Iron detoxification properties of Escherichia coli bacterioferritin. Attenuation of oxyradical chemistry

Bacterioferritin (EcBFR) of Escherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the "ferroxidase center." The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H(2)O(2) as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H(2)O(2) at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H(2)O(2) corresponds to [Fe(II)(2)-P](Z) + H(2)O(2) --> [Fe(III)(2)O-P](Z) + H(2)O, where [Fe(II)(2)-P](Z) represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)(2)O-P](Z) a micro-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2FeOOH((core)) + 4H(+), where two Fe(II) are again oxidized by one H(2)O(2). Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O(2) is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H(2)O(2) produced from O(2) is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H(2)O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H(2)O(2), consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H(2)O(2), thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.     

Reactions of thiocyanate in the mixture of nitrite and hydrogen peroxide under acidic conditions: investigation of the reactions simulating the mixture of saliva and gastric juice

Nitrite and SCN(-) in saliva can mixes with H(2)O(2) in the stomach. The mixing can result in the formation of ONOOH. It is not yet known how salivary SCN(-) reacts with ONOOH. An objective of the present study was to elucidate the reaction between ONOOH and SCN(-). In nitrite/H(2)O(2) systems at pH 2, SCN(-) inhibited the consumption of nitrite and the formation of O(3)(-). SCN(-) enhanced the decomposition of ONOOH and H(2)O(2) in HNO(2)/H(2)O(2) systems. Accompanying the reactions, sulfate was formed, suggesting that ONOOH oxidized SCN(-). SCN(-) inhibited the nitration of phenolics induced by HNO(2)/H(2)O(2). The inhibition is discussed taking SCN(-)-dependent reduction of ONOOH to HNO(2) into consideration. SCN(-) also inhibited H(2)O(2)-induced consumption of nitrite and nitration of phenolics in acidified saliva. The result obtained in this study suggests that salivary SCN(-) can reduce ONOOH to O(2)(-)/HNO(2) inhibiting nitrating reactions in the stomach.     

Modeling of Ni-Fe-center of Ni-CO-dehydrogenases by nickel complexes with thiaazaligands

Three new nickel(II) complexes with ligands 1,8-bis(2'-pyridyl)-3,6-dithiaoctane (Pdto) and dithiosemicarbazone of 4,7-dithiadecane-2,9-dione (DtdtzH2) of composition Ni(Pdto)(H2O)2(ClO4)2, Ni(DtdtzH2)(ClO4)2 and Ni(Dtdtz) were prepared, their molecular structures, spectral and redox-properties were studied. The possibilities of chemical reduction of Ni(Pdto)(H2O)2(ClO4)2 to nickel(I) and nickel(0) species and the reaction of nickel(I) complex with CO were shown, which may be described as the modeling of one of the stages of reactions with CO on active Ni-Fe-site of Ni-CO-dehydrogenases. It was found that Ni(DtdtzH2)(ClO4)2 reacted with (Et4N)2[Fe4S4(SBz)4] (BzSH = C6H5 CH2SH) forming adduct. In the row of studied complexes Ni(Pdto) (H2O)2(ClO4)2 may be described as the best structural model of Ni-Fe-site of Ni-CO-dehydrogenases on the redox properties.     

Inhibition of pyrophosphatase activity of mouse duodenal alkaline phosphatase by magnesium ions

