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Answer added in Western Blot40 What is the best stripping solution recipe (in house) and method for Nitrocellulose and PVDF membrane?we use the Strong stripping solution by Millipore
Publications (27) View all
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Article: Prevalence, clinical, and molecular correlates of KCNJ5 mutations in primary aldosteronism.
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ABSTRACT: Primary aldosteronism is the most common form of secondary hypertension. Mutations in the KCNJ5 gene have been described recently in aldosterone-producing adenomas (APAs). The aim of this study was to investigate the prevalence of KCNJ5 mutations in unselected patients with primary aldosteronism and their clinical, biological and molecular correlates. KCNJ5 sequencing was performed on somatic (APA, n=380) and peripheral (APA, n=344; bilateral adrenal hyperplasia, n=174) DNA of patients with primary aldosteronism, collected through the European Network for the Study of Adrenal Tumors. Transcriptome analysis was performed in 102 tumors. Somatic KCNJ5 mutations (p.Gly151Arg or p.Leu168Arg) were found in 34% (129 of 380) of APA. They were significantly more prevalent in females (49%) than males (19%; P<10(-3)) and in younger patients (42.1±1.0 versus 47.6±0.7 years; P<10(-3)) and were associated with higher preoperative aldosterone levels (455±26 versus 376±17 ng/L; P=0.012) but not with therapeutic outcome after surgery. Germline KCNJ5 mutations were found neither in patients with APA nor those with bilateral adrenal hyperplasia. Somatic KCNJ5 mutations were specific for APA, because they were not identified in 25 peritumoral adrenal tissues or 16 cortisol-producing adenomas. Hierarchical clustering of transcriptome profiles showed that APAs with p.Gly151Arg or p.Leu168Arg mutations were indistinguishable from tumors without KCNJ5 mutations. In conclusion, although a large proportion of sporadic APAs harbors somatic KCNJ5 mutations, germline mutations are not similarly causative for bilateral adrenal hyperplasia. KCNJ5 mutation carriers are more likely to be females; younger age and higher aldosterone levels at diagnosis suggest that KCNJ5 mutations may be associated with a more florid phenotype of primary aldosteronism.Hypertension 03/2012; 59(3):592-8. · 6.21 Impact Factor -
Article: Effects of type 5-phosphodiesterase inhibition on energy metabolism and mitochondrial biogenesis in human adipose tissue ex vivo.
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ABSTRACT: An excess of adipose tissue (AT) in obese individuals is linked to increased cardiovascular risk and mitochondria have been shown to be defective in the muscle and AT of patients with metabolic disorders such as obesity and Type 2 diabetes. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays a role in mitochondrial biogenesis through cyclic-GMP (cGMP). AT harbors the whole molecular signaling pathway of NO, together with type 5-phosphodiesterase (PDE- 5), the main cGMP catabolising enzyme. Our aim was to evaluate the effect of the modulation of NO pathway, through PDE-5 inhibition, on energy metabolism and mitochondria biogenesis in human omental AT. Cultured human omental AT was stimulated with PDE-5 inhibitor, vardenafil, at different concentration for 24 and 72 h. Analysis of the expression of both key-regulator genes of adipocyte metabolism and mitochondria-biogenesis markers was performed. We found an increased gene expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), adiponectin, and proliferator- activated receptor gamma coactivator-1 α (PGC-1α) after a 24-h stimulation with vardenafil at the lowest concentration employed compared to controls (p<0.05). After 72 h of stimulation, a significant increase of mitochondrial DNA was found compared to control samples (p<0.05). Our data suggest that PDE-5 inhibition could have an impact on mitochondrial content of human AT suggesting a positive effect on energy metabolism and adding new elements in the comprehension of AT pathophysiology.Journal of endocrinological investigation 11/2011; 34(10):738-41. · 1.57 Impact Factor -
Article: Analysis of insulin sensitivity in adipose tissue of patients with primary aldosteronism.
