A detailed protocol for RNA cleavage assay in sympathetic neurons

This protocol illustrates the use of an in vitro assay to study the cleavage of the IMPA1 3′UTR by the endonuclease Ago2 in sympathetic neurons. The procedure includes the preparation of cytoplasmic protein extracts and also describes the synthesis and labeling of the RNA probe. The protocol can be applied to other cell systems, RNA transcripts, and endonucleases to confirm the role of known cleavage site(s) and cleavage proteins, or to investigate new ones. For complete details on the use and execution of this protocol, please refer to Andreassi et al. (2021)

Post-transcriptional Processing of mRNA in Neurons: The Vestiges of the RNA World Drive Transcriptome Diversity

Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their extraordinary cytoarchitecture. This formidable task is achieved, at least in part, by targeting mRNA to subcellular compartments where they are rapidly translated. mRNA transcripts are the conveyor of genetic information from DNA to the translational machinery, however, they are also endowed with additional functions linked to both the coding sequence (open reading frame, or ORF) and the flanking 5′ and 3′ untranslated regions (UTRs), that may harbor coding-independent functions. In this review, we will highlight recent evidences supporting new coding-dependent and -independent functions of mRNA and discuss how nuclear and cytoplasmic post-transcriptional modifications of mRNA contribute to localization and translation in mammalian cells with specific emphasis on neurons. We also describe recently developed techniques that can be employed to study RNA dynamics at subcellular level in eukaryotic cells in developing and regenerating neurons

T-786→C polymorphism of the endothelial nitric oxide synthase gene is associated with insulin resistance in patients with ischemic or non ischemic cardiomyopathy

Background: Insulin resistance (IR) and endothelial dysfunction are frequently associated in cardiac disease. The T-786→C variant in the promoter region of the endothelial nitric oxide synthase (eNOS) gene has been associated with IR in both non-diabetic and diabetic subjects. Aim of the study was to assess the reciprocal relationships between T-786→C eNOS polymorphism and IR in ischemic and non-ischemic cardiomyopathy.Method: A group of 132 patients (108 males, median age 65 years) with global left ventricular (LV) dysfunction secondary to ischemic or non-ischemic heart disease was enrolled. Genotyping of T-786→C eNOS gene promoter, fasting glucose, insulin, and insulin resistance (defined as HOMA-IR index > 2.5) were determined in all patients.Results: Genotyping analysis yielded 37 patients homozygous for the T allele (TT), 70 heterozygotes (TC) and 25 homozygous for C (CC). Patients with CC genotype had significantly higher systemic arterial pressure, blood glucose, plasma insulin and HOMA index levels than TT. At multivariate logistic analysis, the history of hypertension and the genotype were the only predictors of IR. In particular, CC genotype increased the risk of IR (CI% 1.4-15.0, p < 0.01) 4.5-fold. The only parameter independently associated with the extent of LV dysfunction and the presence of heart failure (HF) was the HOMA index (2.4 CI% 1.1-5.6, p < 0.04).Conclusions: T-786→C eNOS polymorphism was the major independent determinant of IR in a population of patients with ischemic and non-ischemic cardiomyopathy. The results suggest that a condition of primitive eNOS lower expression can predispose to an impairment of glucose homeostasis, which in turn is able to affect the severity of heart disease. © 2012 Vecoli et al.; licensee BioMed Central Ltd

Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, intakes of folate and related B vitamins and colorectal cancer : a case-control study in a population with relatively low folate intake

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Deoxyribonucleic acid damage in human lymphocytes after percutaneous transluminal coronary angioplasty

AbstractObjectivesWe investigated the presence of oxidative deoxyribonucleic acid (DNA) damage in the peripheral lymphocytes of patients undergoing percutaneous transluminal coronary angioplasty (PTCA) by using the micronucleus test and comet assay, which are sensitive biomarkers of DNA damage.BackgroundAlthough it has recognized that ischemia-reperfusion can induce oxidative DNA damage, its occurrence in patients undergoing PTCA has not yet been demonstrated.MethodsThree groups of patients were enrolled: 30 patients with documented coronary heart disease who underwent elective PTCA (group I); 25 patients who underwent elective coronary angiography for diagnostic purpose (group II); and 27 healthy, age- and gender-matched subjects (group III). For each subject, the frequency of micronucleated binucleated (MNBN) cells, DNA single-strand breaks (SSBs), endonuclease III-sensitive sites, and sites sensitive to formamidopyrimidine glycosylase (FPG) were analyzed before and after diagnostic procedures.ResultsThe mean basal values of MNBN cells (p = 0.04), DNA-SSBs (p = 0.001), endonuclease III-sensitive sites (p = 0.002), and FPG sites (p < 0.0001) were significantly higher in groups I and II than in group III. A high significant increase of MNBN cell frequency was observed in group I after the PTCA procedure (11.0 ± 1.3 vs. 19.8 ± 1.6, p < 0.0001), whereas no significant difference was observed in group II (10.2 ± 1.3 vs. 12.9 ± 1.4, p = 0.18). A significant positive correlation was observed between the increase in the MNBN cell rate and total inflation time during PTCA (R = 0.549, p = 0.0017). The levels of DNA-SSBs (11.7 ± 1.4 vs. 26.5 ± 3.0, p = 0.0003) and FPG sites (13.8 ± 1.8 vs. 22.5 ± 2.4, p = 0.01) were also higher after PTCA.ConclusionsOur results provide evidence for oxidative DNA damage after PTCA, likely related to ischemia-reperfusion injury

