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PubMed2003

Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide.

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of LPS and is virtually complete within 8 h, nodules occurring mainly associated with the dorsal diaphragm on either side of the heart, but sometimes with smaller numbers associated with the ventral diaphragm on either side of the nerve cord. Co-injection of adipokinetic hormone-I (Lom-AKH-I) with LPS stimulates greater numbers of nodules to be formed in larval and adult locusts, and activates phenoloxidase in the haemolymph of mature adults but not of nymphs. The effect of co-injection of Lom-AKH-I with LPS on nodule formation is seen at low doses of hormone; only 0.4 pmol of Lom-AKH-I per adult locust is needed to produce a 50% increase in the number of nodules formed. When different components of LPS from the E. coli Rd mutant are tested, the mono- and the diphosphoryl Lipid A components have similar effects to the intact LPS. Remarkably, detoxified LPS activates phenoloxidase in the absence of Lom-AKH-I, although co-injection with hormone does enhance this response. Both diphosphoryl Lipid A and detoxified LPS induce a level of nodule formation that is enhanced by co-injection of Lom-AKH-I, but monophosphoryl Lipid A does not initiate nodule formation even when injected with hormone. Co-injection of a water-soluble inhibitor of eicosanoid synthesis, diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid), reduces nodule formation in response to injections of LPS (both in the absence and presence of hormone) in a dose-dependent manner, but does not prevent activation of phenoloxidase in adult locusts. It is shown that nodule formation and activation of the prophenoloxidase in locust haemolymph can both be enhanced by Lom-AKH-I, but it is argued that these processes involve distinct mechanisms in which eicosanoid synthesis is important for nodule formation, but not for the increased phenoloxidase activity.

Animals↗
PubMed1999

The effect of an insulin releasing agent, BTS 67582, on advanced glycation end product formation in vitro.

BTS 67582 (1,1-dimethyl-2-(2-morpholinophenyl) guanidine fumarate) is an insulin-releasing agent currently in phase II clinical trials. Its effect on advanced glycation end product (AGE) formation was measured in the BSA/D-glucose and L-lysine/glucose-6-phosphate assay systems and Amadori product formation was measured in the BSA/D-glucose assay system, following a 3 week incubation period. In the BSA/D-glucose assay system, 200 mM BTS 67582 caused an approximate 70% inhibition in AGE formation (p<0.001), whilst at 20 mM and 2 mM it caused a marginal inhibition (21%, (p<0.001) and 8% respectively). 200 mM and 20 mM aminoguanidine-HCl inhibited AGE formation by 95% and 69% (p<0.001), respectively, whereas 2 mM aminoguanidine-HCl had no significant effect. Tolbutamide (200 microM) and glibenclamide (100 microM) had significant, but only marginal, effects on AGE formation (16% and 17%, respectively, p<0.01). In the BSA/D-glucose assay system 200 mM BTS 67582 and 200 mM aminoguanidine-HCl retarded Amadori product formation by 88% (p<0.001) and 60% (p<0.01), respectively. BTS 67582 at 20 mM and 2 mM was shown to inhibit Amadori product formation by 67% and 57%, respectively, (p<0.01). In the lysine and glucose-6-phosphate assay system 200 mM BTS 67582 and 200 mM aminoguanidine-HCl were shown to inhibit AGE formation by about 70% and 96% (p<0.001), respectively. Tolbutamide (200 microM) and glibenclamide (100 microM) had no significant effect on AGE formation.

Ferrous Compounds↗
PubMed2003

Copper catalysis in chloroform formation during water chlorination.

The seminal work of Rook initiated a considerable body of research regarding the formation of trihalomethanes (THMs) and other by-products of chlorine-based disinfection. Since that time, a broad spectrum of compounds has been identified as precursors to THM formation. More recently, it has been demonstrated that the presence of copper in solution enhances THM formation. Copper is known to catalyze a number of reactions that are similar to the conventional haloform reaction. A study was therefore initiated to investigate the specific role played by copper in the formation of chloroform during chlorination of water supplies. Aqueous solutions containing a number of known THM precursors were chlorinated in the presence and absence of copper, and subjected to time-course monitoring of chloroform concentration. The results of experiments with humic acid demonstrated an apparent catalytic effect on the part of copper in chloroform formation. To examine the role of copper in greater detail, a series of experiments involving aqueous solutions of pure compounds of humic substance structural units was conducted. Of the pure compounds investigated as THM precursors, only citric acid demonstrated enhanced chloroform formation in the presence of copper. A detailed matrix of experiments conducted with citric acid as a precursor demonstrated that copper, at environmentally relevant concentrations, can have a profound effect on chloroform formation. Based on previously published information regarding the mechanism of chloroform formation from citric acid and the results of these experiments, it is hypothesized that copper promotes chloroform formation from chlorination of citric acid through catalysis of oxidative decarboxylation, and the subsequent chlorination of beta-ketoglutaric acid.

