylitol remained unaltered. These mutants contained 30% of the xylitol dehydrogenase activity of the parent strain, indicating the presence of a second xylitol dehydrogenase. This activity was shown to be due to lad1-encoded L-arabinitol-4-dehydrogenase, because H. jecorina xdh1 lad1 double-deletion strains failed to grow on D-xylose or xylitol. In contrast, lad1 deletion strains of H. jecorina grew normally on these carbon sources. These results show that H. jecorina contains a single xylitol dehydrogenase which is encoded by xdh1 and is involved in the metabolism of D-xylose and that lad1-encoded L-arabinitol-4-dehydrogenase can compensate for it partially in mutants with a loss of xdh1 function.

Substrate specificity and kinetic mechanism of Escherichia coli ribulokinase.

L-ribulokinase is unusual among kinases since it phosphorylates all four 2-ketopentoses with almost the same k(cat) values. The K(m)'s differ, however, being 0.14 mM for L- and 0.39 mM for d-ribulose and 3.4 mM for l- and 16 mM for d-xylulose. In addition, L-arabitol is phosphorylated at C-5 (K(m) 4 mM) and ribitol (adonitol) is phosphorylated to D-ribitol-5-phosphate (K(m) 5.5 mM), but D-arabitol, xylitol, and aldopentoses are not substrates. The K(m)'s for MgATP depend on the substrates, being 0.02 mM with L-ribulose, 0.027 mM with D-ribulose and L-xylulose, and 0.3-0.5 mM with the other substrates. In the absence of a sugar substrate there is an ATPase with K(m) of 7 mM and k(cat) 1% of that with sugar substrates. The initial velocity pattern is intersecting, and MgAMPPNP is competitive vs MgATP and uncompetitive vs L-ribulose. L-Erythrulose is competitive vs L-ribulose and when MgATP concentration is varied induces substrate inhibition which is partial. These data show that the mechanism is random, but there is a high level of synergism in the binding of sugar and MgATP, and the path in which the sugar adds first is strongly preferred.

Synthesis and antiproliferative activity of some 4'-C-hydroxymethyl-alpha- and -beta-D-arabino-pentofuranosyl pyrimidine nucleosides.

A suitably protected 4-C-hydroxymethyl-arabino-pentofuranose was prepared and condensed with the following nucleobases: uracil, 5-fluorouracil and thymine. The corresponding cytosine and 5-fluorocytosine derivatives have also been obtained respectively from the uracil and 5-fluorouracil nucleosides. Separation of the anomeric mixtures followed by deprotection afforded the target compounds that were found to be non-cytotoxic to CCRF-CEM leukemia cells.

Synthesis of 4-substituted phenyl 3,6-anhydro-1,3-dithio-D-glucofuranosides and -pyranosides as well as 2,6-anhydro-1,2-dithio-alpha-D-altrofuranosides possessing antithrombotic activity.

1,2,5-Tri-O-acetyl-3,6-anhydro-3-thio-D-glucofuranose was synthesised starting from D-glucose and was used as a donor for the glycosidation of 4-cyano- and 4-nitrobenzenethiol. In the latter reaction, besides an anomeric mixture of the 4-nitrophenyl 2,5-di-O-acetyl-3,6-anhydro-1,3-dithio-D-glucofuranosides, the corresponding 2,6-anhydro-1,2-dithio-D-altrofuranosides were also obtained, formed via a rearrangement of the sugar moiety. A similar rearrangement could be observed during the hydrolysis of the glycosidic bond of methyl 3,6-anhydro-2,4-di-O-(4-nitrobenzoyl)-3-thio-alpha-D-glucopyranoside with aqueous trifluoroacetic acid, affording after acetylation besides 1-O-acetyl-3,6-anhydro-2,4-di-O-(4-nitrobenzoyl)-3-thio-alpha-D-glucopyranose (32alpha), 1,1,5-tri-O-acetyl-3,6-anhydro-2,4-di-O-(4-nitrobenzoyl)-3-thio-D-glucose, methyl 3,6-anhydro-2,4-di-O-(4-nitrobenzoyl)-3-thio-beta-D-glucopyranoside and 1,5-di-O-acetyl-2,6-anhydro-3-O-(4-nitrobenzoyl)-2-thio-alpha-D-altrofuranose (40). Glycosidation of 4-cyanobenzethiol with 32alpha in the presence of trimethylsilyl triflate as promoter afforded 4-cyanophenyl 3,6-anhydro-2,4-di-O-(4-nitrobenzoyl)-1,3-dithio-beta-D-glucopyranoside as a minor component only, besides 4-cyanophenyl 3,6-anhydro-2-S-(4-cyanophenyl)-4-O-(4-nitrobenzoyl)-1,2,3-trithio-beta-D-glucopyranoside. When boron trifluoride etherate was used as promoter in the reaction of 32alpha with 4-cyano- and 4-nitrobenzenethiol, the corresponding beta-thioglycosides were obtained, while 40 gave under identical conditions the alpha anomers exclusively. All thioglycosides obtained after deacylation were submitted to biological evaluation. Among these glycosides, the 4-cyanophenyl 3,6-thioanhydro-1,3-dithio-D-glucofuranoside possessed the strongest oral antithrombotic effect.

