A reassessment of semiquantitative analytical procedures for DNA methylation: comparison of bisulfite- and HpaII polymerase-chain-reaction-based methods

Two techniques in particular are used to study site-specific DNA methylation: genomic sequencing after bisulfite modification and polymerase chain reaction after digestion by a methylation-sensitive endonuclease (usually HpaII). Only the former methodology assesses the methylation status of all the cytosine residues in the DNA sequence, but it is so complex and time consuming that the latter procedure, though limited to the restriction sites recognized by the endonuclease(s) used, is often preferred at least for a first analysis. In this work we investigate differences between these two techniques in the assessment of DNA methylation and offer some suggestions on how to avoid uncorrected results. Although there is substantial accordance in the results obtained using these different techniques, we observed a general overestimate for methylation levels above 30% and a general underestimate for methylation levels below this value using the HpaII/PCR technique in the study of methylation of the 5'-flanking region of the mouse myogenin gene in cultured muscle cells and mouse tissues.     

ART2, a T cell surface mono-ADP-ribosyltransferase, generates extracellular poly(ADP-ribose)

NAD functions in multiple aspects of cellular metabolism and signaling through enzymes that covalently transfer ADP-ribose from NAD to acceptor proteins, thereby altering their function. NAD is a substrate for two enzyme families, mono-ADP-ribosyltransferases (mARTs) and poly(ADP-ribose) polymerases (PARPs), that covalently transfer an ADP-ribose monomer or polymer, respectively, to acceptor proteins. ART2, a mART, is a phenotypic marker of immunoregulatory cells found on the surface of T lymphocytes, including intestinal intraepithelial lymphocytes (IELs). We have shown that the auto-ADP-ribosylation of the ART2.2 allelic protein is multimeric. Our backbone structural alignment of ART2 (two alleles of the rat art2 gene have been reported, for simplicity, the ART2.2 protein investigated in this study will be referred to as ART2) and PARP suggested that multimeric auto-ADP-ribosylation of ART2 may represent an ADP-ribose polymer, rather than multiple sites of mono-ADP-ribosylation. To investigate this, we used highly purified recombinant ART2 and demonstrated that ART2 catalyzes the formation of an ADP-ribose polymer by sequencing gel and by HPLC and MS/MS mass spectrometry identification of PR-AMP, a breakdown product specific to poly(ADP-ribose). Furthermore, we identified the site of ADP-ribose polymer attachment on ART2 as Arg-185, an arginine in a crucial loop of its catalytic core. We found that endogenous ART2 on IELs undergoes multimeric auto-ADP-ribosylation more efficiently than ART2 on peripheral T cells, suggesting that these distinct lymphocyte populations differ in their ART2 surface topology. Furthermore, ART2.2 IELs are more resistant to NAD-induced cell death than ART2.1 IELs that do not have multimeric auto-ADP-ribosylation activity. The data suggest that capability of polymerizing ADP-ribose may not be unique to PARPs and that poly(ADP-ribosylation), an established nuclear activity, may occur extracellularly and modulate cell function.     

RHYTHM-AF: design of an international registry on cardioversion of atrial fibrillation and characteristics of participating centers

Background

Cigarette exposure increases brain oxidative stress. The literature showed that increased brain oxidative stress affects cardiovascular regulation. However, no previous study investigated the involvement of brain oxidative stress in animals exposed to cigarette and its relationship with cardiovascular regulation. We aimed to evaluate the effects of central catalase inhibition on baroreflex and cardiovascular responses in rats exposed to sidestream cigarette smoke (SSCS).

Methods

We evaluated males Wistar rats (320-370 g), which were implanted with a stainless steel guide cannula into the fourth cerebral ventricle (4th V). Femoral artery and vein were cannulated for mean arterial pressure (MAP) and heart rate (HR) measurement and drug infusion, respectively. Rats were exposed to SSCS during three weeks, 180 minutes, 5 days/week (CO: 100-300 ppm). Baroreflex was tested with a pressor dose of phenylephrine (PHE, 8 ??g/kg, bolus) to induce bradycardic reflex and a depressor dose of sodium nitroprusside (SNP, 50 ??g/kg, bolus) to induce tachycardic reflex. Cardiovascular responses were evaluated before, 5, 15, 30 and 60 minutes after 3-amino-1,2,4-triazole (ATZ, catalase inhibitor, 0.001 g/100 ??L) injection into the 4th V.

Results

Central catalase inhibition increased basal HR in the control group during the first 5 minutes. SSCS exposure increased basal HR and attenuated bradycardic peak during the first 15 minutes.

Conclusion

We suggest that SSCS exposure affects cardiovascular regulation through its influence on catalase activity.     

