BELFAST centenarians: A Case of Optimal Cardiovascular Risk?

Introduction: Centenarians are reservoirs of genetic and environmental information to successful ageing and local centenarian groups may help us to understand some of the factors that contribute to longevity. The current centenarian cohort in Belfast survived the 1970s epidemic of death from coronary heart disease in Northern Ireland, where cardiovascular mortality was almost highest in the world. These centenarians provided an opportunity to assess biological and genetic factors important in cardiovascular risk and ageing. Methods: Thirty-five (27 female, 8 male) centenarians, participants of the Belfast Elderly Longitudinal Free-living Ageing STudy (BELFAST), were community-living and of good cognition at enrolment. Results: Centenarians showed median Body Mass Index (BMI) at 25.7, systolic blood pressure 140mmHg and diastolic blood pressure 90mmHg, and fasting glucose of 5.54 mmol/l with no sex-related difference. Lipoproteins showed median cholesterol 5.3, High Density Lipoprotein (HDL) 1.10 and Low Density Lipoprotein (LDL) 3.47umol/l respectively. Centenarian smokers showed no different blood pressure or lipid measurements compared with non-smokers. Malondialdehyde, a measure of lipid peroxidation, was low at 1.19 umol/l, and measures of antioxidant status were varied. Male centenarians did not carry any of the vascular risk genotypes studied-ApoE4 for Apolipoprotein E (ApoE), DD for Angiotensinogen Converting Enzyme (ACE) and tt for 5,10-methylenetetrahydrofolate reductase (MTFHR), though this was not true for female centenarians.. Conclusions: This small local study shows that Belfast centenarians carry a reasonably favourable risk profile, except for age, with respect to cardiovascular disease. There is also some evidence that vascular risk factors and genotypes may be tolerated differently between the male and female centenarians. Maintaining a favourable cardiovascular risk profile seems likely to improve the chance of becoming a centenarian, especially for males.

MEASUREMENT OF SUPEROXIDE FORMATION BY MITOCHONDRIAL COMPLEX I OF YARROWIA LIPOLYTICA

Complex I (NADH: ubiquinone oxidoreductase) is generally regarded as one of the major sources of mitochondrial reactive oxygen species (ROS). Mitochondrial membranes from the obligate aerobic yeast Yarrowia lipolytica, as well as the purified and reconstituted enzyme, can be used to measure complex I-dependent generation of superoxide (O-2(center dot-)). The use of isolated complex I excludes interference with other respiratory chain complexes and matrix enzymes during superoxide dismutase-sensitive reduction of acetylated cytochrome c. Alternately. hydrogen peroxide formation can be measured by the Amplex Red/horseradish peroxidase assay. Both methods allow the determination of complex I-generated ROS, depending on substrates (NADH, artificial ubiquinones), membrane potential, and active/deactive transition. ROS production by Yorrowia complex I in the

Identification of the mitochondrial ND3 subunit as a structural component involved in the active/deactive enzyme transition of respiratory complex I

Mitochondrial complex I (NADH: ubiquinone oxidoreductase) undergoes reversible deactivation upon incubation at 30-37 degrees C. The active/deactive transition could play an important role in the regulation of complex I activity. It has been suggested recently that complex I may become modified by S-nitrosation under pathological conditions during hypoxia or when the nitric oxide: oxygen ratio increases. Apparently, a specific cysteine becomes accessible to chemical modification only in the deactive form of the enzyme. By selective fluorescence labeling and proteomic analysis, we have identified this residue as cysteine-39 of the mitochondrially encoded ND3 subunit of bovine heart mitochondria. Cysteine-39 is located in a loop connecting the first and second transmembrane helix of this highly hydrophobic subunit. We propose that this loop connects the ND3 subunit of the membrane arm with the PSST subunit of the peripheral arm of complex I, placing it in a region that is known to be critical for the catalytic mechanism of complex I. In fact, mutations in three positions of the loop were previously reported to cause Leigh syndrome with and without dystonia or progressive mitochondrial disease.

