Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells.

In many diseases, tissue hypoxia occurs in conjunction with other inflammatory processes. Since previous studies have demonstrated a role for leukocytes in ischemia/reperfusion injury, we hypothesized that endothelial hypoxia may "superinduce" expression of an important leukocyte adhesion molecule, E-selectin (ELAM-1, CD62E). Bovine aortic endothelial monolayers were exposed to hypoxia in the presence or absence of tumor-necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS). Cell surface E-selectin was quantitated by whole cell ELISA or by immunoprecipitation using polyclonal anti-E-selectin sera. Endothelial mRNA levels were assessed using ribonuclease protection assays. Hypoxia alone did not induce endothelial E-selectin expression. However, enhanced induction of E-selectin was observed with the combination of hypoxia and TNF-alpha (270% increase over normoxia and TNF-alpha) or hypoxia and LPS (190% increase over normoxia and LPS). These studies revealed that a mechanism for such enhancement may be hypoxia-elicited decrements in endothelial intracellular levels of cAMP (<50% compared with normoxia). Addition of forskolin and isobutyl-methyl-xanthine during hypoxia resulted in reversal of cAMP decreases and a loss of enhanced E-selectin surface expression with the combination of TNF-alpha and hypoxia. We conclude that endothelial hypoxia may provide a novel signal for superinduction of E-selectin during states of inflammation.

Promotion of sleep mediated by the A2a-adenosine receptor and possible involvement of this receptor in the sleep induced by prostaglandin D2 in rats.

A 6-hr continuous infusion of 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenos ine (CGS21680), a selective A2a-adenosine agonist, into the subarachnoid space underlying the ventral surface region of the rostral basal forebrain, which has been defined as the prostaglandin (PG) D2-sensitive sleep-promoting zone, at rates of 0.02, 0.2, 2.0, and 12 pmol/min increased slow-wave sleep (SWS) and paradoxical sleep (PS) in a dose-dependent manner up to 183% and 202% of their respective baseline levels. The increments produced by the infusion of CGS21680 at 0.2 and 2.0 pmol/min were totally diminished when the rats had been pretreated with an i.p. injection of (E)-1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF17837; 30 mg/kg of body weight), a selective A2-adenosine antagonist. In contrast, the infusion of N6-cyclohexyladenosine (CHA), a selective A1-adenosine agonist, at 2 pmol/min significantly suppressed SWS before causing an increase in SWS, and a decrease in PS was also markedly visible. Essentially the same effects of CGS21680 and CHA were observed when these compounds were administered to the parenchymal region of the rostral basal forebrain through chronically implanted microdialysis probes. Thus, we clearly showed that stimulation of A2a-adenosine receptors in the rostral basal forebrain promotes SWS and PS. Furthermore, i.p. injections of KF17837 at 30 and 100 mg/kg of body weight dose-dependently attenuated the magnitude of the SWS increase produced by the infusion of PGD2 into the subarachnoid space of the sleep-promoting zone, thus indicating that the A2a-adenosine receptors are crucial in the sleep-promoting process triggered by PGD2.

Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals.

The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.

Susceptibility of cloned K+ channels to reactive oxygen species.

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.

A numerical approach to modeling the catalytic voltammetry of surface-confined redox enzymes

A finite difference method for simulating voltammograms of electrochemically driven enzyme catalysis is presented. The method enables any enzyme mechanism to be simulated. The finite difference equations can be represented as a matrix equation containing a nonlinear sparse matrix. This equation has been solved using the software package Mathematica. Our focus is on the use of cyclic voltammetry since this is the most commonly employed electrochemical method used to elucidate mechanisms. The use of cyclic voltammetry to obtain data from systems obeying Michaelis-Menten kinetics is discussed, and we then verify our observations on the Michaelis-Menten system using the finite difference simulation. Finally, we demonstrate how the method can be used to obtain mechanistic information on a real redox enzyme system, the complex bacterial molybdoenzyme xanthine dehydrogenase.

