First theophylline-based ratiometric fluorescent synthetic receptor for selective recognition of dihydrogenphosphate and biological phosphate ions

We have developed a new ratiometric fluorescent chemosensor 1 based on xanthine alkaloid theophylline moiety for the detection of dihydrogen phosphate and ATP. The chemosensor 1 selectively recognizes tetrabutylammonium dihydrogen phosphate in CH(3)CN/H(2)O (9:1) by exhibiting a significant decrease in the emission of naphthalene and its sensing properties regarding ATP and other related phosphate species were evaluated. The anion binding properties of 1 were evaluated by (1)H NMR, UV-vis, and fluorescence spectroscopic methods.     

BSA-stabilized Au clusters as peroxidase mimetics for use in xanthine detection

In this paper, we demonstrated that bovine serum albumin (BSA) stabilized Au clusters exhibited highly intrinsic peroxidase-like activity. Unlike nature enzymes, the BSA-Au clusters have strong robustness and can be used over a wide range of pH and temperature. Because of ultra-small size, good stability and high biocompatibility in water solution compare with other kinds of nanoparticles as peroxidase mimetics, such as Fe(3)O(4), FeS or graphene oxide, it is more competent for bioanalysis. Furthermore, we make use of the novel properties of BSA-Au clusters as peroxidase mimetics to detect H(2)O(2). The as-prepared BSA-Au clusters were used to catalyze the oxidation of a peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) by H(2)O(2) to the oxidized colored product, and which provides a colorimetric detection of H(2)O(2). As low as 2.0 × 10(-8)M H(2)O(2) could be detected with a linear range from 5.0 × 10(-7) to 2.0 × 10(-5)M via this method. More importantly, a sensitive and selective method for xanthine detection was developed using xanthine oxidase (XOD) and the as-prepared BSA-Au clusters. The detection limit of this assay for xanthine was 5 × 10(-7)M and the proposed method was successfully applied for the determination of xanthine in urine and human serum sample.     

Xanthine oxidase-catalyzed crosslinking of cell membrane proteins

Isolated erythrocyte membranes exposed to protease-free xanthine oxidase plus xanthine and ferric iron undergo lipid peroxidation and protein crosslinking (appearance of high molecular weight aggregates on sodium dodecyl sulfate (SDS) gel electrophoresis). Spectrin is more susceptible to crosslinking than the other polypeptides. Thiol-reducible bonds (disulfides) as well as nonreducible bonds are generated, the former type relatively rapidly (detected within 10-20 min) and the latter type more slowly (usually detected after 1 h). Reducible crosslinking is inhibited by catalase, but not by superoxide dismutase, desferrioxamine, butylated hydroxyltoluene, and mannitol; whereas nonreducible crosslinking, like free radical lipid peroxidation, is inhibited by all of these agents except mannitol. Zinc(II) also inhibits lipid peroxidation, but stimulates disulfide bond formation to the virtual exclusion of all other crosslinking. Our results indicate that disulfide formation is dependent on H2O2, but not O2- or iron. However, O2-, H2O2, and iron are all required for lipid peroxidation and nondisulfide crosslinking, suggesting the intermediacy of OH generated via the iron-catalyzed Haber-Weiss reaction. The possible role of malonaldehyde (MDA, a by-product of lipid peroxidation) in the latter type of crosslinking was examined. Solubilized samples of xanthine/xanthine oxidase-treated membranes showed a strong visible fluorescence (emission maximum 450 nm; excitation 390 nm). This resembled the fluorescence of membranes treated with authentic MDA, which forms conjugated imine linkages between amino groups. Fluorescence scanning of SDS gels from MDA-treated membranes showed a strong signal coincident with crosslinked proteins and also one in the low molecular weight, nonprotein region, suggestive of aminolipid conjugates. Similar scanning on xanthine/xanthine oxidase-reacted membranes indicated that all fluorescence is associated with the lipid fraction. Thus, nonreducible protein crosslinks in this system do not appear to be of the MDA-derived, Schiff base type.     