Duodenal alkaline phosphatase of juvenile (11-day-old) mice, like other non-specific alkaline phosphatases, has the ability to hydrolyse PP(i). When a constant Mg(2+)/PP(i) concentration ratio is maintained, plots of velocity as a function of PP(i) concentration are consistent with Michaelis-Menten kinetics. Mg(2+) activates pyrophosphate hydrolysis and maximal activity is obtained at a constant Mg(2+)/PP(i) concentration ratio of 0.66. At higher ratios there is strong inhibition. At constant concentrations of Mg(2+) and increasing concentrations of PP(i), the velocity-substrate (PP(i)) concentration plots show sigmoidal dependence. By assuming that the true substrate is MgP(2)O(7) (2-) complex, and using complexity constants, the concentrations of free Mg(2+), Mg(2)P(2)O(7) and MgP(2)O(7) (2-) were calculated in assay mixtures ranging in PP(i) concentration from 0.1 to 2.5mm and in total Mg(2+) concentration from 0.6 to 2.6mm. From these data, the concentrations of added Mg(2+) and PP(i) in the assay mixtures were selected so that the velocity could be measured (1) at three fixed concentrations of free Mg(2+) ions with varied concentrations of MgP(2)O(7) (2-) and (2) at four fixed concentrations of Mg(2)P(2)O(7) with varied concentrations of MgP(2)O(7) (2-). Lineweaver-Burk and Hill plots from these data showed that the inhibition is caused by free Mg(2+) ions, of a mixed type and consistent with Michaelis-Menten kinetics. The sigmoidal dependence observed between velocity and PP(i) concentration at constant concentration of total Mg(2+) is therefore not due to allosteric inhibition. It is due to a combined effect of (1) inhibition by free Mg(2+) ions, (2) depletion of the true substrate, MgP(2)O(7) (2-), owing to the formation of Mg(2)P(2)O(7) and (3) the manner in which the concentrations of these three molecular or ionic species change when PP(i) concentration is increased maintaining the total Mg(2+) concentration constant.     

Mechanistic significance of the preparatory migration of hydrogen atoms around the FeMo-co active site of nitrogenase

The migration of H atoms over S and Fe atoms in the reaction domain of FeMo-co, the active site of nitrogenase, is described and used to explain mechanistic data on the catalyzed reductions of N(2) and C(2)H(2). After electron transfer to FeMo-co, H atoms are generated by fast proton supply to S3B (atom labels from structure 1M1N) and migrate vectorially via several pathways from S3B to locations on the FeMo-co face, specifically Fe6, S2B, Fe2, and S2A (calculated reaction profiles are reported). The E(n)H(n) reduction levels (n = 1-4) in the Thorneley-Lowe kinetic-mechanistic schemes are each potential sequences of substructures with different distributions of H atoms. The positions of H atoms influence the binding of substrates N(2) and C(2)H(2), and the bound substrate subsequently blocks further migration of H atoms past the binding site. This model provides a consistent structural interpretation of (a) the two-site reactivity of C(2)H(2) and the differentiation of the high- and low-affinity sites as due to different preparatory H migration; (b) the differing mutual inhibitions of N(2) and C(2)H(2) in wild-type protein; (c) the modified reactivity of the Azotobacter vinelandii alpha-(Gly)69(Ser) mutant with N(2) and C(2)H(2); and (d) the basis for the stereoselectivity of hydrogenation of C(2)D(2) and its loss in some mutant proteins. Some structures for initially bound N(2) and C(2)H(2), and their hydrogenated intermediates, are presented. The key new concept is that binding sites and binding states for substrates and intermediates are characterized not only by their locations on the FeMo-co face but also by the structural and temporal status of the distribution of H atoms over the FeMo-co reaction domain.     

Studies of antibacterial activity of binuclear rhodium (II) complexes with heterocyclic nitrogen ligands

Binuclear rhodium (II) complexes, [Rh2(OOCPh)2(phen)2(H2O)2] (OOCPh)2 (1), [Rh2(OOCPh)2(bpy)2(H2O)2] (OOCPh)2 (2), [Rh2(OOCBu(n))2 (bpy)2(H2O)2] (OOCBu(n)2 (3), and [Rh2(OOCPr(n)2 (phen)2(H2O)2] (OOCPr(n)2 (4) (Phen = 1,10-phenanthroline and bpy = 2,2'-bipyridine), have been synthesized and characterized using NMR, IR and electronic spectra. Activity of these compounds against Gram-positive bacteria decreases in the order: 1?2?3 > 4. Complex 1 is active against many Staphylococcus strains resistant to commonly used antibiotics. The complexes 1-4 are much less active agents against Gram-negative bacteria.     

