Aging and epigenetics: longitudinal changes in gene-specific DNA methylation

DNA methylation has been associated with age-related disease. Intra-individual changes in gene-specific DNA methylation over time in a community-based cohort has not been well described. We estimated the change in DNA methylation due to aging for nine genes in an elderly, community-dwelling cohort of men. Seven hundred and eighty four men from the Veterans Administration Normative Aging Study who were living in metropolitan Boston from 1999-2009 donated a blood sample for DNA methylation analysis at clinical examinations repeated at approximately 3-5 year intervals. We used mixed effects regression models. Aging was significantly associated with decreased methylation of GCR, iNOS and TLR2 and with increased methylation of IFNγ, F3, CRAT and OGG. Obstructive pulmonary disease at baseline modified the effect of aging on methylation of IFNγ (interaction p = 0.04). For participants who had obstructive pulmonary disease at their baseline visit, the rate of change of methylation of IFNγ was -0.05% 5-methyl-cytosine (5-mC) per year (95% CI: -0.22, 0.13), but was 0.14% 5-mC per year (95% CI: 0.05, 0.24) for those without this condition. Models with random slopes indicated significant heterogeneity in the effect of aging on methylation of GCR, iNOS and OGG. These findings suggest that DNA methylation may reflect differential biological aging.     

Gene promoter methylation is associated with lung function in the elderly: The normative aging study

Lung function is a strong predictor of mortality. While inflammatory markers have been associated with lung function decrease, pathways are still poorly understood and epigenetic changes may participate in lung function decline mechanisms. We studied the cross-sectional association between DNA methylation in nine inflammatory genes and lung function in a cohort of 756 elderly men living in the metropolitan area of Boston. Participants donated a blood sample for DNA methylation analysis and underwent spirometry at each visit every 3 to 5 y from 1999–2006. We used separate multivariate mixed effects regression models to study the association between each lung function measurement and DNA methylation within each gene. Decreased CRAT, F3 and TLR2 methylation was significantly associated with lower lung function. One interquartile range (IQR) decrease in DNA methylation was associated with lower forced vital capacity (FVC) and forced expiratory volume in one second (FEV₁), respectively by 2.94% (p < 10⁻⁴) and 2.47% (p < 10⁻³) for F3 and by 2.10% (p < 10⁻²) and 2.42% (p < 10⁻³) for TLR2. Decreased IFNγ and IL6 methylation was significantly associated with better lung function. One IQR decrease in DNA methylation was associated with higher FEV₁ by 1.75% (p = 0.02) and 1.67% (p = 0.05) for IFNγ and IL6, respectively. These data demonstrate that DNA methylation may be part of the biological processes underlying the lung function decline and that IFNγ and IL6 may have ambivalent roles through activation of negative feedback.Key words: DNA methylation, genes, spirometry, FEV₁, lungs, TLR2, F3, INOS, GCR, OGG1     

Synthesis and pharmacological evaluation of indolinone derivatives as novel ghrelin receptor antagonists

The ghrelin receptor is a G-protein-coupled receptor (GPCR) widely expressed in the brain, stomach and the intestine. It was firstly identified during studies aimed to find synthetic modulators of growth hormone (GH) secretion. GHSR and its endogenous ligand ghrelin were found to be involved in hunger response. Through food intake regulation, they could affect body weight and adiposity. Thus GHSR antagonists rapidly became an attractive target to treat obesity and feeding disorders. In this study we describe the biological properties of new indolinone derivatives identified as a new, chiral class of ghrelin antagonists. Their synthesis as well as the structure-activity relationship will be discussed herein. The in vitro identified compound 14f was a potent GHSR1a antagonist (IC(50)=7nM). When tested in vivo, on gastric emptying model, 14f showed an inhibitory intrinsic effect when given alone and it dose dependently inhibited ghrelin stimulation. Compound 14f also reduced food intake stimulated both by fasting condition (high level of endogenous ghrelin) and by icv ghrelin. Moreover this compound improved glucose tolerance in ipGTT test.     

Patterns of molecular evolution associated with two selective sweeps in the Tb1-Dwarf8 region in maize

We focused on a region encompassing a major maize domestication locus, Tb1, and a locus involved in the flowering time variation, Dwarf8 (D8), to investigate the consequences of two closely linked selective sweeps on nucleotide variation and gain some insights into maize geographical diffusion, through climate adaptation. First, we physically mapped D8 at approximately 300 kb 3' of Tb1. Second, we analyzed patterns of nucleotide variation at Tb1, D8, and seven short regions (400-700 bp) located in the Tb1-D8 region sequenced on a 40 maize inbred lines panel encompassing early-flowering temperate and late-flowering tropical lines. The pattern of polymorphism along the region is characterized by two valleys of depleted polymorphism while the region in between exhibits an appreciable amount of diversity. Our results reveal that a region approximately 100 kb upstream of the D8 gene exhibits hallmarks of divergent selection between temperate and tropical lines and is likely closer than the D8 gene to the target of selection for climate adaptation. Selection in the tropical lines appears more recent than in the temperate lines, suggesting an initial domestication of early-flowering maize. Simulation results indicate that the polymorphism pattern is consistent with two interfering selective sweeps at Tb1 and D8.     