Catalysis↗
PubMedDate not supplied

Carcinogenic metal induced sites of reactive oxygen species formation in hepatocytes.

Severe chronic liver disease results from the hepatic accumulation of copper nickel, cobalt or iron in humans and on the other hand cadmium, dichromate and arsenic may induce lung or kidney cancer. Acute or chronic CdCl2, HgCl2 or dichromate administration induces hepatic and nephrotoxicity in rodents. Oxidative stress is often cited as a possible cause but has not yet been measured. For the first time we have measured the reactive oxygen species (ROS) formation induced when cells are incubated with metals and determined its source. Hepatocytes incubated with 2',7'-dichlorofluorescin diacetate resulted in its rapid uptake and deacetylation by intracellular esterases to form 2',7'-dichlorofluorescin. A marked increase in ROS formation occurred with LD50 concentrations of cadmium [Cd(II)], Hg(II) or arsenite [As(III)] which was released by proton ionophores that uncouple oxidative phosphorylation. Uncouplers or oxidative phosphorylation also inhibited ROS formation induced by these metals, which suggests that mitochondria are major contributors to endogenous ROS formation. Glycolytic substrates also inhibited Cd(II)/Hg(II)/As(III)-induced ROS formation and confirms that mitochondria are the site of ROS formation. By contrast ROS formation by LD50 concentrations of Cu(II), Ni(II), Co(II) or dichromate [Cr(VI)] were not affected by uncouplers or glycolytic substrates. However they were inhibited by lysosomotropic agents or endogenous inhibitors [in contrast to Hg(II), Cd(II) or As(III)]. Furthermore Cu(II), Ni(II), Co(II) or Cr(VI) accumulated in the lysosomes and the ROS formed caused a loss of lysosomal membrane integrity. The release of lysosomal proteases and phospholipases also contributed to hepatocyte cytotoxicity. ROS formation and cytotoxicity induced by added H2O2 or generated by the intracellular redox cycling of nitrofurantoin was also inhibited by lysosomotropic agents and ferric chelators suggesting that lysosomal Fe(II) contributes to H2O2-induced cytotoxicity. In conclusion, lysosomes are sites of cytotoxic ROS formation with redox transition metals (CuII, CrVI, NiII, CoII) whereas mitochondria are the ROS sites for non-redox or poor redox cycling transition metals (CdII, HgII, AsIII).

Animals↗
PubMed2002

Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes.

The rat hepatocyte catalyzed oxidation of 2',7'-dichlorofluorescin to form the fluorescent 2,7'-dichlorofluorescein was used to measure endogenous and xenobiotic-induced reactive oxygen species (ROS) formation by intact isolated rat hepatocytes. Various oxidase substrates and inhibitors were then used to identify the intracellular oxidases responsible. Endogenous ROS formation was markedly increased in catalase-inhibited or GSH-depleted hepatocytes, and was inhibited by ROS scavengers or desferoxamine. Endogenous ROS formation was also inhibited by cytochrome P450 inhibitors, but was not affected by oxypurinol, a xanthine oxidase inhibitor, or phenelzine, a monoamine oxidase inhibitor. Mitochondrial respiratory chain inhibitors or hypoxia, on the other hand, markedly increased ROS formation before cytotoxicity ensued. Furthermore, uncouplers of oxidative phosphorylation inhibited endogenous ROS formation. This suggests endogenous ROS formation can largely be attributed to oxygen reduction by reduced mitochondrial electron transport components and reduced cytochrome P450 isozymes. Addition of monoamine oxidase substrates increased antimycin A-resistant respiration and ROS formation before cytotoxicity ensued. Addition of peroxisomal substrates also increased antimycin A-resistant respiration but they were less effective at inducing ROS formation and were not cytotoxic. However, peroxisomal substrates readily induced ROS formation and were cytotoxic towards catalase-inhibited hepatocytes, which suggests that peroxisomal catalase removes endogenous H(2)O(2) formed in the peroxisomes. Hepatocyte catalyzed dichlorofluorescin oxidation induced by oxidase substrates, e.g., benzylamine, was correlated with the cytotoxicity induced in catalase-inhibited hepatocytes.