The binding of carbon dioxide by horse haemoglobin

1. Three modified horse haemoglobins have been prepared: (i) alpha(c) (2)beta(c) (2), in which both the alpha-amino groups of the alpha- and beta-chains have reacted with cyanate, (ii) alpha(c) (2)beta(2), in which the alpha-amino groups of the alpha-chains have reacted with cyanate, and (iii) alpha(2)beta(c) (2), in which the two alpha-amino groups of the beta-chain have reacted with cyanate. 2. The values of n (the Hill constant) for alpha(c) (2)beta(c) (2), alpha(2)beta(c) (2) and alpha(c) (2)beta(2) were (respectively) 2.5, 2.0 and 2.6, indicating the presence of co-operative interactions between the haem groups for all derivatives. 3. In the alkaline pH range (about pH8.0) all the derivatives show the same charge as normal haemoglobin whereas in the acid pH range (about pH6.0) alpha(c) (2)beta(c) (2) differs by four protonic charges and alpha(c) (2)beta(2), alpha(2)beta(c) (2) by two protonic charges from normal haemoglobin, indicating that the expected number of ionizing groups have been removed. 4. alpha(c) (2)beta(2) and alpha(c) (2)beta(c) (2) show a 25% decrease in the alkaline Bohr effect, in contrast with alpha(2)beta(c) (2), which has the same Bohr effect as normal haemoglobin. 5. The deoxy form of alpha(c) (2)beta(c) (2) does not bind more CO(2) than the oxy form of alpha(c) (2)beta(c) (2), whereas alpha(c) (2)beta(2) and alpha(2)beta(c) (2) show intermediate binding. 6. The results reported confirm the hypothesis that, under physiological conditions, haemoglobin binds CO(2) through the four terminal alpha-amino groups and that the two terminal alpha-amino groups of alpha-chains are involved in the Bohr effect.     

Kinetic evaluation of catalase and peroxygenase activities of tyrosinase

Tyrosinase is a copper monooxygenase containing a coupled dinuclear copper active site (type-3 copper), which catalyzes oxygenation of phenols (phenolase activity) as well as dehydrogenation of catechols (catecholase activity) using O(2) as the oxidant. In this study, catalase activity (conversion of H(2)O(2) to (1/2)O(2) and H(2)O) and peroxygenase activity (H(2)O(2)-dependent oxygenation of substrates) of mushroom tyrosinase have been examined kinetically by using amperometric O(2) and H(2)O(2) sensors. The catalase activity has been examined by monitoring the initial rate of O(2) production from H(2)O(2) in the presence of a catalytic amount of tyrosinase in 0.1 M phosphate buffer (pH 7.0) at 25 degrees C under initially anaerobic conditions. It has been found that the catalase activity of mushroom tyrosinase is three-order of magnitude greater than that of mollusk hemocyanin. The higher catalase activity of tyrosinase could be attributed to easier accessibility of H(2)O(2) to the dinuclear copper site of tyrosinase. Mushroom tyrosinase has also been demonstrated for the first time to catalyze oxygenation reaction of phenols with H(2)O(2) (peroxygenase activity). The reaction has been investigated kinetically by monitoring the H(2)O(2) consumption rate in 0.5 M borate buffer (pH 7.0) under aerobic conditions. Similarity of the substituent effects of a series of p-substituted phenols in the peroxygenase reaction with H(2)O(2) to those in the phenolase reaction with O(2) as well as the absence of kinetic deuterium isotope effect with a perdeuterated substrate (p-Cl-C(6)D(4)OH vs p-Cl-C(6)H(4)OH) clearly demonstrated that the oxygenation mechanisms of phenols in both systems are the same, that is, the electrophilic aromatic substitution reaction by a (micro-eta(2):eta(2)-peroxo)dicopper(II) intermediate of oxy-tyrosinase.     

Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.     

