GCM2 Silencing in Parathyroid Adenoma is associated with Promoter Hypermethylation and Gain of Methylation on Histone 3

PURPOSE: Glial cells missing 2 (GCM2), a zinc finger-transcription factor, is essentially required for the development of parathyroid glands. We sought to identify if the epigenetic alterations in the GCM2 transcription are involved in the pathogenesis of sporadic parathyroid adenoma. In addition, we examined the association between promoter methylation and histone modifications with disease indices. EXPERIMENTAL DESIGN: mRNA and protein expression of GCM2 were analyzed by RT-qPCR and immunohistochemistry in 33 adenomatous and 10 control parathyroid tissues. DNA methylation and histone methylation/acetylation of GCM2 promoter were measured by bisulfite sequencing and ChIP-qPCR. Additionally, we investigated the role of epigenetic modifications on GCM2 and DNA methyltransferase 1 (DNMT1) expression in PTH-C1 cells by treating with 5-aza 2\u27deoxycytidine (DAC) and BRD4770 and assessed for GCM2 mRNA and DNMT1 protein levels. RESULTS: mRNA and protein expression of GCM2 were lower in sporadic adenomatous than in control parathyroid tissues. This reduction correlated with hypermethylation (P\u3c0.001) and higher H3K9me3 levels in GCM2 promoter (P\u3c0.04) in adenomas. In PTH-C1 cells, DAC treatment resulted in increased GCM2 transcription and decreased DNMT1 protein expression, while cells treated with the BRD4770 showed reduced H3K9me3 levels but a non-significant change in GCM2 transcription. CONCLUSION: These findings suggest the concurrent association of promoter hypermethylation and higher H3K9me3 with the repression of GCM2 expression in parathyroid adenomas. Treatment with DAC restored GCM2 expression in PTH-C1 cells. Our results showed a possible epigenetic landscape in the tumorigenesis of parathyroid adenoma and also that DAC may be promising avenues of research for parathyroid adenoma therapeutics

Biochemical and Molecular Mechanisms of Folate Transport in Rat Pancreas; Interference with Ethanol Ingestion

Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency which is due, in part to folate malabsorption. The present study deals with the mechanistic insights of reduced folate absorption in pancreas during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20% solution) orally for 3 months and the mechanisms of alcohol associated reduced folate uptake was studied in pancreas. The folate transport system in the pancreatic plasma membrane (PPM) was found to be acidic pH dependent one. The transporters proton coupled folate transporter (PCFT) and reduced folate carrier (RFC) are involved in folate uptake across PPM. The folate transporters were found to be associated with lipid raft microdomain of the PPM. Ethanol ingestion decreased the folate transport by reducing the levels of folate transporter molecules in lipid rafts at the PPM. The decreased transport efficiency of the PPM was reflected as reduced folate levels in pancreas. The chronic ethanol ingestion led to decreased pancreatic folate uptake. The decreased levels of PCFT and RFC expression in rat PPM were due to decreased association of these proteins with lipid rafts (LR) at the PPM

Effect of harmaline on rat intestinal brush border sucrase activity

119-123The effect of harmaline, a plant alkaloid has been studied on rat intestinal brush border sucrase activity. Stimulation of sucrase activity by Na+ was found to be pH-dependent. At neutral pH, 20 mM Na+ stimulated sucrase activity by reducing Km by 30% while at acidic pH (5.2), the activity increased 4-fold compared to Na+-free enzyme. At 1.0 mM, harmaline markedly inhibited (67%) the enzyme activity at pH 5.2 in the absence of Na+. However, inhibition was reduced in presence of 20 mM sodium, whereas 4.0 mM harmaline was required to inhibit the enzyme activity by 65%. In the absence of Na+ ions, harmaline inhibition of sucrase activity was of competitive type, but it changed to non-competitive type in presence of 20 mM Na+ at pH 5.2. Sucrase-harmaline interactions as a function of pH, both in presence and absence of Na+ revealed a shift in pH optima of the enzyme towards a higher pH in presence of 4 mM and 1 mM harmaline respectively. The observed inhibition was reversible in nature and was only partially overcome by sodium, lithium, potassium, cesium, rubidium and ammonium ions. These findings suggest that harmaline also inhibits rat brush border sucrase and that the presence of Na+ site is not a pre-requisite for the inhibition

Chronic cold stress-induced alterations in brush border membrane composition and enzyme activities in rat intestine

180-185The effect of chronic cold stress on the composition and function of rat intestinal brush border membrane (BBM) was studied. Various lipid fractions from intestinal BBM viz. cholesterol (p<0.01), phospholipids (p<0.01), triglycerides (p<0.05) and gangliosides (p<0.05) were significantly reduced in cold stressed animals, as compared to controls. Analysis of membrane saccharide content revealed a significant increase in sialic acid (25%) and hexosamine (36%) contents and a re-duction in fucose (19%) content in cold stressed rats. Determination of various enzyme activities in BBM showed signifi-cantly enhanced activities of alkaline phosphatase (p<0.01), lactase (p<0.001) and leucine aminopeptidase (p<0.001), whereas sucrase activity was reduced (p<0.05) under these conditions. The magnitude and site of these alterations across the crypt-villus axis varied from enzyme to enzyme. These findings suggest that chronic cold stress results in profound altera-tions in intestinal BBM. Altered structure and function of intestinal BBM may play a role in stress-induced derangements in gastrointestinal tract

Western blots analysis of pancreatic tissue lysate (a) using anti RFC (58 kDa), anti PCFT (54 kDa) antibodies (b) Graph represents summary data of densitometric analysis, lane 1,2: Control; 3,4: ethanol fed.

<p>Western blot analysis of the PPMV (c) using anti RFC (58 kDa), anti PCFT (54 kDa) antibodies (d) Graph represents summary data of densitometric analysis. Data are expressed are means± SD of 4 separate experiments. Lane 1–3: Control; 4–6: ethanol fed. ^**p<0.01, ^***p<0.001 vs. Control.</p

HRM analysis of PCFT and RFC gene (a) upper panel PCFT lower panel RFC, upper/lower panel left side melting profile and upper panel/lower panel right normalized melting curves.

<p>Curves in green; Control, red; Ethanol fed and gray; 100% methylated. (b) Percentage change in methylation of genes in ethanol fed rats compared to control. ^***p<0.001 vs. Control.</p

Uptake of 5-[¹⁴C]-MethylTHF in the rat PPMV as a function of pH optimum.

<p>Uptake was measured by varying the pH of incubation buffer [100 mM NaCl, 80 mM mannitol, 10 mM HEPES, 10 mM 2-morpholinoethanesulfonic acid from 5.0 to 8.0, keeping intravesicular pH 7.4 at 0.5 µM substrate concentration for 10 sec. Each data point is mean± SD of 4 separate uptake determinations. *p<0.05, **p<0.01, ***p<0.001 vs. control.</p