Animals↗
PubMed1998

Basic fibroblast growth factor induces osteoclast formation in murine bone marrow cultures.

We determined the effect of basic fibroblast growth factor (bFGF) on osteoclast-like cell (OCL) formation in bone marrow cultures using C57BL/6 mice. Cells were cultured for 7 days with or without bFGF at various concentrations or 10(-8) mol/L 1,25(OH)2 vitamin D3 [1,25(OH)2D3]. bFGF dose-dependently increased OCL formation per well (10(-10) mol/ L = 40 +/- 2; 10(-9) mol/L = 146 +/- 13; 10(-8) mol/L = 156 +/- 12) compared with control (< 7 per well). The effects of bFGF at 10(-9) and 10(-8) mol/L were similar to that of 10(-8) mol/L 1,25(OH)2D3 (154 +/- 11 per well). OCLs formed by bFGF were multinuclear, tartrate-resistant acid phosphatase (TRAP)-positive, expressed calcitonin receptors, and formed characteristic resorption pits. We also determined whether bFGF enhanced OCL formation during the early proliferative or late differentiating phases of the cultures. When bFGF (10(-8) mol/L) was added only on days 1-2 or days 3-4 of 6 day cultures, there was a significant increase in OCL formation. In contrast, when bFGF was added only on days 5-6 few OCLs formed. Addition of bFGF at days 1-6 or days 1-2 and days 5-6 caused similar increases in OCL formation, which were greater than OCL formation induced by treatment for days 1-2 or days 1-4. We examined the production of prostaglandin E2 (PGE2) in the cultures because bFGF is a potent stimulator of PGE2 synthesis in bone, and PGE2 stimulates OCL formation. bFGF treatment significantly increased PGE2 levels in 7 day cultures (controls = 1.4 +/- 0.1 nmol/L, 10(-8) mol/L bFGF = 132.5 +/- 0.7 nmol/L). In addition, treatment of marrow cultures with the prostaglandin synthesis inhibitors, indomethacin or NS-398 (both at 10(-6) mol/L), completely blocked bFGF-induced OCL formation. We conclude that bFGF stimulates OCL formation in C57BL/6 bone marrow cultures by mechanisms that require prostaglandin synthesis. This pathway is likely to be one mechanism by which bFGF stimulates resorption.

Actins↗
PubMed2001

Probing the mechanism of insulin fibril formation with insulin mutants.

The molecular basis of insulin fibril formation was investigated by studying the structural properties and kinetics of fibril formation of 20 different human insulin mutants at both low pH (conditions favoring monomer/dimer) and at pH 7.4 (conditions favoring tetramer/hexamer). Small-angle X-ray scattering showed insulin to be monomeric in 20% acetic acid, 0.1 M NaCl, pH 2. The secondary structure of the mutants was assessed using far-UV circular dichroism, and the tertiary structure was determined using near-UV circular dichroism, quenching of intrinsic fluorescence by acrylamide and interactions with the hydrophobic probe 1-anilino-8-naphthalene-sulfonic acid (ANS). The kinetics of fibril formation were monitored with the fluorescent dye, Thioflavin T. The results indicate that the monomer is the state from which fibrils arise, thus under some conditions dissociation of hexamers may be rate limiting or partially rate limiting. The insulin mutants were found to retain substantial nativelike secondary and tertiary structure under all conditions studied. The results suggest that fibril formation of the insulin mutants is controlled by specific molecular interactions that are sensitive to variations in the primary structure. The observed effects of several mutations on the rate of fibril formation are inconsistent with a previously suggested model for fibrillation [Brange, J., Whittingham, J., Edwards, D., Youshang, Z., Wollmer, A., Brandenburg, D., Dodson, G., and Finch, J. (1997) Curr. Sci. 72, 470-476]. Two surfaces on the insulin monomer are identified as potential interacting sites in insulin fibrils, one consisting of the residues B10, B16, and B17 and the other consisting of at least the residues A8 and B25. The marked increase in the lag time for fibril formation with mutations to more polar residues, as well as mutations to charged residues, demonstrates the importance of both hydrophobic and electrostatic interactions in the initial stages of fibrillation. A model for insulin fibril formation is proposed in which the formation of a partially folded intermediate is the precursor for associated species on the pathway to fibril formation.