Stereochemical aspects of the formation of double bonds in abscisic acid

The stereochemistry of the hydrogen elimination that occurs during the formation of the Delta(4)- and Delta(2)'-double bonds of abscisic acid has been determined from the (14)C/(3)H ratios in abscisic acid biosynthesized by avocado fruit from [2-(14)C,(2R)-2-(3)H(1)]-, [2-(14)C,(2S)-2-(3)H(1)]- and [2-(14)C,(5S)-5-(3)H(1)]-mevalonate. Setting the (14)C/(3)H ratio at 3:3 for [2-(14)C,(2R)-2-(3)H(1)]mevalonate, the corresponding ratio in derived methyl abscisate was 3:2.28; the analogous ratio for methyl abscisate from [2-(14)C,(2S)-2-(3)H(1)]mevalonate was 3:1.63. Removal of the 3'-hydrogen atom of abscisic acid by base-catalysed exchange altered the ratios to 3:1.55 and 3:1.44 respectively. It was concluded that this 3'-hydrogen atom is derived from the pro-2R-hydrogen atom of mevalonate. Removal of the 4-hydrogen atom from methyl abscisate by formation of a derivative, a lactone, lacking this hydrogen atom changed the ratio to 3:1.04 for material derived from [2-(14)C,(2R)-2-(3)H(1)]-mevalonate and to 3:1.05 for [2-(14)C,(2S)-2-(3)H(1)]mevalonate, showing that this hydrogen atom also is derived from the pro-2R-hydrogen atom of mevalonate. These ratios of the lactones are consistent with their retaining one (3)H atom at the 6'-methyl position of abscisic acid from the [(2R)-2-(3)H(1)]- and [(2S)-2-(3)H(1)]-mevalonate. The presence of some label at positions 3' and 4 when [(2S)-2-(3)H(1)]mevalonate was the precursor is attributed to the action of isopentenyl pyrophosphate isomerase. The hydrogen atom at C-5 of abscisic acid is derived from the pro-5S-hydrogen atom of mevalonate.     

Cellular titration of apoptosis with steady state concentrations of H(2)O(2): submicromolar levels of H(2)O(2) induce apoptosis through Fenton chemistry independent of the cellular thiol state

Apoptosis was studied under conditions that mimic the steady state of H(2)O(2) in vivo. This is at variance with previous studies involving a bolus addition of H(2)O(2), a procedure that disrupts the cellular homeostasis. The results allowed us to define three phases for H(2)O(2)-induced apoptosis in Jurkat T-cells with reference to cytosolic steady state concentrations of H(2)O(2) [(H(2)O(2))(ss)]: (H(2)O(2))(ss) values below 0.7 microM elicited no effects; (H(2)O(2))(ss) approximately 0.7-3 microM induced apoptosis; and (H(2)O(2))(ss) > 3 microM yielded no additional apoptosis and a gradual shift towards necrosis as the mode of cell death were observed. H(2)O(2)-induced apoptosis was not affected by either BCNU, an inhibitor of glutathione reductase, or diamide, a compound that reacts both with low-molecular weight and protein thiols, or selenols. Glutathione depletion, accomplished by incubating cells either with buthionine sulfoximine or in cystine-free medium, rendered cells more sensitive to H(2)O(2)-induced apoptosis, but did not change the threshold and saturating concentrations of H(2)O(2) that induced apoptosis. Two unrelated metal chelators, desferrioxamine and dipyridyl, strongly protected against H(2)O(2)-induced apoptosis. It may be concluded that, under conditions of H(2)O(2) delivery that mimic in vivo situations, the oxidative event that triggers the induction of apoptosis by H(2)O(2) is a Fenton-type reaction and is independent of the thiol or selenium states of the cell.     

Effects of polyamines and analogs on staphylococcal nuclease.

The promoting activity of polyamine analogs (IV approximately XV) on staphylococcal nuclease with DNA as the substrate was compared with that of natural polyamines (I APPROXIMATELY III): I. NH2(CH2)3NH(CH2)4NH(CH2)3NH2(spermine); II. NH2(CH2)3NH(CH2)3NH(CH2)3NH2(thermine); III. NH2(CH2)4NH2 (putrescine); IV. CN(CH2)2NH(CH2)4NH(CH2)2CN; V. HOOC(CH2)2NH(CH2)4NH(CH2)2COOH; VI. C2H5OOC(CH2)2NH(CH2)4NH(CH2)2COOC2H5; VII. HO(CH2)3NH(CH2)4HH(CH2)3OH; VIII. CH3COHH(CH2)3NH(CH2)4NH(CH2)3NHCOCH3; IX. C2H5NH(CH2)3NH(CH2)4NH(CH2)3NHC2H5; X. NH2(CH2)3S(CH2)4S(CH2)3NH2; XI. NH2(CH2)3NH(CH2)2O(CH2)2NH(CH2)3NH2; XII. NH2(CH2)3NCH3(CH2)4HCH3(CH2)3NH2; XIII. CN(CH2)2NCH3(CH2)4NCH3(CH2)2CN; XIV. (CH3)2N(CH2)3NCH3(CH2)4NCH3(CH2)3N(CH3)2; XV. NH2(CH2)2O(CH2)2NH2 Replacement of the terminal groups by CN, COOH, COOEt, NHAc, NHEt, or N(CH3)2 remarkably decreased the activity. The compound VII with terminal hydroxyl groups had a lower promoting activity at low concentrations, but revealed higher activity at higher concentrations and, in contrast to spermine, no inhibition at all even at very high concentrations. Replacement of both internal amino groups by sulfur or NCH3 decreased the activity. The introduction of an ether bond into the internal methylene groups (compound XI) highly decreased the activity. Based upon these findings the possible relationship between structure and activity is discussed.     