Na(+)-dependent transport of D-xylose by bovine intestinal brush border membrane vesicles (BBMV) is inhibited by various pentoses and hexoses.

To detect whether pentoses and hexoses occurring in rumen bacteria or in hemicellulose ingested with feed and partly released in the small intestine have an affinity for the Na(+)-dependent glucose transporter of the bovine intestinal brush border membrane (BBM), we investigated whether these monosaccharides inhibit Na(+)-dependent transport of 14C-labelled D-xylose across the BBM using brush border membrane vesicles (BBMV) isolated from the mid-jejunum of cows. We used D-xylose as the transport substrate, because it has a low affinity for the Na(+)-dependent glucose transporter and thus its uptake into BBMV is more efficiently competitively inhibited by other sugars than that of D-glucose. D-Ribose, D-mannose and L-rhamnose occurring in rumen bacteria significantly inhibited Na(+)-dependent uptake of D-xylose into BBMV, but their inhibitory effect was less than that of D-glucose, D-xylose and phlorizin. This also applied to L-arabinose (and D-arabinose), which is, like D-xylose and D-galactose, a constituent of hemicellulose, and to 2-deoxy-D-glucose. Of all monosaccharides tested, only D-fructose did not affect Na(+)-dependent D-xylose transport. It is concluded that some pentoses and hexoses occurring in rumen bacteria (D-ribose, D-mannose and L-rhamnose) or hemicellulose (L-arabinose and D-xylose) have a low affinity for the Na(+)-dependent glucose transporter of the bovine BBM and may therefore be absorbed from the jejunum when released in the small intestine.

Priority of pentose utilization at the level of transcription: arabinose, xylose, and ribose operons.

When E. coli cells were grown in minimal medium supplemented with D-ribose and D-xylose, a diauxic growth preferring D-xylose was observed. Transcription of the ribose (rbs) operon was repressed in the presence of D-xylose, phenotypically similar to catabolite repression by D-glucose, although D-ribose did not affect transcription of the xylose (xyl) operon. Complementation analysis with xylR revealed that the repression of the rbs operon by D-xylose is exerted at the transcriptional level through XylR, suggesting a novel mechanism for catabolite repression. Furthermore, it was shown that L-arabinose reduced transcriptions of both xyl and rbs operons, whereas the arabinose operon was not affected by D-xylose or D-ribose, suggesting a priority mechanism for pentose utilization.

Design, synthesis, and antiviral activity of alpha-nucleosides: D- and L-isomers of lyxofuranosyl- and (5-deoxylyxofuranosyl)benzimidazoles.