Evidence for free radical formation during the oxidation of 2'-7'-dichlorofluorescin to the fluorescent dye 2'-7'-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements.

The oxidation of 2'-7'-dichlorofluorescin (DCFH) to the fluorescent 2'-7'-dichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated by fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H2O2 oxidized DCFH to the highly fluorescent DCF. However, DCF fluorescence was still observed when H2O2 was omitted, although its intensity was reduced by 50%. Surprisingly, the fluorescence increase, in the absence of exogenous H2O2, was still strongly inhibited by catalase, demonstrating that H2O2 was present and necessary for DCF formation. H2O2 was apparently formed during either chemical or enzymatic deacetylation of 2'-7'-dichlorofluorescin diacetate (DCFH-DA), probably by auto-oxidation. Spectrophotometric measurements clearly showed that DCFH could be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF*-). Oxidation of DCF*- to DCF by oxygen would yield superoxide (O2*-). ESR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of both superoxide and hydroxyl radicals in the DCFH/H2O2/HRP system. Both radicals were also detected in the absence of added H2O2, although the intensities of the resultant adducts were decreased. This work demonstrates that DCF fluorescence cannot be used reliably to measure O2*- in cells because O2*- itself is formed during the conversion of DCFH to DCF by peroxidases. The disproportionation of superoxide forms H2O2 which, in the presence of peroxidase activity, will oxidize more DCFH to DCF with self-amplification of the fluorescence. Because the deacetylation of DCFH-DA, even by esterases, can produce H2O2, the use of this probe to measure H2O2 production in cells is problematic.     

Stimulation of mixed-function oxidation of 7-ethoxycoumarin in periportal and pericentral regions of the perfused rat liver by xylitol

Rates of O-deethylation of 7-ethoxycoumarin by perfused livers from fasted, phenobarbital-treated rats were 3.7 mumol X g-1 X h-1. Approximately 50% of the product was conjugated. When rates of 7-ethoxycoumarin O-deethylation were varied by infusing different concentrations of substrate, a good correlation (r = 0.91) was found between rates of O-deethylation of 7-ethoxycoumarin and fluorescence of 7-hydroxycoumarin detected from the liver surface. Micro-light guides (tip diameter 170 microns) placed on periportal and pericentral regions on the liver surface were used to monitor the conversion of nonfluorescent 7-ethoxycoumarin to fluorescent 7-hydroxycoumarin. The O-deethylation of 7-ethoxycoumarin to 7-hydroxycoumarin increased fluorescence 64% and 28% in pericentral and periportal regions of the liver lobule, respectively. Rates of 7-ethoxycoumarin O-deethylation estimated from these increases in fluorescence were 5.2 mumol X g-1 X h-1 in pericentral and 2.2 mumol X g-1 X h-1 in periportal regions of the liver. During mixed-function oxidation of 7-ethoxycoumarin, the oxidation:reduction state of NADP(H) was similar in both regions of the liver lobule. Xylitol (2 mM) decreased the NADP+/NADPH ratio and stimulated rates of drug metabolism in both regions of the liver lobule. This indicates that conditions exist where the supply of NADPH is an important rate-determining factor for 7-ethoxycoumarin metabolism in both periportal and pericentral regions of the liver lobule.     

Mechanism of O2- (-) and H2O2-induced stimulation of sugar transport in mouse fibroblast BALB/3T3 cells

Xanthine/xanthine oxidase and H2O2 stimulated sugar transport. Application of superoxide dismutase and catalase to the cells showed an inhibitory effect on these agent-stimulated sugar transports. Addition of amiloride and 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), which abolish the cytoplasmic alkalinization, inhibited the stimulation of sugar transport by xanthine/xanthine oxidase in the presence of catalase. The calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and trifluoperazine inhibited H2O2-stimulated sugar transport. These results suggest that O2- stimulates sugar transport in an intracellular pH-dependent manner and that H2O2 stimulates sugar transport in a calcium-calmodulin-dependent manner. These mechanisms may be involved in sugar-transport stimulation in mouse fibroblast BALB/3T3 cells by the tumor-promoting phorbol ester phorbol-12,13-dibutyrate and insulin, since the stimulatory effects of these agents were inhibited by scavengers of oxygen radicals.     