Amino Acid Sequence↗
PubMed2004

Kinetic characterization of thiolate anion formation and chemical catalysis of activated microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) displays the unique ability to be activated, up to 30-fold, by the reaction with sulfhydryl reagents, e.g., N-ethylmaleimide. Analysis of glutathione (GSH) thiolate formation, which occurs upon mixing activated MGST1 with GSH, reveals biphasic kinetics, where the rapid phase dominated at higher GSH concentrations. The kinetic behavior suggests a two-step mechanism consisting of a rapid GSH-binding step (K(D)(GSH) approximately 10 mM), followed by slower formation of thiolate (k(2) approximately 10 s(-1)). The release rate (or protonation of the enzyme GSH thiolate complex) of GS(-) was slow (k(-2) = 0.016 s(-1)), consistent with overall tight binding of GSH. Electrophilic second substrates react rapidly with the E*GS(-) complex, and again, a two-step mechanism is suggested. In comparison to the unactivated enzyme [Morgenstern et al. (2001) Biochemistry 40, 3378-3384], the mechanisms of GSH thiolate formation and electrophile interaction are similar; however, thiolate anion formation is enhanced 30-fold in the activated enzyme, contributing to an increased k(cat) (3.6 s(-1)). Interestingly, in the activated enzyme, thiolate formation and proton release from the enzyme are not strictly coupled, because proton release (as well as k(cat)) was found to be approximately 4 times slower than GSH thiolate formation in an unbuffered system. Solvent kinetic isotope effect measurements demonstrated a 2-fold decrease in the rate constant (k(2)) for thiolate formation and k(cat) (in the reaction with 1-chloro-2,4-dinitrobenzene) for both unactivated and activated MGST1. This indicates that thiolate formation contributes to k(cat) for the activated enzyme, as suggested previously for unactivated MGST1. The stoichiometry of thiolate formation, proton release, and burst kinetics suggested utilization of one GSH molecule per enzyme trimer.

Animals↗
PubMed2005

Correlation of acrylamide generation in thermally processed model systems of asparagine and glucose with color formation, amounts of pyrazines formed, and antioxidative properties of extracts.

The relations between the formation of acrylamide and color, pyrazines, or antioxidants in an asparagine/d-glucose browning model system under various conditions were investigated. The highest level of acrylamide was produced in the asparagine/glucose (1:3) system heated at 170 degrees C for 30 min (2629 microg/g asparagine). Color intensity increased with temperature and heating time. The formation of pyrazines increased steadily with an increase of temperature (140-170 degrees C) and heating time (15-60 min). Antioxidant formation varied among the samples heated under different conditions. A clear correlation between formation of acrylamide and browning color was obtained. The formation of acrylamide was linearly correlated with the formation of total pyrazines during the initial stages of the Maillard reaction. No obvious correlation between formation of acrylamide and antioxidants was observed. However, excess amounts of asparagine increased the formation of antioxidants, whereas excess amounts of glucose reduced its formation.

Acrylamides↗
PubMed2006

Selection criteria for potato tubers to minimize acrylamide formation during frying.

A number of parameters linked to the selection of potato tubers were evaluated with regard to their potential to influence acrylamide formation in French fries. The formation of acrylamide, which is a potential human carcinogen, can be minimized for a big extent by the selection of an appropriate tuber. This study focused on the following selection criteria: variety as influenced by storage time and soil type, underwater weight, and tuber size. A total of 16 varieties were compared, concerning their potential for acrylamide formation. From that survey, certain varieties, such as Tebina and Quincy, could be appointed as unsuitable for frying. The differences in the potential of acrylamide formation between the varieties could mainly be explained by the reducing sugar content of the potato (R2 = 0.82, n = 96). The investigated type of soil and storage time at 8 degrees C appeared to have a minor influence on the acrylamide formation during frying. On the other hand, the tuber size of the potato did contribute in a significant manner to the acrylamide formation. Smaller tubers were more susceptible to acrylamide formation and should be avoided in the frying process. The last selection parameter, the underwater weight, appeared to be of minor importance in the acrylamide formation. On the basis of these simple selection criteria, it is possible to make a first screening of potatoes to reduce the acrylamide formation during frying.