Role of phosphatidylinositol 4,5-bisphosphate in the activation of cytosolic phospholipase A2-alpha

Cytosolic phospholipase A(2)-alpha (cPLA(2)) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA(2) is dependent on a rise in cytosolic Ca(2+) concentration, membrane association via the Ca(2+)-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA(2) via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), including the role of a "pleckstrin homology (PH)-like" region of cPLA(2) (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate+the Ca(2+) ionophore, A23187, or epidermal growth factor+A23187, expression of the PH domain of phospholipase C-delta1 (which sequesters membrane PIP(2)) attenuated cPLA(2) activity. Stimulated cPLA(2) activity was also attenuated by the expression of cPLA(2) 135-366, or cPLA(2) 2-366, and expression of a PIP(2)-specific 5'-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP(2), with high affinity, only cPLA(2) 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA(2)s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA(2) activity can be modulated by sequestration or depletion of cellular PIP(2), although the interaction of cPLA(2) with membrane PIP(2) appears to be indirect, or of weak affinity.     

Investigation of the mechanism of the methylmalonyl-CoA mutase reaction with the substrate analogue: ethylmalonyl-CoA.

1. Ethylmalonyl-CoA was found to be a substrate for methylmalonyl-CoA mutase from Propionibacterium shermanii, the product being mainly (2R)-methylsuccinyl-CoA along with some (2S)-diastereoisomer. 2. The relevant 1H-nuclear magnetic resonance signals of methylsuccinic acid and of its dimethyl ester were assigned to the diastereotopic methylene hydrogens using sterospecifically dideuterated specimens of known configuration. 3. [2(-2)H1]Ethylmalonyl-CoA was converted by methylmalonyl-CoA mutase in 2H2O mainly to (2R, 3S)-[3(-2)H1]methylsuccinyl-CoA. No dideuterated product was observed. 4. Starting from (1R)-[1(-2)H1]-ethathanol, (1S)-[1(-2)H1]ethanol and [2H6] ethanol the following deuterated specimens of ethylmalonic acid were synthesised and characterised: (3S)-[3(-2)H1], (3R)-[3(-2)H1] and [3(-2)H2, 4(-2)H3], respectively. 5. Conversion of (3S)-[3(-2)H1]-ethylmalonyl-CoA (70% 2H1 and 2% 2H2 species) on the mutase in water afforded mainly (2R)-[2(-2)H1]methylsuccinyl-CoA along with some (2S)-diastereoisomer. No deuterium loss was observed. 6. Methylmalonyl-CoA mutase converted (3R)-[3(-2)H1]ethylmalonyl-CoA (81% 2H1 and 2% 2H2 species) to the following methylsuccinyl-CoA species: 33% [3(-2)H1], the deuterium being in the threo position with respect to the methyl group; 21% [2(-2)H1]; 46% unlabelled. The ratio of the species with (2R) and (2S) configuration was about 60:40. 7. Reaction of [3(-2)H2, 4(-2)H3]ethylmalonyl-CoA (94.5% [2H5] species) with the mutase gave the following labelled methylsuccinyl-CoA species:53.4% [methyl-2H3, 2(-2)H1, 3(-2)H1], the 3-deuterium being in the threo position with respect to the methyl group; 37.6% [methyl-2H3, 2(-2)H1]; 5% [methyl(-2)H3, 2(-2)H1, 2(-2)H1, 3(-2)H1] the 3-deuterium being in erythro position with respect to the methyl group; 4% [methyl(-2)H3, 3(-2)H1]. The ratio of the species with (2R) and (2S) configuration was about 70:30. 8. Implications of these findings for the mechanism of the rearrangements catalysed by coenzyme B12 are discussed.     