Several 2-substituted alpha-D- and alpha-L-lyxofuranosyl and 5-deoxylyxofuranosyl derivatives of 5,6-dicholro-2-(isopropylamino)-1-(beta-L-ribofuranosyl) benzimidazole (1263W94) and 2,5,6-trichloro-1(beta-D-ribofuranosyl)benzimidazole (TCRB) were synthesized and evaluated for activity against two herpesviruses (HSV-1 and HCMV) and for their cytotoxicity against HFF and KB cells. Condensation of 1,2,3,5-tetra-O-acetyl-L-lyxofuranose (2a) with 2,5,6-trichlorobenzimidazole (1) yielded the alpha-nucleoside 3a. The 2-bromo derivative and 2-methylamino derivative were prepared by treatment of 3a with HBr followed by deprotection or from methylamine, respectively. Compound 3a was deprotected and the resultant nucleoside used to prepare the 2-cyclopropylamino and 2-isopropylamino derivatives. The 2-alkylthio nucleosides were prepared by condensing 2a with 5,6-dichlorobenzimidazole-2-thione followed by deprotection. Alkylation of this adduct gave the 2-methylthio and 2-benzylthio derivatives. Condensation of 5-deoxy-1,2,3-tri-O-acetyl-L-lyxofuranosyl, prepared from L-lyxose, with 1 or 2-bromo-5,6-dichlorobenzimidazole (15), followed by deprotection, gave the 2-chloro or 2-bromo-5'-deoxylyxo-furanosyl derivative, respectively. The cyclopropylamino derivative was prepared from the 2-chloro derivative. All D-isomers were prepared in an analogous fashion from D-lyxose. Either compounds were inactive against HSV-1 or weak activity was poorly separated from cytotoxicity. In contrast, the 2-halogen derivatives in both the alpha-lyxose and 5-deoxy-alpha-lyxose series were active against the Towne strain of HCMV. The 5-deoxy alpha-L analogues were the most active, IC50's = 0.2-0.4 microM, plaque assay; IC90's = 0.2-2 microM, yield reduction assay. All of the 2-isopropylamino or 2-cyclopropylamino derivatives were less active (IC50's = 60-100 microM, plaque assay; IC90's = 17-100 microM, yield reduction assay) and were not cytotoxic. The methylamino, thio, and methylthio derivatives were neither active nor cytotoxic. The benzylthio derivatives were weakly active, but this activity was poorly separated from cytotoxicity. The alpha-lyxose L-isomers were more active in a plaque assay against the AD169 strain of HCMV compared to the Towne strain, thereby providing additional evidence of antiviral specificity.

Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435.

In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on D-xylose or L-arabinose as well as on the structurally related sugars D-arabinose or D-lyxose. Mixtures of D-xylose with the more rapidly metabolizable sugar D-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of D-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control.

Galactose-induced dimerization of blocked ricin at acidic pH: evidence for a third galactose-binding site in ricin B-chain.

Blocked ricin is a glycoconjugate formed by covalent modification of each of the two galactose-binding sites of ricin with affinity ligands derived by modification of glycopeptides containing galactose-terminated, triantennary, N-linked oligosaccharides. Blocked ricin undergoes a pH-dependent reversible self-association, being predominantly dimeric at neutral pH and monomeric at acidic pH. The shift in the monomer-dimer equilibrium towards the monomeric form at acidic pH (pH 4) is inhibited by lactose, as shown by size-exclusion chromatography. This behavior of blocked ricin can be reproduced in studies with isolated blocked B-chain. The effect, which is dependent on the concentration of the sugar, is specific for sugars having terminal galactose moieties, or sugars having the same orientation of hydroxyl groups at C2 and C4 as galactose. These results are interpreted as providing further support for the notion that ricin B-chain has a third galactose-binding site, which may be important for the intracellular trafficking of ricin during intoxication of cells.

Kinetics of the cooperative binding of glucose to dimeric yeast hexokinase P-I.

