Effects of two types of energy restriction on methylation levels of adiponectin receptor 1 and leptin receptor overlapping transcript in a mouse mammary tumour virus-transforming growth factor-α breast cancer mouse model.

The role of adiponectin and leptin signalling pathways has been suggested to play important roles in the protective effects of energy restriction (ER) on mammary tumour (MT) development. To study the effects of ER on the methylation levels in adiponectin receptor 1 (AdipoR1) and leptin receptor overlapping transcript (Leprot) genes using the pyrosequencing method in mammary fat pad tissue, mouse mammary tumour virus-transforming growth factor-α (MMTV-TGF-α) female mice were randomly assigned to ad libitum (AL), chronic ER (CER, 15 % ER) or intermittent ER (3 weeks AL and 1 week 60 % ER in cyclic periods) groups at 10 weeks of age until 82 weeks of age. The methylation levels of AdipoR1 in the CER group were higher than those in the AL group at week 49/50 (P < 0·05), while the levels of methylation for AdipoR1 and Leprot genes were similar among the other groups. Also, the methylation levels at CpG2 and CpG3 regions of the promoter region of the AdipoR1 gene in the CER group were three times higher (P < 0·05), while CpG1 island of Leprot methylation was significantly lower compared with the other groups (P < 0·05). Adiponectin and leptin gene expression levels were consistent with the methylation levels. We also observed a change with ageing in methylation levels of these genes. These results indicate that different types of ER modify methylation levels of AdipoR1 and Leprot in different ways and CER had a more significant effect on methylation levels of both genes. Epigenetic regulation of these genes may play important roles in the preventive effects of ER against MT development and ageing processes.

Cytotoxicity of 5 endodontic sealers on L929 cell line and human dental pulp cells.

AIM: To investigate the cytotoxicity of five root canal sealers on L929 mouse fibroblasts and primary human dental pulp cells. METHODOLOGY: Cylindrical specimens of AH Plus (Dentsply De Trey GmbH, Konstanz, Germany), RoekoSeal (Coltène Whaledent, Langenau, Germany), EndoREZ (Ultradent Products Inc., South Jordan, UT, USA), Epiphany (Pentron Clinical Technologies, LLCC, Wallingford, CT, USA) and Activ GP (Brasseller Inc., USA, Savannah, GA, USA) were kept at 37 °C in a humidified atmosphere of 5% CO(2) for thrice the length of the setting time given by the manufacturer. Extraction of specimens was performed after setting in cell growth medium for 1, 4 and 7 days. Undiluted, 50% and 25% diluted eluates were incubated with cultured cells for 24 and 72 h. Cytotoxicity was assessed using MTS colorimetric bioassay. Kruskal-Wallis test and post hoc Dunn's multiple comparison test were used to compare the sealers and diluted/undiluted eluates in terms of cell viability (% of control). Friedman test and post hoc Dunn's multiple comparison test were performed to compare extraction periods. Wilcoxon test was utilized in comparing 24- and 72-h readings. RESULTS: Undiluted 1-day eluate of Activ GP was significantly more cytotoxic than all other sealers (P < 0.0001). Undiluted 4- and 7-day eluates of Epiphany and Activ GP were significantly more cytotoxic than the other three sealers (P < 0.0001). Diluted eluates of Activ GP and Epiphany were generally less toxic than the undiluted ones. The cytotoxicity of Epiphany significantly increased as the extraction period increased (P < 0.0001). Epiphany became more toxic with time of exposure to cells. No or minimal cytotoxicity was observed with RoekoSeal, AH Plus and EndoREZ. CONCLUSIONS: The sealers exhibited varying degrees of cytotoxicity dependent on their chemical composition.

Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis.

A number of studies have reported in the last decade that human tooth germs contain multipotent cells that give rise to dental and peri-odontal structures. The dental pulp, third molars in particular, have been shown to be a significant stem cell source. In this study, we isolated and characterized human tooth germ stem cells (hTGSCs) from third molars and assessed the expression of developmentally important transcription factors, such as oct4, sox2, klf4, nanog and c-myc, to determine their pluri-potency. Flow-cytometry analysis revealed that hTGSCs were positive for CD73, CD90, CD105 and CD166, but negative for CD34, CD45 and CD133, suggesting that these cells are mesenchymal-like stem cells. Under specific culture conditions, hTGSCs differentiated into osteogenic, adipogenic and neurogenic cells, as well as formed tube-like structures in Matrigel assay. hTGSCs showed significant levels of expression of sox2 and c-myc messenger RNA (mRNA), and a very high level of expression of klf4 mRNA when compared with human embryonic stem cells. This study reports for the first time that hTGSCs express developmentally important transcription factors that could render hTGSCs an attractive candidate for future somatic cell re-programming studies to differentiate germs into various tissue types, such as neurons and vascular structures. In addition, these multipotential hTGSCs could be important stem cell sources for autologous transplantation.

Angiotensin-converting enzyme insertion/deletion gene polymorphism in patients with familial multiple cerebral cavernous malformations.

Cavernous malformations can occur in both sporadic and autosomal dominant forms. The aim of this study was to investigate the potential role of insertion/deletion (I/D) polymorphisms of the angiotensin-converting enzyme (ACE) gene in the development of cerebral cavernous malformations (CCM). Forty-one members of two families affected by familial CCM were included in this study. DNA was isolated from peripheral venous blood, and polymerase chain reaction analysis was used to detect I/D polymorphisms of the ACE gene, using HACE3s and HACE3as as primers. Only 10 participants had MRI-confirmed CCM. Of these 10 subjects, seven had the I/D, two had the D/D, and one had the I/I genotype. Of the remaining 31 subjects, 14 had the I/I, 13 had the I/D, and four had the D/D genotype. There was a greater proportion of subjects with the D allele among those with MRI-confirmed CCM than among those without (p<0.05). These results suggest that the D polymorphism of the ACE gene may be involved in the pathogenesis of familial CCM.