Role of hydrogen peroxide in the cytotoxicity of the xanthine/xanthine oxidase system.

1. The survival of mammalian epithelial cells exposed in vitro to the xanthine/xanthine oxidase system in phosphate-buffered saline (PBS) or serum-containing medium (SCMEM) was investigated. 2. The cytotoxic effect observed depended on the composition of the medium in which the enzymic reaction was carried out; a surviving fraction of 5 x 10(-5) was found for cells exposed in PBS and 5.2 x 10(-1) for those in SCMEM. 3. The cytotoxic product(s) formed by the xanthine/xanthine oxidase system was relatively stable in PBS; survival of cells incubated after completion of the enzymic reaction was always less than that found for cells exposed during the reaction in the same system. 4. Superoxide dismutase or mannitol present during the enzymic reaction did not inhibit the cytotoxic effect. 5. NaN3 (a single-oxygen quencher and a catalase inhibitor) added to the system in SCMEM caused a reduction in survival to the level observed for cells exposed to the enzymic reaction in PBS. 6. Catalase completely protected cells, but no protection was observed when both catalase and NaN3 were present in the reaction mixture. 7. A similar cytotoxic effect was produced when cells were treated with H2O2 alone. 8. The rate of H2O2 decomposition in medium was accelerated by the presence of serum, but this was completely inhibited by NaN3. 9. It is concluded that H2O2 is the major cytotoxic product formed by the xanthine/xanthine oxidase system.     

Inactivation of xanthine oxidase by hydrogen peroxide involves site-directed hydroxyl radical formation.

The mechanism of xanthine oxidase (XO) inactivation by hydrogen peroxide (H2O2) and its biologic significance are unclear. We found that addition of increasing concentrations of H2O2 progressively decreased xanthine oxidase activity in the presence but not the absence of xanthine in vitro. Inactivation of XO by H2O2 was also enhanced by anaerobic reduction of XO by xanthine. Inactivation of XO by H2O2 was accompanied by production of hydroxyl radical (.OH), measured as formation of formaldehyde from dimethylsulfoxide (DMSO). In contrast, addition of H2O2 to deflavo XO did not produce .OH. Inactivation of XO by H2O2 was decreased by simultaneous addition of the .OH scavenger, DMSO. However, inactivation of XO by H2O2 and formation of .OH were not decreased following addition of the metal chelator. DETAPAC, and/or the O2 scavenger, superoxide dismutase. The results suggest that inactivation of XO by H2O2 occurs by production of .OH following direct reduction of H2O2 by XO at the flavin site.     

Effect of glucose on 7-hydroxycoumarin glucuronide production in periportal and pericentral regions of the liver lobule.

The effect of starvation and glucose addition on glucuronidation was assessed in sublobular regions of the lobule in perfused livers from phenobarbital-treated rats. Fibre-optic micro-light guides were placed on periportal and pericentral areas on the surface of livers to monitor the fluorescence (excitation 366 nm, emission 450 nm) of free 7-hydroxycoumarin from the tissue surface. After infusion of 7-hydroxycoumarin (80 microM) under normoxic conditions, steady-state increases in fluorescence were reached in 6-8 min in both regions. Subsequently, the formation of non-fluorescent 7-hydroxycoumarin glucuronide was inhibited completely by perfusion with N2-saturated perfusate containing 20 mM-ethanol. The difference in fluorescence between anoxic and normoxic perfusions was due to glucuronidation under these conditions. In livers from fed rats, rates of glucuronidation in periportal and pericentral regions of the liver lobule were 8 and 19 mumol/h per g, respectively. In contrast, rates of glucuronidation were 3 and 9 mumol/h per g, respectively, in periportal and pericentral regions of livers from starved rats. Infusion of glucose (20 mM) had no effect on rates of glucuronidation in livers from fed rats; however, glucose increased rates of glucuronidation rapidly (half-time, t0.5 = 1.5 min) in periportal and pericentral regions to 7 and 17 mumol/h per g, respectively in livers from starved rats. These results indicate that the rapid synthesis of the cofactor UDP-glucuronic acid derived from glucose is an important rate-determinant for glucuronidation of 7-hydroxycoumarin in both periportal and pericentral regions of livers from starved rats.     