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ABSTRACT: The objective of the study was to assess the effect of high aldosterone levels on insulin sensitivity of adipose tissue in humans. Visceral adipose tissue (VAT) was obtained from patients with aldosterone-producing adenoma (APA; n=14) and, as controls, nonfunctioning adenoma (NFA; n=14) undergoing laparoscopic adrenalectomy. Homeostasis model assessment index was higher and potassium was lower in APA than NFA (P<0.05). Immunohistochemistry, Western blotting, and real-time PCR were used to detect and quantify mineralocorticoid receptor (MR) expression. Transcript levels of peroxisome proliferative-activated receptor-gamma, insulin receptor, glucose transporter 4, insulin receptor substrate-1 and -2, leptin, adiponectin, IL-6, monocyte chemoattractant protein-1, glucocorticoid receptor (GR)-alpha, 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1, and HSD11B2 were quantified. The effect of increasing aldosterone concentrations on 2-deoxy-[3H]d-glucose uptake was tested in human sc abdominal adipocytes. Expression of MR was demonstrated in VAT, with no difference between APA and NFA as to mRNA levels of MR, GRalpha, HSD11B1, and glucose metabolism and inflammation factors. In cultured adipocytes, basal and insulin-stimulated glucose uptake were unaffected by 1-100 nM (normal/hyperaldosteronism) and impaired only by much higher, up to 10 microM, aldosterone concentrations. The impairment was prevented by RU486 but not by eplerenone. Gene expression of insulin signaling/inflammatory molecules was similar in VAT of APA and NFA patients, not supporting an effect of aldosterone excess on insulin sensitivity of adipose tissues. Only at pharmacological concentrations and through GR activation, aldosterone reduced glucose uptake in adipocytes. Insulin resistance in primary aldosteronism might occur in compartments other than fat and/or depend on concurrent environmental factors.The Journal of clinical endocrinology and metabolism 08/2010; 95(8):4037-42. · 6.50 Impact Factor -
Article: Evidence for osteocalcin production by adipose tissue and its role in human metabolism.
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ABSTRACT: The adipose tissue (AT), which is an endocrine organ, is linked to several metabolic abnormalities. Undercarboxylated osteocalcin (ucOCN) regulates insulin and adiponectin secretion. Our objective was to investigate the involvement of OCN in obesity and to evaluate, in vitro and ex vivo, the role of AT in the modulation of this endocrine circuit. This transversal study involved 83 male subjects, divided according to the World Health Organization body mass index classification, evaluated at Padova's Obesity Outpatient Clinic. OCN, both undercarboxylated (ucOCN) and carboxylated (cOCN) forms, was measured in serum by ELISA. OCN mRNA expression and protein production were measured by quantitative RT-PCR and immunohistochemistry during in vitro adipogenesis and in sc AT (SAT) and omental AT (OAT) from normal adult men. cOCN and ucOCN release by AT in a simple growth medium was verified by ELISA. Overweight and obese patients had a lower ucOCN and ucOC/OCN ratio. In the whole cohort, ucOCN/OCN ratio was negatively correlated to body mass index (rho = -0.233; P < 0.05). OCN mRNA was present in SAT and OAT and during all stages of adipogenesis, with higher expression in the first steps. Immunohistochemistry confirmed the expression of OCN protein. Both SAT and OAT were able to release cOCN and ucOCN. Our data support a pathophysiological link between ucOCN and cOCN balance and obesity. OCN is present in the first phases of adipogenesis but also in human AT ex vivo. AT releases, in vitro, both ucOCN and cOCN, suggesting a possible link between AT and OCN in the regulation of metabolism.The Journal of clinical endocrinology and metabolism 07/2010; 95(7):3502-6. · 6.50 Impact Factor -
Article: Teratocarcinoma-derived growth factor-1 is upregulated in aldosterone-producing adenomas and increases aldosterone secretion and inhibits apoptosis in vitro.
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ABSTRACT: Aldosterone-producing adenomas (APA) are a frequent cause of secondary hypertension characterized by autonomous hypersecretion of aldosterone. However, the molecular mechanisms involved in adrenal tumorigenesis and deregulated aldosterone secretion are currently unknown. To identify putative functional genes, a transcriptional screening was performed on 8 APA and 3 normal adrenals (NA) using oligonucleotide microarrays. Data were next validated on an expanded set of samples by real-time PCR (APA, n=19; NA, n=10). The epidermal growth factor-like teratocarcinoma-derived growth factor-1 (TDGF-1) was upregulated in APA compared with NA (14.7-fold and 21.4-fold by microarray and real-time PCR, respectively). In vitro studies and Western blot analysis using the NCI H295R adrenocortical cell line showed that TDGF-1 increased Akt phosphorylation on Thr308 and Ser473, consistent with activation of phosphatidylinositol 3-kinase/Akt signaling, and also demonstrated a concomitant inactivation of the Akt substrate glycogen synthesis kinase-3beta via Ser9 phosphorylation. Furthermore, TDGF-1 mediated a 3.8+/-0.4-fold increase in aldosterone secretion (n=4) that was specifically blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin (50 nmol/L) and LY294002 (20 micromol/L). Finally, TDGF-1 protected H295R cells from apoptosis induced by staurosporine, causing a decrease in caspase-3 activity, a reduction in the inactivation of poly(ADP-ribose) polymerase, and an inhibition of DNA fragmentation, detected by the TUNEL reaction and fluorescence microscopy that was blocked by LY294002. Taken together, our data suggest that TDGF-1, which is significantly upregulated in APA and mediates aldosterone hypersecretion and deregulated growth in adrenocortical cells in vitro, may represent a key player in the development and pathophysiology of primary aldosteronism.Hypertension 04/2010; 55(6):1468-75. · 6.21 Impact Factor