Cytoplasmic cleavage of IMPA1 3' UTR is necessary for maintaining axon integrity

The 3′ untranslated regions (3′ UTRs) of messenger RNAs (mRNAs) are non-coding sequences involved in many aspects of mRNA metabolism, including intracellular localization and translation. Incorrect processing and delivery of mRNA cause severe developmental defects and have been implicated in many neurological disorders. Here, we use deep sequencing to show that in sympathetic neuron axons, the 3′ UTRs of many transcripts undergo cleavage, generating isoforms that express the coding sequence with a short 3′ UTR and stable 3′ UTR-derived fragments of unknown function. Cleavage of the long 3′ UTR of Inositol Monophosphatase 1 (IMPA1) mediated by a protein complex containing the endonuclease argonaute 2 (Ago2) generates a translatable isoform that is necessary for maintaining the integrity of sympathetic neuron axons. Thus, our study provides a mechanism of mRNA metabolism that simultaneously regulates local protein synthesis and generates an additional class of 3′ UTR-derived RNAs

Expression level of CCR5 chemokine receptor on blood CD4+ and CD8+ T-cells plays an important role in the Ascending Aortic Aneurysm pathophysiology.

Background and aim: The CC chemokine receptor 5 (CCR5) is involved in the migration of circulating NK and Th1 cells towards inflammatory sites. CCR5 expression has also been demonstrated on endothelial cells, aortic smooth muscle cells and implicated in the development of abdominal aortic aneurysm. Thoracic aortic aneurysm (TAA) is a lethal disease burdened by complications such as aortic dissection/rupture. The risk of these acute events has been related to the severity of aortic enlargement. The aim of our study is to investigate a possible role of CCR5 expression on peripheral blood CD4+ and CD8+ T-lymphocytes in the pathogenesis of TAA. Methods: We have studied 14 patients (8 female, 6 male) with mean age of 67.35?7.70, undergoing isolated aortic valve replacement (AVR) and/or TAA surgery. Preoperatively, venous blood samples were obtained. A three colors flow cytometric analysis was performed by appropriate combinations of monoclonal antibodies directed against the following surface molecules: CD3, CD4, CD8, CCR5. Data are expressed in terms of percentage of positivity. Maximal aortic diameter (MAD) was determined by transesophageal echocardiography. For each patient we calculated the aortic size index (ASI), defined as MAD/BSA (mm/m2). Results: Aortic index was 21.52?3.14 mm/m2. Nine patients underwent isolated AVR (group 1) and five patients underwent TAA surgery (group 2). The percentage of CCR5+ on CD4+ was significantly higher in group 2 (17.03?3.08 vs 13.03?2.72, p=0.0269). A trend towards a higher percentage of CCR5+ on CD8+ was observed in group 2 (22.74?8,39 vs 16.26?3.75, p=0.0653). A significant correlation between aortic index and the percentage of CD4+ and CD8+ T-cells expressing CCR5 was observed (p=0.048, R2=0.287 and p=0.0067, R2=0.471 respectively). Conclusions: The correlation between the percentage of CD4+ and CD8+ T-cells expressing CCR5 and aortic index suggests the role of a T-cell immune-mediated cytotoxic mechanism in the progression of TAA disease

Proteomic analysis of S-nitrosylated nuclear proteins in rat cortical neurons

The ability of neurons to modulate gene expression in response to extrinsic signals is necessary for proper brain function. S-nitrosylation is the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols and is critical for transducing extracellular stimuli into specific patterns of gene expression. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is required for gene transcription during neuronal development, however only few nuclear targets of Snitrosylation have been identified to date. Here, we used S-nitrosothiol Resin Assisted Capture (SNORAC) coupled with mass spectrometry analysis to identify 614 S-nitrosylated nuclear proteins. Of these, 131 proteins had never been shown to be S-nitrosylated in any system, and 612 are new targets of S-nitrosylation in neurons. The site(s) of S-nitrosylation were identified for 59% of the targets, and motifs containing single lysines found at 33% of these sites. In addition, lysine motifs were found to be necessary for promoting S-nitrosylation of HDAC2 and Methyl-CpG Binding Protein 3 (MBD3). Moreover, S-nitrosylation of the histone binding protein RBBP7 was found to be necessary for dendritogenesis. Overall, our study provides the first extensive characterization of Snitrosylated nuclear proteins in neurons and identifies putative S-nitrosylation motifs that may be shared with other targets of nitric oxide signaling