The effects of spacer length on the fluorescence quantum yields of the benzofurazan compounds bearing a donor–acceptor system

We studied the effects of spacer length on the fluorescence quantum yields (Φ) of photoinduced electron transfer (PET) reagents, using nitrobenzoxadiazole (NBD) derivatives that have the –NMe₂ moiety and NBD–NH– fluorophore as electron donor (D) and electron acceptor (A), respectively. The Φ values were reduced as the spacer length became shorter (n ≤ 4; n is the number of methylene units of the spacer) and the fluorescence recovered by suppression of the PET process. It is necessary for the useful PET reagents to link D and A with a short spacer to obtain a difference in the Φ values between fluorescent ‘off‐state’ and ‘on‐state’. Copyright © 2002 John Wiley & Sons, Ltd.     

Inhibition of bovine phenol sulfotransferase (bSULT1A1) by CoA thioesters. Evidence for positive cooperativity and inhibition by interaction with both the nucleotide and phenol binding sites

Previous work with the bovine phenol sulfotransferase (bSULT1A1, EC ) demonstrated inhibition by CoA that was competitive with respect to the sulfuryl donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (Leach, M., Cameron, E., Fite, N., Stassinopoulos, J., Palmreuter, N., and Beckmann, J. D. (1999) Biochem. Biophys. Res. Commun. 261, 815-819). Here we report that long chain acyl-CoAs are more potent inhibitors of bSULT1A1 and also of human dopamine sulfotransferase (SULT1A3) when compared with unesterified CoA and short chain-length acyl-CoAs. A complex pattern of inhibition was revealed by systematic variation of palmitoyl-CoA, PAPS, and 7-hydroxycoumarin, the acceptor substrate. Convex plots of apparent K(m)/V(max) versus [palmitoyl-CoA] were adequately modeled using an ordered rapid equilibrium scheme with PAPS as the leading substrate and by accounting for the possible binding of two equivalents of inhibitor to the dimeric enzyme. Interestingly, the first K(i) of 2-3 microm was followed by a second K(i) of only 0.01-0.05 microm, suggesting that positive subunit cooperativity enhances binding of long chain acyl-CoAs to this sulfotransferase. Simultaneous interaction of palmitoyl-CoA with both the nucleotide and phenol binding sites is suggested by two experiments. First, the acyl-CoA displaced 7-hydroxycoumarin from the highly fluorescent bSULT1A1.PAP.7-HC complex in a cooperative manner. Second, palmitoyl-CoA prevented the quenching of bSULT1A1 fluorescence observed with pentachlorophenol. Finally, titrations of bSULT1A1-pentachlorophenol complex with palmitoyl-CoA caused the return of protein fluorescence, and the binding of palmitoyl-CoA was highly cooperative (Hill constant of 1.9). Overall, these results suggest a model of sulfotransferase inhibition in which the 3'-phosphoadenosine-5'-diphosphate moiety of CoA docks to the PAPS domain, and the acyl-pantetheine group docks to the hydrophobic phenol binding domain.     