Acrylamides↗
PubMed2006

Enthalpies of formation, bond dissociation energies, and molecular structures of the n-aldehydes (acetaldehyde, propanal, butanal, pentanal, hexanal, and heptanal) and their radicals.

Aldehydes are important intermediates and products in a variety of combustion and gas-phase oxidation processes, such as in low-temperature combustion, in the atmosphere, and in interstellar media. Despite their importance, the enthalpies of formation and bond dissociation energies (BDEs) for the aldehydes are not accurately known. We have determined enthalpies of formation for acetaldehyde, propanal, and butanal from thermodynamic cycles, using experimentally measured reaction and formation enthalpies. All enthalpy values used for reference molecules and reactions were first verified to be accurate to within around 1 kcal mol-1 using high-level ab initio calculations. Enthalpies of formation were found to be -39.72 +/- 0.16 kcal mol-1 for acetaldehyde, -45.18 +/- 1.1 kcal mol-1 for propanal, and -49.27 +/- 0.16 kcal mol-1 for butanal. Enthalpies of formation for these three aldehydes, as well as for pentanal, hexanal, and heptanal, were calculated using the G3, G3B3, and CBS-APNO theoretical methods, in conjunction with bond-isodesmic work reactions. On the basis of the results of our thermodynamic cycles, theoretical calculations using isodesmic work reactions, and existing experimental measurements, we suggest that the best available formation enthalpies for the aldehydes acetaldehyde, propanal, butanal, pentanal, hexanal, and heptanal are -39.72, -45.18, -50.0, -54.61, -59.37, and -64.2 kcal mol-1, respectively. Our calculations also identify that the literature enthalpy of formation of crotonaldehyde is in error by as much as 1 kcal mol-1, and we suggest a value of -25.1 kcal mol-1, which we calculate using isodesmic work reactions. Bond energies for each of the bonds in the aldehydes up to pentanal were calculated at the CBS-APNO level. Analysis of the BDEs reveals the R-CH(2)CH=O to be the weakest bond in all aldehydes larger than acetaldehyde, due to formation of the resonantly stabilized vinoxy radical (vinyloxy radical/formyl methyl radical). It is proposed that the vinoxy radical as well as the more commonly considered formyl and acetyl radicals are important products of aldehyde combustion and oxidation, and the reaction pathways of the vinoxy, formyl, and acetyl radicals are discussed. Group additivity values for the carbon-oxygen-hydrogen groups common to the aldehydes are also determined. Internal rotor profiles and electrostatic potential surfaces are used to study the dipole induced dipole-dipole interaction in the synperiplanar conformation of propanal. It is proposed that the loss of this dipole-dipole interaction in RC(.-)HCH(2)CH=O radicals causes a ca. 1-2 kcal mol-1 decrease in the aldehyde C-H and C-C bond energies corresponding to RC(.-)HCH(2)CH=O radical formation.

Journal Article↗
PubMed2007

Air-facilitated three-phase contact formation at hydrophobic solid surfaces under dynamic conditions.

The paper presents results documenting the mechanism of facilitation of the three-phase contact (TPC) formation due to gas entrapped during immersion of hydrophobic (Teflon) plates into distilled water and n-octanol solutions. Collisions, bouncing, the time scale of the TPC formation, and bubble attachment to Teflon plates of different surface roughness were studied using a high-speed camera. Processes occurring during the microscopic wetting film formation at the Teflon plates were monitored using the microinterferometric method (Scheludko-Exerowa cell). A strong relation between the time necessary to form a stable TPC and the roughness of the Teflon was observed. The higher the Teflon roughness was the shorter the time for the TPC formation. This effect can be attributed to two factors: (i) local differences in the thickness of the thinning intervening liquid layer (quicker attainment of rupture thickness at pillars of rough surface) and/or (ii) the presence of gas at the hydrophobic surface. Experimental findings, that (i) prolongation of the plate immersion time resulted in quicker TPC formation, (ii) white irregular and disappearing spots (air pockets) were recorded during the wetting film formation, and (iii) high n-octanol concentration caused prolongation of the time of the TPC formation, show that attachment (TPC formation) of the colliding bubble to hydrophobic surfaces was facilitated by air entrapped at the Teflon plates (and re-distributed) during their immersion into water phase. Thus, on collision instead of solid/gas wetting liquid film a thin gas/liquid/gas foam film was formed which facilitated the TPC formation.