Effects of nomegestrol acetate administration on central and peripheral beta-endorphin and allopregnanolone in ovx rats

The aim of this study was to investigate the effects of nomegestrol acetate (NOMAc) on the central nervous system by analyzing the neurosteroid allopregnanolone and the opioid beta-endorphin (beta-endorphin). 104 Wistar female rats were used in this study; one group of fertile and one group of ovariectomized rats were used as control. The others were ovariectomized and they underwent a 2-week oral treatment of NOMAc (0.05, 0.1, 0.2, 0.5, 1mg/kg/day), alone or with 0.05 mg/kg/day of estradiol valerate (E2V). Allopregnanolone and beta-endorphin were assessed in different brain areas and in circulation. Ovariectomy decreased allopregnanolone anywhere except in the adrenal gland and E2V reversed the effects of ovariectomy. 0.5 and 1mg/kg/day of NOMAc increased allopregnanolone levels in hippocampus. Combined administration of 1mg/kg/day of NOMAc plus E2V induced a further increase of allopregnanolone levels in hippocampus, hypothalamus, and anterior pituitary. NOMAc (1mg/kg/day) decreased the adrenal content of allopregnanolone, both by itself and associated with E2V. NOMAc increased hippocampal and hypothalamic content of beta-endorphin at the highest doses, and it increased positively E2V action, at 1mg/kg/day, also in anterior pituitary and plasma. These findings reinforce the clinical data regarding the capability of NOMAc to modulate the pathways involved in mood and behaviour. In fact, due to the NOMAc action on hippocampus, hypothalamus, and anterior pituitary, our results highlight the selectivity of NOMAc on part of the limbic system and the anterior pituitary, regarding both allopregnanolone and beta-endorphin.     

Plasma adrenomedullin concentrations in patients with adrenal pheochromocytoma.

BACKGROUND: The hypotensive peptide adrenomedullin was first isolated in extracts of human pheochromocytoma. There is, however, no information available on the behaviour of circulating adrenomedullin or on the correlation with catecholamines in patients with pheochromocytoma. OBJECTIVES: 1) to investigate whether plasma adrenomedullin levels were changed in 10 patients with pheochromocytoma when compared to 21 healthy subjects and 16 patients with essential hypertension; 2) to determine whether or not adrenomedullin has a counter-regulatory role in catecholamine excess in pheochromocytoma or is responsible for hemodynamic modifications before and after tumour resection; 3) to determine tissue distribution of iradrenomedullin in the pheochromocytoma. METHODS: Plasma adrenomedullin and catecholamine levels were measured in all patients with pheochromocytoma before and four weeks after tumour removal. In the four patients undergoing resection of tumours, plasma levels of adrenomedullin were measured at different time-points during surgery. RESULTS: The mean plasma adrenomedullin concentrations ( SD) in patients with pheochromocytoma (37.9 +/- 6pg/ml) were significantly higher (p<0.0001) than those in normal subjects (13.7 +/- 6.1 pg/mI) and patients with essential hypertension (22.5 +/- 9.lpg/ml). Adrenomedullin levels correlated with plasma noradrenaline (r = 0.516, p = 0.0124). In all patients with pheochromocytoma, plasma adrenomedullin concentrations decreased after removal of tumours (from 37.9 +/- 6 to 10.9 +/- 4.6 pg/ml; p < 0.0001). In the four patients studied during surgery, baseline plasma adrenomedullin and noradrenaline levels were markedly elevated, and increased significantly with tumour manipulation, decreasing 24 hours after operation. Adrenal medulla cells surrounding the pheochromocytoma site stained for ir-adrenomedullin, whereas only isolated cells of pheochromocytoma stained for the peptide. CONCLUSIONS: This study demonstrates that circulating adrenomedullin is increased in pheochromocytoma, and is also correlated with plasma noradrenaline levels. Adrenomedullin may represent an additional biochemical parameter for clinical monitoring of patients with pheochromocytoma.     

Renal cell cultures for the study of growth factor interactions underlying kidney organogenesis

Summary The present study was performed in four renal cell lines to evaluate their capability to: (1) produce and express transforming growth factor α (TGFα), its respective receptor, the epidermal growth factor receptor (EGFr) and the small G protein, RhoA, and (2) exhibit morphogenetic properties when grown on Matri-cell substrates. The cell lines were derived from normal (Madin-Darby canine kidney cells), embryonic (SK-NEP-1 and 293 cells), and cancerous (human renal adenocarcinoma cells) kidneys. TGFα messenger ribonucleic acid evaluated by a nonradioactive in situ hybridization technique, was found to be expressed in all the cell lines. Large amounts of TGFα peptide were observed in all four cell lines, while EGFr was highly expressed only in cancerous ACHN and embryonic-tumor SK-NEP-1 cells. RhoA peptide was found in appreciable amounts in SK-NEP-1 and 293 cells (compared to the other two cell lines). The morphogenetic properties of the four cell lines were assessed, by culturing them on Matri-cell dishes: SK-NEP-1 cells alone were found to grow in three-dimensional structures forming clusters and worm-like cellular aggregates. This feature was displayed by SK-NEP-1 cells but not by the other three cell lines, and may be connected with the contemporary presence of RhoA, EGFr, and TGFα found in significant amounts only in the SK-NEP-1 cell line.