Epigenetic regulation of maspin expression in human ovarian carcinoma cells

OBJECTIVE: Maspin expression is often deregulated in human cancer cells compared to their normal cells due to loss of epigenetic control. In contrast to normal human ovarian surface epithelial (HOSE) cells, ovarian carcinoma cells display a gain of maspin mRNA expression. The objective of this study was to determine whether gain of maspin expression in ovarian cancer is governed by epigenetic mechanisms. METHODS: We examined the cytosine methylation and chromatin accessibility status of the maspin promoter in normal HOSE cells and ovarian carcinoma cells with real-time RT-PCR, sodium bisulfite genomic sequencing, and chromatin accessibility assays. 5-Aza-2'-deoxycytidine (5-aza-dC) was used to induce demethylation of the maspin promoter. Ad p53 was used to induce transient overexpression of wild-type p53. RESULTS: Normal HOSE cells were maspin-negative in association with methylation of the maspin promoter. In the maspin-positive ovarian cancer cell lines, the maspin promoter was unmethylated. Increased maspin expression in ovarian carcinoma cells was accompanied by a more accessible chromatin structure in the maspin promoter. In the maspin-negative ovarian cancer cell line A222, maspin could be induced following 5-aza-dC treatment or by forced overexpression of p53. CONCLUSIONS: These results suggest that changes in cytosine methylation and chromatin accessibility play an important role in maspin expression in human ovarian carcinoma. Deregulation of maspin expression in ovarian cancer is due to loss of epigenetic control as has been shown in other cancers. This observation provides further evidence of the strict epigenetic control of the maspin gene.     

Short-duration enzymatic treatment promotes integration of a cartilage graft in a defect

OBJECTIVES: Surgical manipulation of cartilage tissue is associated with chondrocyte death in the wound edges that hinders integration. The objective of this study was to evaluate the effect of a short course of treatment of a cartilage graft with a combination of hyaluronidase and collagenase on chondrocyte density and integrative capacity. METHODS: Cartilage explants were treated with enzymes for various time periods and at various concentrations. A central core was punched out of a larger explant, treated with enzymes, reimplanted, and placed subcutaneously in athymic mice. The number of chondrocytes in the wound edges was counted, and the integrative capacity of the grafts was evaluated by histology. RESULTS: Treatment with collagenase for 48 hours led to a significant increase in the number of vital chondrocytes and restored it to normal after 14 days of culture. Treatment with hyaluronidase and collagenase for 48 hours further increased chondrocyte densities to supranormal values. Shortening the treatment to 1 hour restored the chondrocyte density to normal after 14 days of culture. In vivo integration experiments showed increased chondrocyte densities in treated wound edges and extracellular matrix fibers crossing over from enzyme-treated parts to untreated parts. CONCLUSIONS: Short-duration treatment of a cartilage graft with a combination of hyaluronidase and collagenase increases cell density at wound edges and promotes integrative repair.     

Hot stage extrusion of p-amino salicylic acid with EC using CO2 as a temporary plasticizer

The aim of the current research project was to explore the possibilities of combining pressurized carbon dioxide with hot stage extrusion during manufacturing of solid dispersions of the thermally labile p-aminosalicylic acid (p-ASA) and ethylcellulose 20cps (EC 20cps) and to evaluate the ability of the pressurized gas to act as a temporary plasticizer. The thermal stability of the p-ASA was investigated using DSC, TGA and HPLC. The compound decomposes completely upon melting. Below 110 degrees C and under atmospheric conditions, the compound is thermally stabile for 10min. Pressurized carbon dioxide was injected into a Leistritz Micro 18 intermeshing co-rotating twin-screw melt extruder using an ISCO 260D syringe pump. Carbon dioxide acted as plasticizer for p-ASA/EC 20cps, reducing the processing temperature during the hot stage extrusion process. HPLC showed that without carbon dioxide injection, approximately 17% of p-ASA degraded, while less than 5% degraded with CO(2) injection. The experiments clearly showed that injecting pressurized carbon dioxide broadens the application of hot stage extrusion to thermally labile compounds in a one step process.     

Protonation of non-Watson-Crick base pairs and encapsidation of turnip yellow mosaic virus RNA

The 5' UTR of turnip yellow mosaic virus RNA contains two conserved hairpins with internal loops consisting of C.C and C.A mismatches. In this article, evidence is presented indicating that the 5' proximal hairpin functions as an encapsidation initiation signal. Extensive mutagenesis studies on this hairpin and sequencing of virus progeny showed a clear preference for C.C and C.A mismatches within the internal loop. The importance of these mismatches lies in their pH-dependent protonation and stable base pair formation. Encapsidation efficiency was found to be severely affected for several mutants lacking the protonatable mismatches in the internal loop of the 5' proximal hairpin. Furthermore, gel mobility-shift assays were performed with various RNA hairpins and empty capsids with a hole. Protonatable hairpins containing C.C and/or C.A pairs were found to bind specifically to the interior of the protein shell under acidic conditions (pH 4.5) in the presence of spermidine. Based on these results we propose that this binding of protonated cytosines to the coat protein of turnip yellow mosaic virus may represent a new motif in RNA-protein interactions.     

Punctate white matter lesions in full-term infants with neonatal seizures associated with SLC13A5 mutations

Introduction

Early-onset epileptic encephalopathy caused by biallelic SLC13A5 mutations is characterized by seizure onset in the first days of life, refractory epilepsy and developmental delay. Little detailed information about the brain MRI features is available in these patients.