Journal Article↗
PubMed1969

Lipid peroxide formation in microsomes. General considerations.

1. Liver microsomes form lipid peroxide when incubated with ascorbate or NADPH, but not with NADH. Increasing the concentration of ascorbate beyond the optimum (0.5mm) decreases the rate of lipid peroxide formation, but this effect does not occur with NADPH. Other reducing agents such as p-phenylenediamine or ferricyanide were not able to replace ascorbate and induce lipid peroxide formation. 2. The rate of ascorbate-induced peroxidation is optimum at pH6.0 whereas the rate of the NADPH system is optimum at pH7.0. Both systems require phosphate for maximum activity. 3. Lipid peroxide formation occurs at the maximum specific rate in very dilute microsome suspensions (0.15mg. of protein/ml.). 4. Treatment of microsomes with deoxycholate and other detergents causes membrane disintegration and inhibits lipid peroxide formation. 5. Lipid peroxide formation is accompanied by a rapid uptake of oxygen and there is a large excess of oxygen utilized for each molecule of malonaldehyde measured in the peroxide method. 6. Boiled microsomes form lipid peroxide in the presence of ascorbate, but not if NADPH is added. 7. Lipid peroxide formation induced by NADPH is strongly inhibited by p-chloromercuribenzoate, weakly inhibited by N-ethylmaleimide and unaffected by iodoacetamide. Ascorbate-induced peroxidation in untreated microsomes is unaffected by p-chloromercuribenzoate, but inhibited if boiled microsomes are used. These experiments may be interpreted on the basis that a ferredoxin-type protein forms part of the system in which NADPH induces lipid peroxide formation. 8. Most heavy-metal ions, with the exception of inorganic iron (Fe(2+) or Fe(3+)), which activates, inhibit both ascorbate-induced and NADPH-induced peroxidation. Mg(2+) increases the rate of peroxidation whereas Ca(2+) inhibits it. 9. Lipid peroxide formation is inhibited strongly by GSH and weakly by cysteine. Ascorbate-induced peroxidation is much more sensitive than NADPH-induced peroxidation. 10. Peroxidation is strongly inhibited by addition of low concentrations (0.01-0.1mm) of cytochrome c or of haemoglobin. 11. It is considered that lipid peroxide formation occurs as a result of the operation of the microsomal electron-transport chain switching from hydroxylation to oxidize unsaturated lipids of the endoplasmic reticulum.

Animals↗
PubMed1993

Inhibition of iron-catalysed hydroxyl radical formation by inositol polyphosphates: a possible physiological function for myo-inositol hexakisphosphate.

1. The ability of myo-inositol polyphosphates to inhibit iron-catalysed hydroxyl radical formation was studied in a hypoxanthine/xanthine oxidase system [Graf, Empson and Eaton (1987) J. Biol. Chem. 262, 11647-11650]. Fe3+ present in the assay reagents supported some radical formation, and a standard assay, with 5 microM Fe3+ added, was used to investigate the specificity of compounds which could inhibit radical generation. 2. InsP6 (phytic acid) was able to inhibit radical formation in this assay completely. In this respect it was similar to the effects of the high affinity Fe3+ chelator Desferral, and dissimilar to the effects of EDTA which, even at high concentrations, still allowed detectable radical formation to take place. 3. The six isomers of InsP5 were purified from an alkaline hydrolysate of InsP6 (four of them as two enantiomeric mixtures), and they were compared with InsP6 in this assay. Ins(1,2,3,4,6)P5 and D/L-Ins(1,2,3,4,5)P5 were similar to InsP6 in that they caused a complete inhibition of iron-catalysed radical formation at > 30 microM. Ins(1,3,4,5,6)P5 and D/L-Ins(1,2,4,5,6)P5, however, were markedly less potent than InsP6, and did not inhibit radical formation completely; even when Ins(1,3,4,5,6)P5 was added up to 600 microM, significant radical formation was still detected. Thus InsP5s lacking 2 or 1/3 phosphates are in this respect qualitatively different from InsP6 and the other InsP5s. 4. scyllo-Inositol hexakisphosphate was also tested, and although it caused a greater inhibition than Ins(1,3,4,5,6)P5, it too still allowed detectable free radical formation even at 600 microM. 5. We conclude that the 1,2,3 (equatorial-axial-equatorial) phosphate grouping in InsP6 has a conformation that uniquely provides a specific interaction with iron to inhibit totally its ability to catalyse hydroxyl radical formation; we suggest that a physiological function of InsP6 might be to act as a 'safe' binding site for iron during its transport through the cytosol or cellular organelles.