S-nitrosation of mitochondrial complex I depends on its structural conformation

Nitric oxide is known to cause persistent inhibition of mitochondrial respiration as a result of S-nitrosation of NADH: ubiquinone oxidoreductase (complex I) (Clementi, E., Brown, G. C., Feelisch, M., and Moncada, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7631-7636). Little is known about whether such nitrosation occurs in physiological conditions and, if so, what are the possible cellular mechanisms. We have now found that the conformational state (active/deactive transition (Vinogradov, A. D. (1998) Biochim. Biophys. Acta 1364, 169-185)) of mitochondrial complex I is an important factor for the interaction of the enzyme with nitrosothiols and peroxynitrite. Only the deactivated, idle form of complex I was susceptible to inhibition by nitrosothiols and peroxynitrite. In contrast, the active form of the enzyme was insensitive to such treatment. Neither form of complex I was inhibited by nitric oxide itself. Our data suggest that the process of active/deactive transition plays an important role in the regulation of complex I activity and cellular respiration by nitric oxide. The implications of this finding for hypoxic or pathophysiological conditions in vivo are discussed.

The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of 3.8H(->+)/2e(-) indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of 3.6H(->+)/2e(-). This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme. (c) 2006 Elsevier B.V. All rights reserved.

The redox-Bohr group associated with iron-sulfur cluster N2 of complex I

Proton pumping respiratory complex I (NADH: ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H+/2e(-). This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.

Rapid colorimetric assay for antimicrobial susceptibility testing of Pseudomonas aeruginosa

A colorimetric assay based on the reduction of a tetrazolium salt {2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT)} for rapidly determining the susceptibility of Pseudomonas aeruginosa isolates to bactericidal antibiotics is described. There was excellent agreement between the tobramycin and ofloxacin MICs determined after 5 h using the XTT assay and after 18 h using conventional methods. The data suggests that an XTT-based assay could provide a useful method for rapidly determining the susceptibility of P. aeruginosa to bactericidal antibiotics.

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 K-m = 26.7 mu M, V-max = 0.43 mu M/mg protein/min; RIF-1 K-m = 33.5 mu M, V-max = 0.42 mu M/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 K-m = 37.5 mu M; V-max = 1.4 mu M/mg protein/min; RIF-1 K-m = 37.5 mu M; V-max = 1.2 mu M/mg protein/min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (K-m = 4 mu M, V-max = 3.5 pmol/mg protein/min) and normal kidney (K-m = 4 mu M, V-max = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

Statins in Heart Failure-Where Do We Stand?

HMG Co-A reductase inhibitors (statins) are a group of drugs which lower cholesterol by inhibiting the conversion of HMG Co-A to mevalonate early in the cholesterol synthetic pathway. They are used in the primary and secondary prevention of cardiovascular events in patients deemed to be at increased risk and their benefit in patients with ischaemic heart disease is well supported. Their use in patients with heart failure (HF) however, is controversial. Evidence from observational and mechanistic studies suggests that statins should benefit patients with HF. However, larger randomised controlled trials have failed to demonstrate these expected benefits. The aim of this review article is to summarise the data from trials of statin use in patients with HF and attempt to explain the apparent conflict between recent placebo controlled trials and earlier observational and mechanistic studies.

Glucose lowers the threshold for human aortic vascular smooth muscle cell migration: inhibition by protein phosphatase-2A

AIMS/HYPOTHESIS: Atherosclerosis, which occurs prematurely in individuals with diabetes, incorporates vascular smooth muscle cell (VSMC) chemotaxis. Glucose, through protein kinase C-beta(II) signalling, increases chemotaxis to low concentrations of platelet-derived growth factor (PDGF)-BB. In VSMC, a biphasic response in PDGF-beta receptor (PDGF-betaR) level occurs as PDGF-BB concentrations increase. The purpose of this study was to determine whether increased concentrations of PDGF-BB and raised glucose level had a modulatory effect on the mitogen-activated protein kinase/extracellular-regulated protein kinase pathway, control of PDGF-betaR level and chemotaxis.

METHODS: Cultured aortic VSMC, exposed to normal glucose (NG) (5 mmol/l) or high glucose (HG) (25 mmol/l) in the presence of PDGF-BB, were assessed for migration (chemotaxis chamber) or else extracted and immunoblotted.

RESULTS: At concentrations of PDGF-BB <540 pmol/l, HG caused an increase in the level of PDGF-betaR in VSMC (immunoblotting) versus NG, an effect that was abrogated by inhibition of aldose reductase or protein kinase C-beta(II). At higher concentrations of PDGF-BB (>540 pmol/l) in HG, receptor level was reduced but in the presence of aldose reductase or protein kinase C-beta(II) inhibitors the receptor levels increased. It is known that phosphatases may be activated at high concentrations of growth factors. At high concentrations of PDGF-BB, the protein phosphatase (PP)2A inhibitor, endothall, caused an increase in PDGF-betaR levels and a loss of biphasicity in receptor levels in HG. At higher concentrations of PDGF-BB in HG, the chemoattractant effect of PDGF-BB was lost (chemotaxis chamber). Under these conditions inhibition of PP2A was associated with a restoration of chemotaxis to high concentrations of PDGF-BB.

CONCLUSION/INTERPRETATION: The biphasic response in PDGF-betaR level and in chemotaxis to PDGF-BB in HG is due to PP2A activation.

Functional effect of the C242T polymorphism in the NAD(P)H oxidase p22phox gene on vascular superoxide production in atherosclerosis.

BACKGROUND:

Increased superoxide anion production increases oxidative stress and reduces nitric oxide bioactivity in vascular disease states. NAD(P)H oxidase is an important source of superoxide in human blood vessels, and some studies suggest a possible association between polymorphisms in the NAD(P)H oxidase CYBA gene and atherosclerosis; however, no functional data address this hypothesis. We examined the relationships between the CYBA C242T polymorphism and direct measurements of superoxide production in human blood vessels.

METHODS AND RESULTS:

Vascular NAD(P)H oxidase activity was determined in human saphenous veins obtained from 110 patients with coronary artery disease and identified risk factors. Immunoblotting, reverse-transcription polymerase chain reaction, and DNA sequencing showed that p22phox protein, mRNA, and 242C/T allelic variants are expressed in human blood vessels. Vascular superoxide production, both basal and NADH-stimulated, was highly variable between patients, but the presence of the CYBA 242T allele was associated with significantly reduced vascular NAD(P)H oxidase activity, independent of other clinical risk factors for atherosclerosis.

CONCLUSIONS:

Association of the CYBA 242T allele with reduced NAD(P)H oxidase activity in human blood vessels suggests that genetic variation in NAD(P)H oxidase components may play a significant role in modulating superoxide production in human atherosclerosis.

UV dosimeters based on neotetrazolium chloride

A novel UV dosimeter is described comprising a tetrazolium dye, neotetrazolium chloride (NTC), dissolved in a film of polymer, polyvinyl alcohol (PVA). The dosimeter is pale yellow/colourless in the absence of UV light, and turns red upon exposure to UV light. The spectral characteristics of a typical UV dosimeter film and the mechanism through which the colour change occurs are detailed. The NTC UV dosimeter films exhibit a response to UV light that is related to the intensity and duration of UV exposure, the level of dye present in the films and the thickness of the films themselves. The response of the dosimeter is temperature independent over the range 20-40 degrees C and, like most UV dosimeters, exhibits a cosine-like response dependence upon irradiance angle. The introduction of a layer of a UV-screening compound which slows the rate at which the dosimeter responds to UVR enables the dosimeter response to be tailored to different UV doses. The possible use of these novel dosimeters to measure solar UV exposure dose is discussed. (c) 2008 Elsevier B.V. All rights reserved.