Ultra-endurance exercise and oxidative damage: Implications for cardiovascular health

At least 30 minutes of moderate-intensity physical activity accumulated on most, preferably all days is considered the minimum level necessary to reduce the risk of developing cardiovascular disease. Despite an unclear explanation, some epidemiological data paradoxically suggest that a very high volume of exercise is associated with a decrease in cardiovascular health. Although ultra-endurance exercise training has been shown to increase antioxidant defences (and therefore confer a protective effect against oxidative stress), an increase in oxidative stress may contribute to the development of atherosclerosis via oxidative modification of low-density lipoprotein (LDL). Research has also shown that ultra-endurance exercise is associated with acute cardiac dysfunction and injury, and these may also be related to an increase in free radical production. Longitudinal studies are needed to assess whether antioxidant defences are adequate to prevent LDL oxidation that may occur as a result of increased free radical production during very high volumes of exercise. In addition, this work will assist in understanding the accrued effect of repeated ultra-endurance exercise-induced myocardial damage.

Lead compounds for antimalarial chemotherapy: Purine base analogs discriminate between human and P-falciparum 6-oxopurine phosphoribosyltransferases

The malarial parasite Plasmodium falciparum depends on the purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) to convert purine bases from the host to nucleotides needed for DNA and RNA synthesis. An approach to developing antimalarial drugs is to use HGXPRT to convert introduced purine base analogs to nucleotides that are toxic to the parasite. This strategy requires that these compounds be good substrates for the parasite enzyme but poor substrates for the human counterpart, HGPRT. Bases with a chlorine atom in the 6-position or a nitrogen in the 8-position exhibited strong discrimination between P. falciparum HGXPRT and human HGPRT. The k(cat)/K-m values for the Plasmodium enzyme using 6-chloroguanine and 8-azaguanine as substrates were 50-80-fold and 336-fold higher than for the human enzyme, respectively. These and other bases were effective in inhibiting the growth of the parasite in vitro, giving IC50 values as low as 1 mu M.

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

Pentoxifylline and the behaviour of 29-month female mice

The dimethyl-xanthine derivative pentoxifylline (PTX) increases blood flow through capillaries. In elderly humans the drug leads to improvement in a number of imapired neuropsychological parameters. We now report that oral administration to 29-month female mice (C57, black and tan) over six days induced four different patterns of behavioural reponse: (1) consistent improvement in grooming behaviour throughout the six day trial; (2) significant improvement in light/dark zone curiosity and curiosity towards a strange object on day three, which declined but remained significantly above pre-treatment levels at day 6; (3) an improvement in general activity which only becomes detectable on day six; (4) a significant improvement in rod-walking, rearing an shuttle-box crosses on day three which returned to pre-treatment levels by day 6. Age-related deficits in general activity, grooming and curiosity were completely eliminated by the drug - the mean group performance levels attained those seen in 9-12 month individuals of this strain.

Base modified peptidic nucleic acids: synthesis and crystallographic analysis of intermediates

Peptidic Nucleic Acids (PNAs) are achiral, uncharged nucleic add mimetics, with a novel backbone composed of N-(2-aminoethyl)glycine units attached to the DNA bases through carboxymethylene linkers. With the aim of extending and improving upon the molecular recognition properties of PNAs, the aim of this work was to synthesjse PNA building block intermediates containing a series of substituted purine bases for subsequent use in automated PNA synthesis. Four purine bases: 2,6~diaminopurine (D), isoGuanine (isoG), xanthine (X) and hypoxanthine (H) were identified for incorporation into PNAs targeted to DNA, with the promise of increased hybrid stability over extended pH ranges together with improvements over the use of adenine (A) in duplex formation, and cytosine (C) in triplex formation. A reliable, high-yielding synthesis of the PNA backbone component N -('2- butyloxycarbonyl-aminoethyl)glycinate ethyl ester was establishecl. The precursor N~(2-butyloxycarbonyl)amino acetonitrile was crystallised and analysed by X-ray crystallography for the first time. An excellent refinement (R = 0.0276) was attained for this structure, allowing comparisons with known analogues. Although chemical synthesis of pure, fully-characterised PNA monomers was not achieved, chemical synthesis of PNA building blocks composed of diaminopurine, xanthine and hypoxanthine was completely successful. In parallel, a second objective of this work was to characterise and evaluate novel crystalline intermediates, which formed a new series of substituted purine bases, generated by attaching alkyl substituents at the N9 or N7 sites of purine bases. Crystallographic analysis was undertaken to probe the regiochemistry of isomers, and to reveal interesting structural features of the new series of similarly-substituted purine bases. The attainment of the versatile synthetic intermediate 2,6-dichloro~9- (carboxymethyl)purine ethyl ester, and its homologous regioisomers 6-chloro~9- (carboxymethyl)purine ethyl ester and 6-chloro-7-(carboxymethyl)purine ethyl ester, necessitated the use of X-ray crystallographic analysis for unambiguous structural assignment. Successful refinement of the disordered 2,6-diamino-9-(carboxymethyl) purine ethyl ester allowed comparison with the reported structure of the adenine analogue, ethyl adenin-9-yl acetate. Replacement of the chloro moieties with amino, azido and methoxy groups expanded the internal angles at their point of attachment to the purine ring. Crystallographic analysis played a pivotal role towards confirming the identity of the peralkylated hypoxanthine derivative diethyl 6-oxo-6,7-dihydro-3H-purlne~3,7~djacetate, where two ethyl side chains were found to attach at N3 and N7,