Ferric Compounds↗
PubMed1994

Intracellular Ca2+, inositol 1,4,5-trisphosphate and additional signalling in the stimulation by platelet-activating factor of prostaglandin E2 formation in P388D1 macrophage-like cells.

In the P388D1 macrophage-like cell line, phospholipase A2 activity and prostaglandin production are stimulated by platelet-activating factor (PAF) and bacterial lipopolysaccharide (LPS). We have investigated the role of Ins(1,4,5)P3 and Ca2+ in signal transduction of PAF-induced prostaglandin E2 (PGE2) formation in these cells. The role of Ca2+ in the activation mechanism was studied by fluorescence imaging of intracellular Ca2+ in individual adherent cells and by determining the PGE2 production in the same population of cells. This new approach enabled us to correlate directly events on the single-cell level with a physiologically relevant response of the cell population. Priming the cells with LPS was required for PAF to stimulate PGE2 formation, yet LPS affected neither the intracellular free Ca2+ concentration ([Ca2+]i) nor the PAF-induced rise in [Ca2+]i. In addition, basal and PAF-stimulated Ins(1,4,5)P3 levels were not affected by LPS priming. However, the Ca2+ transient, the release of Ins(1,4,5)P3 and the formation of PGE2 induced by PAF were inhibited in cells pretreated with pertussis toxin. Buffering the [Ca2+]i with intracellular BAPTA [bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid] blocked the PAF-stimulated rise in [Ca2+]i and PGE2 formation. Removal of extracellular Ca2+ during PAF stimulation prevented the influx of Ca2+, but did not affect the initial [Ca2+]i transient, nor did it inhibit PGE2 formation. Under the same conditions, ionomycin stimulated an identical [Ca2+]i transient, but, in contrast with PAF stimulation, no PGE2 formation was observed. PGE2 production could be rescued by prompt subsequent addition of PAF, which caused no further [Ca2+]i change on its own. These results show that the transient initial rise in [Ca2+]i, produced either by PAF via the formation of Ins(1,4,5)P3 or directly by ionomycin, is necessary, but not sufficient for the formation of PGE2 in LPS-primed P388D1 cells. Furthermore, we have demonstrated for the first time that PAF triggers a second signal that is not mediated by a change in [Ca2+]i. However, both signals are required to induce PGE2 formation.

Calcium↗
PubMed2002

The influence of CYP2B6, CYP2C9 and CYP2D6 genotypes on the formation of the potent antioestrogen Z-4-hydroxy-tamoxifen in human liver.

AIMS: To investigate in a large panel of 50 human liver samples the contribution of CYP2C9, CYP2D6, and CYP3A4 to the overall formation of the potent antioestrogen Z-4-hydroxy-tamoxifen, and how various genotypes affect its formation from tamoxifen. METHODS: The formation of Z-4-hydroxy-tamoxifen from 10 microm tamoxifen was studied in human liver microsomes (n=50), characterized for CYP2B6, CYP2C9, CYP2D6 and CYP3A4 expression, and CYP2B6, CYP2C9 and CYP2D6 genotype. The effect of chemical and monoclonal antibody inhibitors, and the formation in supersomes expressing recombinant CYP isoforms was also investigated. Z-4-hydroxy-tamoxifen was quantified using LC-MS analysis. RESULTS: Z-4-hydroxy-tamoxifen was formed by supersomes expressing CYP2B6, CYP2C9, CYP2C19 and CYP2D6, but not CYP3A4. In agreement with these data, the mean formation of Z-4-hydroxy-tamoxifen was inhibited 49% by sulphaphenazole (P=0.001), 38% by quinidine (P<0.05) and 13% by monoclonal antibody against CYP2B6 (MAB-2B6, P<0.05). Furthermore, Z-4-hydroxy-tamoxifen formation significantly correlated with both CYP2C9 expression (r(s)=0.256, P<0.05) and CYP2D6 expression (r(s)=0.309, P<0.05). Genotypes of CYP2D6, CYP2B6 and CYP2C9 had an effect on metabolite formation in such a way that samples with two nonfunctional CYP2D6, or two variant CYP2C9 or CYP2B6 alleles, showed lower enzyme activity compared with those with two functional or wild-type alleles, (5.0 vs 9.9 pmol mg(-1) protein min(-1), P=0.046, 5.1 vs 9.9 pmol mg(-1) protein min(-1), P=0.053, and 6.8 vs 9.4 pmol mg(-1) protein min(-1), P=0.054, respectively). CYP2D6 and CYP2C9 contribute on average 45 and 46%, respectively, to the overall formation of Z-4-hydroxy-tamoxifen. CONCLUSIONS: CYP2B6, CYP2C9 and CYP2D6 genotypes all affected Z-4-hydroxy-tamoxifen formation and can predict individual ability to catalyse this reaction.