Iron and hydrogen peroxide detoxification properties of DNA-binding protein from starved cells. A ferritin-like DNA-binding protein of Escherichia coli

The DNA-binding proteins from starved cells (Dps) are a family of proteins induced in microorganisms by oxidative or nutritional stress. Escherichia coli Dps, a structural analog of the 12-subunit Listeria innocua ferritin, binds and protects DNA against oxidative damage mediated by H(2)O(2). Dps is shown to be a Fe-binding and storage protein where Fe(II) oxidation is most effectively accomplished by H(2)O(2) rather than by O(2) as in ferritins. Two Fe(2+) ions bind at each of the 12 putative dinuclear ferroxidase sites (P(Z)) in the protein according to the equation, 2Fe(2+) + P(Z) --> [(Fe(II)(2)-P](FS)(Z+2) + 2H(+). The ferroxidase site (FS) bound iron is then oxidized according to the equation, [(Fe(II)(2)-P](FS)(Z+2) + H(2)O(2) + H(2)O --> [Fe(III)(2)O(2)(OH)-P](FS)(Z-1) + 3H(+), where two Fe(II) are oxidized per H(2)O(2) reduced, thus avoiding hydroxyl radical production through Fenton chemistry. Dps acquires a ferric core of approximately 500 Fe(III) according to the mineralization equation, 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(III)OOH((core)) + 4H(+), again with a 2 Fe(II)/H(2)O(2) stoichiometry. The protein forms a similar ferric core with O(2) as the oxidant, albeit at a slower rate. In the absence of H(2)O(2) and O(2), Dps forms a ferrous core of approximately 400 Fe(II) by the reaction Fe(2+) + H(2)O + Cl(-) --> Fe(II)OHCl((core)) + H(+). The ferrous core also undergoes oxidation with a stoichiometry of 2 Fe(II)/H(2)O(2). Spin trapping experiments demonstrate that Dps greatly attenuates hydroxyl radical production during Fe(II) oxidation by H(2)O(2). These results and in vitro DNA damage assays indicate that the protective effect of Dps on DNA most likely is exerted through a dual action, the physical association with DNA and the ability to nullify the toxic combination of Fe(II) and H(2)O(2). In the latter process a hydrous ferric oxide mineral core is produced within the protein, thus avoiding oxidative damage mediated by Fenton chemistry.     

Synthesis of an octasaccharide fragment of high-mannose-type glycans of glycoproteins.

O-(alpha-D-Mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----3)- O- [(alpha-D-mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----6)]- O- (alpha-D-mannopyranosyl)-(1----6)-O-(beta-D-mannopyranosyl)-(1----4)-O-( 2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- glucopyranose, an octasaccharide fragment of high-mannose type glycan of glycoproteins, was synthesized. Crucial glycosylation of trisaccharide intermediate, benzyl O-(2,4-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(2-acetamido-3,6-di -O- benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-3,6-di-O-benz yl-2- deoxy-beta-D-glucopyranoside, was successful only with a di-O-acetyltetradeca-O-benzyl-D-mannopentaosyl chloride. The use of the corresponding hexadeca-O-acetyl-D-mannopentaosyl bromide did not give the desired product.     

Synthesis of novel heterobranched beta-cyclodextrins from 4(2)-O-beta-D-galactosyl-maltose and beta-cyclodextrin by the reverse action of pullulanase, and isolation and characterization of the products

From the mixture of 4(2)-O-beta-D-galactosyl-maltose (Gal-G2) and beta-cyclodextrin (betaCD), novel heterobranched betaCDs, (Gal-G2)-betaCD and (Gal-G2)2-betaCDs, were synthesized by the reverse action of debranching enzyme. The optimum conditions for the production of (Gal-G2)2-betaCDs were examined. A mixture of (Gal-G2)2-betaCDs was produced in about 4% yield when Aerobacter aerogenes pullulanase (64 units per 1 g of Gal-G2) was incubated with 1.6 M Gal-G2 and 0.16 M betaCD at 50 degrees C for 4 days. The reaction products, (Gal-G2)2-betaCDs, were separated into three peaks by HPLC analysis on a Hypercarb S column. Their structures were analyzed by fast atom bombardment mass spectroscopy and NMR spectroscopies, and confirmed by comparison of their hydrolyzates by beta-galactosidase with the authentic (G2)2 -betaCDs. The structures of (Gal-G2)-betaCD and three components of (Gal-G2)2-betaCDs were identified as 6-O-(GalG2)-betaCD, 6(1),6(2)-, 6(1),6(3)- and 6(1),6(4)-di-O-(Gal-G2)2-betaCD, respectively.     