Mechanisms of anticholinesterase-induced myopathy and its prevention

In the introduction a brief outline of the possible mechanisms involved in the process of cellular necrosis with particular emphasis on skeletal muscle necrosis after antiChE is discussed. Ecothiopate (ECO), an antiChE, was shown to produce dose-dependent inhibition of both AChE and BuChE in diaphragm and blood of mice. Inhibition of AChE resulted in dose-dependent influx of calcium at the junctional region with the consequent development of morphological and biochemical alterations. Non-necrotising doses of ECO caused hypercontractions of varying severity, distorted end plate and slight elevation of serum creatine kinase (CK). Necrotising doses of ECO further caused contraction clumps, loss of striations and procion staining with high serum CK. The extent of ECO-induced myopathy depended on entry of extracellular calcium rather than the degree of AChE inhibition. The essential Ca2+ mediated process(es) in ECO-induced myopathy was thought to be the generation of superoxide and superoxide-derived free radicals and/or lipid peroxidation. Mitochondria and xanthine oxidase may be the major contributors to the generation of superoxide. No evidence was found for the depletion of high energy phosphates. ECO-induced myopathy could be successfully prevented by prior administration of pyridostigmine or various antioxidants, the most effective being Vit E or Vit E + N-acetylcysteine. Allopurinol or N-acetylcysteine alone were also effective. However, the use of a wide range of membrane end plate channel blockers or non-quantal release blockers were unsuccessful in the prevention of ECO-induced myopathy.

Interactions between oligoamines and superoxide anion

1- Oligoamines and EDTA inhibited the reduction of cytochrome-C and nitrobule tetrazolium (NBT) induced by the hypoxanthine/xanthine oxidase superoxide anion generating system in the following order of effectiveness: putrescine > diaminopropane > spermidine > EDTA > spermine > cadaverine. 2- Oligoamines and EDTA did not affect the rate of urate formation from the hypoxanthine/xanthine oxidase system. 3- Oligoamines and EDTA inhibited the reduction of cytochrome-C induced by stimulated PMNL's in the same order of effectiveness as mentioned before. 4- Oligoamines and EDTA inhibited luminol dependent stimulated PMNL's chemiluminescence. 5- Oligoamines and EDTA inhibited the aerobic photoreduction of NBT. 6- Oligoamines-copper sulphate complexes inhibited the reduction of cytochrome-C induced by the hypoxanthine/xanthine oxidase system more effectively than oligoamines or copper sulphate individually. 7- Superoxide anion, hydrogen peroxide and hydroxyl radical induced breakdown of isolated intact guinea pig liver lysosomes. 8- Oligoamines and EDTA protected isolated intact guinea pig liver lysosomes from the lytic effect of superoxide anion generated either by the hypoxanthine/xanthine oxidase system or by stimulated PMNL's. 9- Oligoamines and EDTA have no stabilizing effect on isolated intact guinea pig liver lysosomes. 10- The uptake of oligoamines by lysosomes was in the following order: putrescine > spermidine > spermine. 11- Oligoamines were metabolised into aldehyde compounds either by the hypoxanthine/xanthine oxidase system or stimulated PMNL's. 12- Oligoamines and EDTA have no effect on the activities of free lysosomal enzymes (acid phosphatase and -glucosaminidase). 13- Oligoamines and EDTA inhibited lipid peroxidation in guinea pig liver lysosomes induced either by the hypoxanthine/xanthine oxidase or ascorbic acid-ferrous sulphate. 14- Oligoamines and EDTA have no effect on the release of PGE_2 from stimulated peritoneal guinea pig PMNL's. 15- Oligoamines increased the uptake of (^3H)thymidine and (^3H)leucine by stimulated peritoneal guinea pig macrophages in the following order of effectiveness: spermine > spermidine > putrescine > cadaverine. 16- PGE_2, dibutyryl Cyclic AMP, and theophylline inhibited luminol dependent stimulated peritoneal guinea pig PMNL's chemiluminescence.