Hepatic steatosis,carotid plaques and achieving MDA in psoriatic arthritis patients starting TNF-α blockers treatment: a prospective study

Introduction

We prospectively evaluated whether hepatic steatosis (HS) and the presence of carotid plaques (CPs) impacts on achieving minimal disease activity (MDA) in psoriatic arthritis (PsA) patients starting tumor necrosis factor (TNF)-α blockers treatment.

Methods

Before starting treatment with TNF-α blockers, consecutive PsA subjects with an active disease were evaluated for the presence of the metabolic syndrome (MetS), HS and CPs. The incidence of MDA was evaluated 12 and 24 months later.

Results

Among 270 PsA subjects, 91 (33.7%) exhibited the MetS, 58 (21.5%) CPs and 76 (28.1%) HS. At the 12-month follow-up, 98 (36.3%) individuals achieved MDA. Compared with those who did, a higher prevalence of the MetS, HS and CPs was found in subjects who did not achieve the MDA (P always < 0.001). After adjusting for the MetS and for all the other demographic/clinical characteristics analyzed, the presence of HS and CPs at baseline independently predicted the risk of not achieving MDA (Hazard Ratio: 1.91, 95% confidence interval (CI): 1.04 to 3.38, P = 0.035 and Hazard Ratio: 3.21, 95%CI: 1.64 to 6.29, P = 0.001, respectively). Separate Kaplan-Meier survival models confirmed this (Log-Rank: 12.894, P < 0.001 and Log-Rank: 12.849, P < 0.001, respectively). Compared with those without, progressively increasing Hazard Ratios of not achieving MDA were found in those with HS, CPs or HS + CPs at baseline. Moreover, the presence of HS and/or CPs predicted the risk of relapse during the additional 12-month follow-up (Hazard Ratio: 2.85, 95%CI: 1.27 to 6.37, P = 0.011 and Hazard Ratio: 3.17, 95%CI: 1.57 to 6.41, P = 0.001 respectively).

Conclusions

HS and/or CPs at baseline are negative predictors of achieving and maintaining MDA.     

IRS2 signaling in LepR-b neurons suppresses FoxO1 to control energy balance independently of leptin action

Irs2-mediated insulin/IGF1 signaling in the CNS modulates energy balance and glucose homeostasis; however, the site for Irs2 function is unknown. The hormone leptin mediates energy balance by acting on leptin receptor (LepR-b)-expressing neurons. To determine whether LepR-b neurons mediate the metabolic actions of Irs2 in the brain, we utilized Lepr(cre) together with Irs2(L/L) to ablate Irs2 expression in LepR-b neurons (Lepr(ΔIrs2)). Lepr(ΔIrs2) mice developed obesity, glucose intolerance, and insulin resistance. Leptin action was not altered in young Lepr(ΔIrs2) mice, although insulin-stimulated FoxO1 nuclear exclusion was reduced in Lepr(ΔIrs2) mice. Indeed, deletion of Foxo1 from LepR-b neurons in Lepr(ΔIrs2) mice normalized energy balance, glucose homeostasis, and arcuate nucleus gene expression. Thus, Irs2 signaling in LepR-b neurons plays a crucial role in metabolic sensing and regulation. While not required for leptin action, Irs2 suppresses FoxO1 signaling in LepR-b neurons to promote energy balance and metabolism.     

Red blood cells protect albumin from cigarette smoke-induced oxidation

Different studies reported the presence of oxidized (carbonylated) albumin in the extravascular pool, but not in the intravascular one of cigarette smokers. In this study we attempted to explain this apparent discrepancy exposing human serum albumin (HSA) to aqueous cigarette smoke extract (CSE). CSE induces HSA carbonylation and oxidation of the HSA Cys34 sulfhydryl group. An antioxidant action of glutathione, cysteine, and its synthetic derivative N-acetylcysteine was observed only at supra-physiological concentrations, suggesting that physiological (plasma) concentrations of glutathione and cysteine in the low micromolar range are ineffective in preventing cigarette smoke-induced oxidation of HSA. Differently, human erythrocytes resulted to be protective towards CSE-induced oxidation (carbonylation and thiol oxidation) of both HSA and total human plasma proteins.     

Analysis of drought responsive proteins in wheat (Triticum durum) by 2D-PAGE and MALDI-TOF mass spectrometry

Drought is an abiotic stress that strongly influences plant growth, development and productivity. By proteomics study it is possible to identify the complex mechanism of water-stress response. The aim of this research was the analysis of wheat (Triticum durum) proteome changes under stress conditions by applying a severe water deficit treatment for 7 days. Stress-induced proteome changes were analyzed by two-dimensional gel electrophoresis in conjunction with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Thirty-six protein spots showed a reproducible significant change between control and stressed samples. The reasonable implications in drought response of the identified proteins were discussed. Results provide new insights that can lead to a better understanding of the molecular basis of drought-sensitivity in plants. Therefore, the obtained data could suggest the development of drought resistant wheat varieties, in order to improve agricultural production in dry regions.