Hydrolysis of phosphatidylinositol 4,5-bisphosphate mediates calcium-induced inactivation of TRPV6 channels

TRPV6 is a member of the transient receptor potential superfamily of ion channels that facilitates Ca(2+) absorption in the intestines. These channels display high selectivity for Ca(2+), but in the absence of divalent cations they also conduct monovalent ions. TRPV6 channels have been shown to be inactivated by increased cytoplasmic Ca(2+) concentrations. Here we studied the mechanism of this Ca(2+)-induced inactivation. Monovalent currents through TRPV6 substantially decreased after a 40-s application of Ca(2+), but not Ba(2+). We also show that Ca(2+), but not Ba(2+), influx via TRPV6 induces depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2) or PIP(2)) and the formation of inositol 1,4,5-trisphosphate. Dialysis of DiC(8) PI(4,5)P(2) through the patch pipette inhibited Ca(2+)-dependent inactivation of TRPV6 currents in whole-cell patch clamp experiments. PI(4,5)P(2) also activated TRPV6 currents in excised patches. PI(4)P, the precursor of PI(4,5)P(2), neither activated TRPV6 in excised patches nor had any effect on Ca(2+)-induced inactivation in whole-cell experiments. Conversion of PI(4,5)P(2) to PI(4)P by a rapamycin-inducible PI(4,5)P(2) 5-phosphatase inhibited TRPV6 currents in whole-cell experiments. Inhibiting phosphatidylinositol 4 kinases with wortmannin decreased TRPV6 currents and Ca(2+) entry into TRPV6-expressing cells. We propose that Ca(2+) influx through TRPV6 activates phospholipase C and the resulting depletion of PI(4,5)P(2) contributes to the inactivation of TRPV6.     

Synthesis of methyl glycoside derivatives of tri- and penta-saccharides related to the antithrombin III-binding sequence of heparin, employing cellobiose as a key starting-material

Two key synthons for the title pentasaccharide derivative, methyl O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-acetyl- 2-azido - 3-O- benzyl-2-deoxy-beta-D-glucopyranoside and O-(methyl 2,3-di-O-benzyl-4-O- chloroacetyl-beta-D-glucopyranosyluronate)-(1----4)-3,6-di-O-acetyl-2-az ido-2- deoxy-alpha-D- glucopyranosyl bromide, were prepared from a common starting material, cellobiose. They were coupled to give a tetrasaccharide derivative that underwent O-dechloroacetylation to the corresponding glycosyl acceptor. Its condensation with the known 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide afforded a 77% yield of suitably protected pentasaccharide, methyl O-(6-O- acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)- O- (methyl 2,3- di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-O-(3,6-di-O-acetyl-2- azido-2 - deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L- idopyranosyluronate)- (1----4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranoside. Sequential deprotection and sulfation gave the decasodium salt of methyl O-(2- deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1----4)-O-(be ta-D- glucopyranosyl-uronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gluco pyranosyl)- (1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1----4)-2-deoxy-2- sulfamido-6-O- sulfo-beta-D-glucopyranoside (3). In a similar way, the trisaccharide derivative, the hexasodium salt of methyl O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)- (1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-2-deoxy-2-sulfamido-3,6- di-O- sulfo-alpha-D-glucopyranoside (4) was synthesized from methyl O-(6-O-acetyl-2- azido- 3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2,3-di-O- benzyl-beta- D-glucopyranosyluronate)-3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D- glucopyranoside. The pentasaccharide 3 binds strongly to antithrombin III with an association constant almost equivalent to that of high-affinity heparin, but the trisaccharide 4 appears not to bind.     

A nuclear magnetic resonance spectroscopic investigation of Kdo-containing oligosaccharides related to the genus-specific epitope of Chlamydia lipopolysaccharides.

The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2----8)-linked Kdo (unit b) in alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcN-(1--- -6)- GlcNol, alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1- ---O)- allyl, and alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl relative to that preferred in alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, alpha-Kdo-(2----8)-alpha-Kdo-(2----O)-allyl, alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl, and alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, irrespective of the size of the aglycon, e.g., allyl or beta-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach.