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.

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.

Mechanisms underlying the metabolic actions of galegine that contribute to weight loss in mice

Background and purpose: Galegine and guanidine, originally isolated from Galega officinalis, led to the development of the biguanides. The weight-reducing effects of galegine have not previously been studied and the present investigation was undertaken to determine its mechanism(s) of action.

Experimental approach: Body weight and food intake were examined in mice. Glucose uptake and acetyl-CoA carboxylase activity were studied in 3T3-L1 adipocytes and L6 myotubes and AMP activated protein kinase (AMPK) activity was examined in cell lines. The gene expression of some enzymes involved in fat metabolism was examined in 3T3-L1 adipocytes.

Key results: Galegine administered in the diet reduced body weight in mice. Pair-feeding indicated that at least part of this effect was independent of reduced food intake. In 3T3-L1 adipocytes and L6 myotubes, galegine (50 µm-3 mm) stimulated glucose uptake. Galegine (1–300 µm) also reduced isoprenaline-mediated lipolysis in 3T3-L1 adipocytes and inhibited acetyl-CoA carboxylase activity in 3T3-L1 adipocytes and L6 myotubes. Galegine (500 µm) down-regulated genes concerned with fatty acid synthesis, including fatty acid synthase and its upstream regulator SREBP. Galegine (10 µm and above) produced a concentration-dependent activation of AMP activated protein kinase (AMPK) in H4IIE rat hepatoma, HEK293 human kidney cells, 3T3-L1 adipocytes and L6 myotubes.

Conclusions and implications: Activation of AMPK can explain many of the effects of galegine, including enhanced glucose uptake and inhibition of acetyl-CoA carboxylase. Inhibition of acetyl-CoA carboxylase both inhibits fatty acid synthesis and stimulates fatty acid oxidation, and this may to contribute to the in vivo effect of galegine on body weight.

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.

A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers

Thioacetamide (TAA) administration is an established technique for generating rat models of liver fibrosis and cirrhosis. Oxidative stress is believed to be involved as TAA-induced liver fibrosis is initiated by thioacetamide S-oxide, which is derived from the biotransformation of TAA by the microsomal flavine-adenine dinucleotide (FAD)-containing monooxygense (FMO) and cytochrome P450 systems. A two-dimensional gel electrophoresis-mass spectrometry approach was applied to analyze the protein profiles of livers of rats administered with sublethal doses of TAA for 3, 6 and 10 weeks respectively. With this approach, 59 protein spots whose expression levels changed significantly upon TAA administration were identified, including three novel proteins. These proteins were then sorted according to their common biochemical properties and functions, so that pathways involved in the pathogenesis of rat liver fibrosis due to TAA-induced toxicity could be elucidated. As a result, it was found that TAA-administration down-regulated the enzymes of the primary metabolic pathways such as fatty acid beta-oxidation, branched chain amino acids and methionine breakdown. This phenomenon is suggestive of the depletion of succinyl-CoA which affects heme and iron metabolism. Up-regulated proteins, on the other hand, are related to oxidative stress and lipid peroxidation. Finally, these proteomics data and the data obtained from the scientific literature were integrated into an

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.

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.

Effect of B-group vitamins and antioxidant vitamins on hyperhomocysteinemia:a double-blind, randomized, factorial-design, controlled trial

Mild hyperhomocysteinemia is accepted as a risk factor for premature cardiovascular disease. In a population with a high prevalence of cardiovascular disease, we screened a group of clinically healthy working men aged 30-49 y (n = 509) for plasma homocysteine and 5,10-methylene tetrahydrofolate reductase (MTHFR) genotype status. Those with mildly elevated homocysteine concentrations (> or = 8.34 micromol/L) were selected for intervention. In a randomized, factorial-design, controlled trial we assessed the effects of B-group vitamins and antioxidant vitamin supplementation on homocysteine concentrations. The 132 men were randomly assigned to one of four groups: supplementation with B-group vitamins alone (1 mg folic acid, 7.2 mg pyridoxine, and 0.02 mg cyanocobalamin), antioxidant vitamins alone (150 mg ascorbic acid, 67 mg RRR-alpha-tocopherol, and 9 mg beta-carotene), B-group vitamins with antioxidant vitamins, or placebo. Intervention was double-blind. A total of 101 men completed the 8-wk intervention. When homocysteine concentrations were analyzed by group, significant (P <0.001) decreases (32.0% and 30.0%, respectively) were observed in both groups receiving B-group vitamins either with or without antioxidants. The effect of B-group vitamins alone over 8 wk was a reduction in homocysteine concentrations of 27.9% (95% CI: 22.0%, 33.3%; P <0.001) whereas antioxidants alone produced a nonsignificant increase of 5.1% (95% CI: -2.8%, 13.6%; P = 0.21). There was no evidence of any interaction between the two groups of vitamins. The effect of B-group vitamin supplementation seemed to depend on MTHFR genotype. Supplementation with the B-group vitamins with or without antioxidants reduced homocysteine in the men with mildly elevated concentrations, and hence may be effective in reducing cardiovascular risk.

Arsenic uptake and metabolism in plants

Arsenic (As) is an element that is nonessential for and toxic to plants. Arsenic contamination in the environment occurs in many regions, and, depending on environmental factors, its accumulation in food crops may pose a health risk to humans.Recent progress in understanding the mechanisms of As uptake and metabolism in plants is reviewed here. Arsenate is taken up by phosphate transporters. A number of the aquaporin nodulin26-like intrinsic proteins (NIPs) are able to transport arsenite,the predominant form of As in reducing environments. In rice (Oryza sativa), arsenite uptake shares the highly efficient silicon (Si) pathway of entry to root cells and efflux towards the xylem. In root cells arsenate is rapidly reduced to arsenite, which is effluxed to the external medium, complexed by thiol peptides or translocated to shoots. One type of arsenate reductase has been identified, but its in planta functions remain to be investigated. Some fern species in the Pteridaceae family are able to hyperaccumulate As in above-ground tissues. Hyperaccumulation appears to involve enhanced arsenate uptake, decreased arsenite-thiol complexation and arsenite efflux to the external medium, greatly enhanced xylem translocation of arsenite, and vacuolar sequestration of arsenite in fronds. Current knowledge gaps and future research directions are also identified.

Resolving candidate genes of mouse skeletal muscle QTL via RNA-Seq and expression network analyses

BACKGROUND:

We have recently identified a number of Quantitative Trait Loci (QTL) contributing to the 2-fold muscle weight difference between the LG/J and SM/J mouse strains and refined their confidence intervals. To facilitate nomination of the candidate genes responsible for these differences we examined the transcriptome of the tibialis anterior (TA) muscle of each strain by RNA-Seq.

13,726 genes were expressed in mouse skeletal muscle. Intersection of a set of 1061 differentially expressed transcripts with a mouse muscle Bayesian Network identified a coherent set of differentially expressed genes that we term the LG/J and SM/J Regulatory Network (LSRN). The integration of the QTL, transcriptome and the network analyses identified eight key drivers of the LSRN (Kdr, Plbd1, Mgp, Fah, Prss23, 2310014F06Rik, Grtp1, Stk10) residing within five QTL regions, which were either polymorphic or differentially expressed between the two strains and are strong candidates for quantitative trait genes (QTGs) underlying muscle mass. The insight gained from network analysis including the ability to make testable predictions is illustrated by annotating the LSRN with knowledge-based signatures and showing that the SM/J state of the network corresponds to a more oxidative state. We validated this prediction by NADH tetrazolium reductase staining in the TA muscle revealing higher oxidative potential of the SM/J compared to the LG/J strain (p<0.03).

Thus, integration of fine resolution QTL mapping, RNA-Seq transcriptome information and mouse muscle Bayesian Network analysis provides a novel and unbiased strategy for nomination of muscle QTGs.

Phase I and II biotransformation enzymes and polycyclic aromatic hydrocarbons in the Mediterranean mussel (Mytilus galloprovincialis, Lamarck, 1819) collected in front of an oil refinery

The aim of the present study was to investigate the responses of phase I and II biotransformation enzymes and levels of PAHs in the Mediterranean mussel (Mytilus galloprovincialis, Lamarck, 1819) collected from three sites at different distance from an oil refinery. Phase I enzyme activities as NAD(P)H-cyt c red, NADH ferry red, B(a)PMO and phase II as UDPGT. GST were measured in digestive gland while 16 PAHs (US-EPA) in whole soft tissue. An added value to the data obtained in the present study rely on the RDA analysis which showed close correlations between PAHs levels and phase I enzyme activities in mussels collected in front of the refinery. And again a significant spatial correlation between B(a)P levels and NADPH-cyt c red activities was observed using linear models. No differences among sites for B(a) PMO and phase II GST activities were observed, while the application of UDPGT as biomarkers requires further investigation. (C) 2012 Elsevier Ltd. All rights reserved.

Chemisorption and reactivity of furan on Pd{111}

XPS, HREELS, ARUPS and Delta phi data show that furan chemisorbs non-dissociatively on Pd{111} at 175 K, the molecular plane being significantly tilted with respect to the surface normal. Bonding involves both the oxygen lone pair and significant a interaction with the substrate. The degree of decomposition that accompanies molecular desorption is a strong function of coverage: similar to 40% of the adsorbate desorbs molecularly from the saturated monolayer. Decomposition occurs via decarbonylation to yield COa and H-a followed by desorption rate limited loss of H-2 and CO. It seems probable that an adsorbed C3H3 species formed during this process undergoes subsequent stepwise dehydrogenation ultimately yielding H-2 and C-a.

Influences of phosphorus starvation on OsACR2.1 expression and arsenic metabolism in rice seedlings

The effects of phosphorus (P) status on arsenate reductase gene (OsACR2.1) expression, arsenate reductase activity, hydrogen peroxide (H(2)O(2)) content, and arsenic (As) species in rice seedlings which were exposed to arsenate after -P or +P pretreatments were investigated in a series of hydroponic experiments. OsACR2.1 expression increased significantly with decreasing internal P concentrations; more than 2-fold and 10-fold increases were found after P starvation for 30 h and 14 days, respectively. OsACR2.1 expression exhibited a significant positive correlation with internal root H(2)O(2) accumulation, which increased upon P starvation or exposure to H(2)O(2) without P starvation. Characterization of internal and effluxed As species showed the predominant form of As was arsenate in P-starved rice root, which contrasted with the +P pretreated plants. Additionally, more As was effluxed from P-starved rice roots than from non-starved roots. In summary, an interesting relationship was observed between P-starvation induced H(2)O(2) and OsACR2.1 gene expression. However, the up-regulation of OsACR2.1 did not increase arsenate reduction in P-starved rice seedlings when exposed to arsenate.

Phosphorylation of plant actin-depolymerising factor by calmodulin-like domain protein kinase

actin-depolymerising factor (ADF)/cofilin group of proteins are stimulus-responsive actin-severing proteins, members of which are regulated by reversible phosphorylation. The phosphorylation site on the maize ADF, ZmADF3, is Ser-6 but the kinase responsible is unknown [Smertenko et al,, Plant J. 14 (1998) 187-193]. We have partially purified the ADF kinase(s) and found it to be calcium-regulated and inhibited by N-(6-aminohesyl)-[H-3]5-chloro-1-naphthalenesulphonamide. Immunoblotting reveals that calmodulin-like domain protein kinase(s) (CDPK) are enriched in the purified preparation and addition of anti-CDPK to in vitro phosphorylation assays results in the inhibition of ADF phosphorylation, These data strongly suggest that plant ADP is phosphorylation by CDPK(s), a class of protein kinases unique to plants and protozoa. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Editorial

Stoddart, S.and C. Malone . Editorial. Antiquity, (Topics Include funding for archaeology, British Museum finances and heritage finances, conferences, Portugal, Coa and Palaeolithic matters, deep time, David Coombs).