Kinetic studies of the cooperative binding of glucose to yeast hexokinase P-I at pH 6.5 have been carried out using the fluorescence temperature-jump technique. Three relaxation effects were observed: a fast low-amplitude effect which could only be resolved at low glucose concentrations (tau 1(-1) = 500-800 s-1), an intermediate effect (tau 2) which showed a linear dependence of reciprocal relaxation time on concentration, and a slow effect (tau 3) which showed a curved dependence on glucose concentration, increasing from approximately 28 s-1 at low concentrations to 250 s-1 at high levels. The findings are interpreted in terms of the concerted Monod-Wyman-Changeux mechanism, the two faster relaxations being assigned to binding to the R and T states, and the slow relaxation to isomerization between the states. Quantitative fitting of the kinetic data to the mechanism has been carried out using independent estimates of the equilibrium parameters of the model; these have been derived from equilibrium dialysis data and by determining the enhancement of the intrinsic ATPase activity of the enzyme by the non-phosphorylatable sugar lyxose, which switches the conformation of the enzyme to the active R state.

Proton-linked L-rhamnose transport, and its comparison with L-fucose transport in Enterobacteriaceae.

1. An alkaline pH change occurred when L-rhamnose, L-mannose or L-lyxose was added to L-rhamnose-grown energy-depleted suspensions of strains of Escherichia coli. This is diagnostic of sugar-H+ symport activity. 2. L-Rhamnose, L-mannose and L-lyxose were inducers of the sugar-H+ symport and of L-[14C]rhamnose transport activity. L-Rhamnose also induced the biochemically and genetically distinct L-fucose-H+ symport activity in strains competent for L-rhamnose metabolism. 3. Steady-state kinetic measurements showed that L-mannose and L-lyxose were competitive inhibitors (alternative substrates) for the L-rhamnose transport system, and that L-galactose and D-arabinose were competitive inhibitors (alternative substrates) for the L-fucose transport system. Additional measurements with other sugars of related structure defined the different substrate specificities of the two transport systems. 4. The relative rates of H+ symport and of sugar metabolism, and the relative values of their kinetic parameters, suggested that the physiological role of the transport activity was primarily for utilization of L-rhamnose, not for L-mannose or L-lyxose. 5. L-Rhamnose transport into subcellular vesicles of E. coli was dependent on respiration, was optimal at pH 7, and was inhibited by protonophores and ionophores. It was insensitive to N-ethylmaleimide or cytochalasin B. 6. L-Rhamnose, L-mannose and L-lyxose each elicited an alkaline pH change when added to energy-depleted suspensions of L-rhamnose-grown Salmonella typhimurium LT2, Klebsiella pneumoniae, Klebsiella aerogenes, Erwinia carotovora carotovora and Erwinia carotovora atroseptica. The relative rates of subsequent acidification varied, depending on both the organism and the sugar. L-Fucose promoted an alkaline pH change in all the L-rhamnose-induced organisms except the Erwinia species. No L-rhamnose-H+ symport occurred in any organism grown on L-fucose. 7. All these results showed that L-rhamnose transport into the micro-organisms occurred by a system different from that for L-fucose transport. Both systems are energized by the trans-membrane electrochemical gradient of protons. 8. Neither steady-state kinetic measurements nor binding-protein assays revealed the existence of a second L-rhamnose transport system in E. coli.

Feeding modulation by pentose and hexose analogues.

D-Glucosamine (GlcN), N-acetyl-D-glucosamine (GlcNAc) and 2,5-anhydro-D-mannitol (2,5-AM) were infused into the rat third cerebroventricle (icv) to compare their effects on food intake. GlcN (24 mumols/L) accelerated eating, and concomitantly increased plasma glucose, free fatty acids, and glycerol without affecting plasma insulin. GlcN accelerated lateral hypothalamic (LHA), and reciprocally decreased ventromedial hypothalamic (VMH) neuronal activity. Infusion of 12 mumols GlcNAc icv did not affect feeding, but oral administration (1200 mumols/L) induced feeding. The GlcNAc-induced feeding was completely abolished by bilateral truncal vagotomy. Infusion of 2,5-AM dose-dependently induced feeding (P less than 0.01). A maximal dose (24 mumols/L) did not substantially change plasma glucose or insulin. Unilateral 2,5-AM microinfusion (1.2 mumols/L) into the VMH, but not into the LHA, elicited feeding. The characteristic actions of these analogues are useful to clarify central control of food intake and also as probes to examine relations between feeding modulation and energy metabolism in the central nervous system.