Adolescent↗
PubMed1996

Dose dependent effect of aprotinin on rate of clot formation.

Forty-eight patients scheduled for elective cardiac surgery were randomly assigned to receive aprotinin in the following doses: 0.2 mg kg-1 (group A), 0.7 mg kg-1 (group B), 1 mg kg-1 (group C) and 1.4 mg kg-1 (group D). Clot formation was analysed by thromboelastography immediately after induction of anaesthesia and again 30 min after administration of aprotinin. Rate of clot formation was assessed using R (reaction time = rate of initial fibrin formation), K (clot formation time = rate of fibrin build-up and cross linking) and angle of clot formation (denoting speed at which solid clot forms). Strength of the clot was assessed by maximal amplitude of clot formation (MA) and % lysis after 30 and 60 min. Significant reduction of R and K times and increase in angle of clot formation was observed in groups A and B. This effect was not apparent in the other groups. In group D, an increase in R time was noted. These findings indicate a dose-dependent effect of aprotinin on rate of clot formation with an earlier clot formation at low doses.

Aged↗
PubMed2000

N-carboxymethanofuran (carbamate) formation from methanofuran and CO2 in methanogenic archaea. Thermodynamics and kinetics of the spontaneous reaction.

N-carboxymethanofuran (carbamate) formation from unprotonated methanofuran (MFR) and CO2 is the first reaction in the reduction of CO2 to methane in methanogenic archaea. The reaction proceeds spontaneously. We address here the question whether the rate of spontaneous carbamate formation is high enough to account for the observed rate of methanogenesis from CO2. The rates of carbamate formation (v1) and cleavage (v2) were determined under equilibrium conditions via 2D proton exchange NMR spectroscopy (EXSY). At pH 7.0 and 300 K the second order rate constant k1* of carbamate formation from 'MFR'(MFR + MFRH+) and 'CO2' (CO2 + H2CO3 + HCO3-+ CO32-) was found to be 7 M-1.s-1 (v1 = k1* ['MFR'] ['CO2']) while the pseudo first order rate constant k2* of carbamate cleavage was 12 s-1 (v2 = k2* [carbamate]). The equilibrium constant K* = k1*/k2* = [carbamate]/['MFR']['CO2'] was 0.6 M-1 at pH 7.0 corresponding to a free energy change DeltaG degrees ' of + 1.3 kJ.mol-1. The pH and temperature dependence of k1*, of k2* and of K* were determined. From the second order rate constant k1* it was calculated that under physiological conditions the rate of spontaneous carbamate formation is of the same order as the maximal rate of methane formation and as the rate of spontaneous CO2 formation from HCO3- in methanogenic archaea, the latter being important as CO2 is mainly present as HCO3- which has to be converted to CO2 before it can react with MFR. An enzyme catalyzed carbamate formation thus appears not to be required for methanogenesis from CO2. Consistent with this conclusion is our finding that the rate of carbamate formation was not enhanced by cell extracts of Methanosarcina barkeri and Methanobacterium thermoautotrophicum or by purified formylmethanofuran dehydrogenase which catalyzes the reduction of N-carboxymethanofuran to N-formylmethanofuran. From the concentrations of 'CO2' and of 'MFR' determined by 1D-NMR spectroscopy and the pKa of H2CO3 and of MFRH+ the concentrations of CO2 and of MFR were obtained, allowing to calculate k1 (v1 = k1 [MFR] [CO2]). The second order rate constant k1 was found to be approximately 1000 M-1 x s-1 at 300 K and pH values between 7.0 and 8. 0 which is in the order of k1 values determined for other carbamate forming reactions by stopped flow.

Aldehyde Oxidoreductases↗