Protective effects of green tea polyphenol against reactive oxygen species-induced oxidative stress in cultured rat calvarial osteoblast

The injurious effects of reactive oxygen species on osteoblasts and the potential protective role played by green tea polyphenols (GtPP) were investigated using primarily cultured rat calvarial osteoblasts. Oxidative stress was induced in cultured osteoblasts, either by adding 100 mmol/L H2O2 or by the action of 40 U/L xanthine oxidase (XO) in the presence of xanthine (250 micromol/L). After incubation, the cellular viability, function and morphology were evaluated. Both treatments produced a significant reduction in osteoblast viability, as assessed by a two-colored fluorescence staining method combined with flow cytometric analysis and MTT assay. A significant reduction in the alkaline phosphatase activity was observed after H2O2 addition, whereas XO did not have the same effect. On the microscopic observations, the morphological changes and intracellular ultrastructural damages were remarkably induced by both treatments. The H2O2-induced alterations were prevented by pre-incubating the osteoblasts with 200 microg/ml GtPP for 1 h. When the oxidative stress was induced by XO, the cellular viability and morphology was also maintained at the same polyphenol concentration. These results demonstrate that GtPP can act as a biological antioxidant in a cell culture experimental model and protect cells from oxidative stress-induced toxicity.     

Mechanism of dilation to reactive oxygen species in human coronary arterioles

We tested whether reactive oxygen species (ROS) generated from treatment with xanthine (XA) and xanthine oxidase (XO) alter vascular tone of human coronary arterioles (HCA). Fresh human coronary arterioles (HCA) from right atrial appendages were cannulated for video microscopy. ROS generated by XA (10(-4) M) + XO (10 mU/ml) dilated HCA (99 +/- 1%, 20 min after application of XA/XO). This dilation was not affected by denudation or superoxide dismutase (150 U/ml). Catalase (500 U/ml or 5,000 U/ml) attenuated the dilation early on, but a significant latent vasodilation appeared after 5 min peaking at 20 min (51 +/- 1%, 20 min after application of XA/XO + 500 U/ml catalase, P < 0.01 vs. control). KCl (40 mM) reduced the early and sustained vasodilation to XA/XO in the absence of catalase but 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 x 10(-5) M), diethyldithiocarbamate trihydrate (DDC, 10(-2) M), and deferoxamine (DFX, 10(-3) M) had no effect. In contrast, the catalase-resistant vasodilation was significantly attenuated by DDC, ODQ, and DFX as well as polyethylene-glycolated catalase (5,000 U/ml), but KCl had no effect. Confocal microscopy revealed that even in the presence of catalase, 2',7'-dichlorodihydrofluoresein diacetate fluorescence was observed in the vascular smooth muscle, but this was abolished by DDC. These data indicate that the exogenously generated superoxide anion (O2-*) by XA/XO is spontaneously converted to H2O2, which dilates HCA through vascular smooth muscle hyperpolarization. O2-* is also converted to H2O2 likely by superoxide dismustase within vascular cells and dilates HCA through a different pathway involving the activation of guanylate cyclase. These findings suggest that exogenously and endogenously produced H2O2 may elicit vasodilation by different mechanisms.     

Theoretical and spectroscopic evidence for coordination ability of 3,3'-benzylidenedi-4-hydroxycoumarin. New neodymium (III) complex and its cytotoxic effect

Theoretical and spectroscopic studies of 3,3'-benzylidenedi-4-hydroxycoumarin (bhc) have been performed. B3LYP/6-31G* calculations reproduced the experimental molecular structure of bhc and showed two O-H...O asymmetrical intramolecular hydrogen bonds with O...O distances 2.638 and 2.696 A. The calculated Fukui functions and Molecular Electrostatic Potential for bhc and its deprotonated form, bhc(2-), predicted that the most probable reactive sites for electrophilic attack and hydrogen bonds are the carbonyl oxygens, followed by the hydroxyl oxygens. The coordination ability of 3,3'-benzylidenedi-4-hydroxycoumarin has been proved in a complexation reaction with neodymium (III) ion. The new neodymium (III) complex of bhc was studied by elemental analyses, conductivity and other physical properties, mass spectra, (1)H, (13)C NMR, UV-Vis and IR spectroscopy. The data obtained are in agreement with the metal:ligand ratio of 1:1, and the formula Nd(bhc(2-))(OH)(H(2)O), where bhc(2-)=C(25)H(14)O(6)(2-). The vibrational analysis of the neodymium (III) complex, free bhc, and its monomeric building block, 4-hydroxycoumarin, showed that in the Nd(III) complex the ligand coordinates to the metal ion through both deprotonated hydroxyl groups. The participation of both carbonyl groups in coordination to the metal ion was confirmed by the significant shift of nu(C=O) to lower wavenumber. The evaluation of the cytotoxic activity of the new Nd(III) complex on SKW-3 and HL-60/Dox cells revealed, that it is a potent cytotoxic agent and should be subset further to more detailed pharmacological and toxicological study.     