Studies on the anti-inflammatory effect of S-Adenosyl-L-methionine

1. S-adenosyl-L-methionine (SAMe) had no effect on cytochrome C reduction by superoxide generated from xanthine oxidase except at high concentrations. This was due to direct inhibition of the enzyme. 2. SAMe inhibited the neutrophil respiratory burst , measured by luminol enhanced chemiluminescence, to FMLP and zymosan A but not to PMA. 3. Adenosine and methylthioadenosine (MTA) inhibited the respiratory burst elicited by FMLP. 4. SAMe inhibited the phagocytosis of latex particles by neutrophils at high concentrations but methionine and S-adenosyl L-homocysteine had no effect. 5. Treatment with SAMe had no effect on cell infiltration or PGE2 production in 6-day air pouches. 6. Treatment with SAMe at the optimum dose of 50mg/kg inhibited the early phases of carrageenan induced rat hind paw inflammation but had a lesser effect on the secondary response. The antiinflammatory effect was sustained after inhibiton of polyamine synthesis. 7. SAMe increased liver putrescine levels in the presence and absence of inflammation Spermidine levels were increased in the presence of inflammation but spermine levels were unaffected by any of the treatments. 8. MT A and adenosine increased liver putrescine and spermidine levels 9. Treatment with SAMe had no effect on the polyamine status of blood. lO.Treatment with SAMe had no effect on the levels of glutathione in liver or blood. 11.SAMe and MTA inhibited histamine and platelet-activating factor (PAF) induced hind paw inflammation but had no effect on inflammation induced by dextran, zymosan, compound 48/80, 5-hydroxytryptamine, arachidonic acid or glucose oxidase. MTA was more effective than SAMe. 12. PAP-induced rat hind paw inflammation was inhibited by isoprenaline and verapamil. Combinations of these drugs with SAMe or MT A had no further enhancement of effect. 13. Incubation of rat PMNLs with [14c ] SAMe increased the intracellular levels of S-adenosyl-L-homocysteine in a dose dependent manner, but had no effect on the intracellular levels of SAMe, adenosine or MT A. 14. Pharmacokinetic studies of plasma SAMe following a single dose of the drug (50mg/kg) i.p. demonstrated that SAMe is rapidly absorbed and metabolised

Folate metabolism in the female weanling rat

The metabolism of a mixture of [2-14C] and [3',5',7,9-3H] folic acid was studied in female weanling rats. Intact folates and folate catabolites were excreted in the urine. Folate polyglutamates were found in the tissues. Rats treated with the oestrogen diethylstilbestrol and 17 -ethynyloestradiol exhibited marked changes in the metabolic handling of folic acid and folate catabolism was greatly increased compared to controls. Allopurinol treatment gave greater label retention in the gut, with a substantial increase in catabolism compared to normals. A dose response relationship was illustrated between allopurinol dose and folate catabolism. After lead acetate dosing there was little radioactivity in the urine and tissues over 72h and more radioactivity was retained in the faeces compared to normals. Excretion of intact folates was depressed, especially 5MeTHF and 10CHOTHF. A tenfold increase in both lead and folic acid dosage resulted in an even further decrease of radioactivity in the tissues and urine over 72h. Excretion in the faeces was further elevated. Ferrous sulphate administration resulted in increased catabolism. The retention of radioactivity in the liver, kidney and gut was greatly reduced. A new method of folate analysis; Sephadex LH-20 was introduced. In vitro superoxide anion formation was illustrated using an allopurinol/xanthine oxidase system. Histological studies were employed to qualitatively and quantitatively illustrate the oxidative status in livers and brains of allopurinol and ferrous sulphate dosed rats. Increased dose related formazan deposition was observed when livers of pretreated animals were incubated with nitroblue tetrazolium. Formazan deposition was reduced in pretreated animals also treated with the anti-oxidants vitamin E, mannitol or 4-hydroxy-methyl-4,6-ditertiary-butylphenol. A possible route of folate catabolism is scission by a non-enzymic oxidation involving active oxygen species.