EFFECT OF IN-VIVO AND IN-VITRO LOVASTATIN TREATMENT ON MAST-CELL ACTIVATION

The hydroxymethylglutaryl coenzmye A (HMG CoA) reductase inhibitor lovastatin is used to treat hyperlipidaemia. This agent prevents the isoprenylation of some proteins involved in signal transduction processes and inhibits IgE-receptor-linked mediator release from RBL-2H3 cells. In this study the effect of in vivo and in vitro administration of lovastatin on histamine release from rat peritoneal mast cells was examined. Lovastatin (4 mg/kg/day for 2 weeks) inhibited histamine release induced by concanavalin A (con A) from rat peritoneal mast cells of Hooded-Lister rats and both homozygous lean and obese Zucker rats. In contrast, release induced by antirat IgE (anti-IgE) was only significantly inhibited in cells derived from Hooded-Lister rats and that induced by compound 48/ 80 was not altered. Lovastatin (20 mu M, 24 h, in vitro) caused a significant inhibition of the subsequent histamine release to con A, anti-IgE and compound 48/80 but not to the calcium ionophore A 23187. It is important to determine whether such inhibitory effects are also observed after the chronic, clinical administration of lovastatin and other HMG CoA reductase inhibitors.

Synthesis and in Vitro and in Vivo Evaluation of a Series of Dihydroisocoumarin Derivatives Conjugated with Fatty Acids, Alcohols, and Amines as Potential Anticancer Agents

In this paper, we report the synthesis and biological activity of a series of dihydroisocoumarin analogues Conjugated with fatty acids, alcohols, or amines, of varying hydrocarbon chain length and degree of unsaturation, to (he dihydroisocoumarins, kigelin and mellein, at the C-7 and C-8 positions on the core dihydroisocoumarin structure. These compounds were evaluated for their antiproliferative activity against human breast cancer (MCF-7 and MDA-MB-468) and melanoma cells (SK-MEL-28 and Malme-3M) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Two compounds Conjugated with gamma-linolenyl alcohol (18:3 n-6) demonstrated potent antiproliferative activity in vitro with one of these 4-hydroxy-3-oxo-1.3-dihydro-isobenzofuran-5-carboxylic acid octadeca-6,9,12-trienyl ester, demonstrating significant antitumor activity in vivo ill a number of human tumor xenograft models.

Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations

Elevation in plasma homocysteine concentration has been associated with vascular disease and neural tube defects. Methionine synthase is a vitamin B(12)-dependent enzyme that catalyses the remethylation of homocysteine to methionine. Therefore, defects in this enzyme may result in elevated homocysteine levels. One relatively common polymorphism in the methionine synthase gene (D919G) is an A to G transition at bp 2,756, which converts an aspartic acid residue believed to be part of a helix involved in co-factor binding to a glycine. We have investigated the effect of this polymorphism on plasma homocysteine levels in a working male population (n = 607) in which we previously described the relationship of the C677T "thermolabile" methylenetetrahydrofolate reductase (MTHFR) polymorphism with homocysteine levels. We found that the methionine synthase D919G polymorphism is significantly (P = 0.03) associated with homocysteine concentration, and the DD genotype contributes to a moderate increase in homocysteine levels across the homocysteine distribution (OR = 1.58, DD genotype in the upper half of the homocysteine distribution, P = 0.006). Unlike thermolabile MTHFR, the homocysteine-elevating effects of the methionine synthase polymorphism are independent of folate and B(12) levels; however, the DD genotype has a larger homocysteine-elevating effect in individuals with low B(6) levels. This polymorphism may, therefore, make a moderate, but significant, contribution to clinical conditions that are associated with elevated homocysteine.