Effects of reactive oxygen metabolites on erythropoietin production in renal carcinoma cells

The present studies were undertaken to determine the effects of reactive oxygen metabolites on erythropoietin (Ep) biosynthesis in Ep-producing renal carcinoma (RC) cells using a sensitive radioimmunoassay for Ep. Xanthine (10-5M) and increasing concentrations of xanthine oxidase (8 x 10(-7) to 5 x 10(-4) units/ml) produced a significant dose-related increase in Ep production at a concentration of greater than or equal to 4 x 10(-6) units/ml, whereas xanthine alone had no effect. Catalase, a scavenger of hydrogen peroxide (H2O2), in concentrations of 50 to 500 micrograms/ml produced a significant inhibition of the increase in Ep production induced by xanthine-xanthine oxidase; while no effect was seen on basal levels of Ep production and the growth of RC cells. Glucose oxidase (greater than or equal to 0.032 mU/ml), a direct H2O2 generator, and exogenous H2O2 (greater than or equal to 4 x 10(-6)M) added to the incubation mixture, caused a significant enhancement of Ep production in a dose-dependent manner. Xanthine-xanthine oxidase, glucose oxidase, and H2O2 in the above concentrations did not produce significant cytotoxicity (51Cr release or trypan blue dye exclusion). The present data suggests that H2O2, a reactive oxygen metabolite may play a significant role in Ep production.     

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha.

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.     

APPLICATIONS OF TIME-RESOLVED FLUOROIMMUNOASSAY TO DETECT MAGNETIC BEAD CAPTURED ESCHERICHIA COLI O157:H7

A time-resolved fluorescence technique was developed to detect Escherichia coli O157:H7 in ground beef burger. After a 4.5 h enrichment period, streptavidin coated magnetic beads conjugated with biotin-labeled anti E. coli O157:H7 were used to capture the bacteria. The bacteria were, at the same time, also labeled by a nonfluorescent, europium (Eu)-tagged anti-E. coli O157:H7 antibody. The sandwiched bacterial complexes were then concentrated using a magnetic particle concentrator and washed to remove other solution components. Upon addition of an enhancement buffer, the Eu-labels were then released from the antibodies and chelated to nitrilo-triacetic acid (NTA) and trioctylphosphine oxide (TOPO) to form highly fluorescent Eu-(2-NTA)₃(TOPO)_2–3 micellar complexes. Delayed fluorescence associated with these complexes was measured and its intensity was used to estimate the original bacterial concentration spiked in hamburger. This approach was applied to detect E. coli O157:H7 spiked in hamburgers. The results indicated this method is able to detect 1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37C. Specificity studies indicated that the approach exhibited no or limited cross reactivity to Salmonella typhimurium, E. coli K-12 or Shigella dysenteriae spiked in hamburgers. Thus, the developed approach may be used as a rapid screening procedure for E. coli O157 bacteria in foods.     

Crystal structure of N-(1-deoxy-beta-D-fructopyranos-1-yl)-L-proline-an Amadori compound

Here we report the crystal structure data on N-(1-deoxy-beta-D-fructopyranos-1-yl)-L-proline (Fru-Pro)-an Amadori compound. X-ray crystal and molecular structures of its two isomorphous crystalline forms, (Fru-Pro)xMeOH, C(11)H(19)NO(7)xCH(4)O (1a) and (Fru-Pro)x2H(2)O, C(11)H(19)NO(7)x2H(2)O (1b) were determined. In 1a and 1b the compound crystallizes as the beta-anomer with the overall geometry of Fru-Pro zwitterions being very similar. Fructose ring adopts the chair (2)C(5) conformation with the proline moiety bonded to equatorial C-1 atom and remaining in a trans-gauche (tg) orientation with respect to the sugar ring. The five-membered pyrrolidine ring adopts an envelope conformation, with the Cbeta atom puckered. Fructosyl and carboxylate groups are in bisectional and axial positions of pyrrolidine ring, respectively. The overall molecular geometry of Fru-Pro zwitterions, especially the relative orientation of sugar and amino acid moieties, is stabilized by intramolecular, three-centred N-H...O(Fru)/O(Pro) hydrogen bonds (with bifurcated acceptor) formed between aminium and hydroxyl/carboxylate groups. The packing diagrams are very similar in both 1a and 1b with the adjacent zwitterions linked to each other by the extensive network of O-H...O and C-H...O hydrogen bonds to form channels along the a-axis, filled up with solvent molecules.     

Oxidative stress in vitiligo: photo-oxidation of pterins produces H(2)O(2) and pterin-6-carboxylic acid

Patients with vitiligo accumulate millimolar levels of H(2)O(2) in their epidermis. The recycling process of (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin in these patients is disrupted due to deactivation of 4a-OH-BH(4) dehydratase by H(2)O(2). The H(2)O(2) oxidation products 6- and 7-biopterin lead to the characteristic fluorescence of the affected skin upon Wood's light examination (UVA 351 nm). Here we report for the first time the presence and accumulation of pterin-6-carboxylic acid (P-6-COOH) in the epidermis of these patients. Exploring potential sources for P-6-COOH revealed that sepiapterin and 6-biopterin are readily photo-oxidised to P-6-COOH by UVA/UVB irradiation. Photolysis of sepiapterin and 6-biopterin produces stoichiometric H(2)O(2) under aerobic conditions, where O(2) is the electron acceptor, thus identifying an additional source for H(2)O(2) generation in vitiligo. A detailed analysis utilising UV/visible spectrophotometry, HPLC, TLC, and mass spectroscopy showed for sepiapterin direct oxidation to P-6-COOH, whereas 6-biopterin formed the intermediate 6-formylpterin (P-6-CHO) which is then further photo-oxidised to P-6-COOH.     

Xanthine oxidase-catalyzed reductive debromination of 6-(bromomethyl)-9H-purine with concomitant covalent modification of the FAD prosthetic group

Bovine milk xanthine oxidase was potently inhibited by 6-(bromomethyl)-9H-purine in a time-dependent process with O2 as the electron acceptor. If the enzyme were assayed with phenazene ethosulfate as an electron acceptor, 6-(bromomethyl)-9H-purine was not an inhibitor. The rate of formation of inhibited enzyme increased with increasing concentrations of 6-(halomethyl)-9H-purine, decreased with increasing concentrations of O2, and increased in the presence of xanthine. The inhibited enzyme regained activity nonactinically at pH 7 with a t1/2 of 31 h. The optical difference spectrum between native enzyme and inhibited enzyme suggested that the enzyme-bound FAD was modified. This conclusion was confirmed by demonstrating that activity was restored to the inhibited enzyme if the enzyme-bound flavin was removed by treatment with CaCl2 and the resulting apoenzyme was reconstituted with FAD. Aerobically, 6-(bromomethyl)-9H-purine was oxidized by the enzyme to a species having a UV spectrum consistent with hydroxylation of the purine ring to form a urate analogue. Anaerobically, the enzyme reduced 6-(bromomethyl)-9H-purine to 6-methylpurine with 1 mol of enzyme being completely inhibited after reduction of 23 mol of 6-(bromomethyl)-9H-purine. Thus, 6-(bromomethyl)-9H-purine was not only oxidized by xanthine oxidase but was also reduced by the enzyme in a reaction that partitioned between formation of 6-methylpurine and inhibition of the enzyme by modification of the enzyme-bound flavin. Similar results were found when 6-(chloromethyl)-9H-purine was the inhibitor.