AUF1 cell cycle variations define genomic DNA methylation by regulation of DNMT1 mRNA stability

DNA methylation is a major determinant of epigenetic inheritance. DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division, and deregulated expression of DNMT1 leads to cellular transformation. We show herein that AU-rich element/poly(U)-binding/degradation factor 1 (AUF1)/heterogeneous nuclear ribonucleoprotein D interacts with an AU-rich conserved element in the 3' untranslated region of the DNMT1 mRNA and targets it for destabilization by the exosome. AUF1 protein levels are regulated by the cell cycle by the proteasome, resulting in cell cycle-specific destabilization of DNMT1 mRNA. AUF1 knock down leads to increased DNMT1 expression and modifications of cell cycle kinetics, increased DNA methyltransferase activity, and genome hypermethylation. Concurrent AUF1 and DNMT1 knock down abolishes this effect, suggesting that the effects of AUF1 knock down on the cell cycle are mediated at least in part by DNMT1. In this study, we demonstrate a link between AUF1, the RNA degradation machinery, and maintenance of the epigenetic integrity of the cell.     

Measurement of [<Superscript>18</Superscript>F]-fluorodeoxyglucose incorporation into human osteoblast–An experimental method

An evaluation of human osteoblast metabolism usually involves measurements of the by-products of bone matrix elaboration. The assessment of glycolytic activity of osteoblasts is not a standard tool in most of the reports, but might be of value by providing a direct indicator of cellular metabolism. Measurement of the incorporation of [¹⁸F]-fluorodeoxyglucose, which is not further degradable following its conversion into glycose-6-phosphate during glycolysis and is trapped in this form within the cells, can be used as an effective research tool for estimation of osteoblast metabolism. In order to estimate the [¹⁸F]-fluorodeoxyglucose incorporation we used cultured human osteoblast-like cells. Following incubation of the culture samples in a glucose free medium with 5 μ Ci [¹⁸F]-fluorodeoxyglucose we measured the radioactivity of the cell fraction, as a percent from the initial dose, and compared to the incorporation values in cells treated by protoporphyrine IX (10⁻⁵ M), an endogenous pro-apoptotic agent. To compare the response of [¹⁸F]-fluorodeoxyglucose incorporation studies, following treatment of cells with the protoporphyrine IX, to other experimental cell metabolism evaluation methods, we performed a parallel comparison of alkaline phospatase activity, which is a standard measurement tool of osteoblast metabolism, in the control and treatment groups. A narrow range of 0.22–1.36% of [¹⁸F]-fluorodeoxyglucose incorporation per million cells was found. Additionally in the protoporphyrine IX treated cells a significant 62% decrease of [¹⁸F]-fluorodeoxyglucose incorporation was observed (p < .05). A parallel significant decrease in alkaline phosphatase activity (p < .001) was found in the cells treated by the protoporphyrine IX. Therefore we suggest that the presented method of [¹⁸F]-fluorodeoxyglucose incorporation measurement can be utilized as an effective research tool for estimation of the cellular glycolitic activity in human osteoblast-like cells in vitro.     

Effect of oxazaborolidines on immobilized fructosyltransferase analyzed by surface plasmon resonance

Dental diseases are among the most prevalent afflictions of humankind. These diseases are associated with the formation of biofilms harboring pathogenic bacteria. Fructosyltransferases (FTF) are extra cellular enzymes of several oral bacteria. FTF are associated with the formation of extracellular polysaccharide matrix (fructans) which play a role in biofilm formation and oral bacteria physiology. Oxazaborolidines have been shown to inhibit biofilm formation. The purpose of this study was to examine if the anti-biofilm effect is, in part, an effect on the immobilized enzymes synthesizing the extra cellular polysaccharide participating in biofilm formation. Eight different oxazaborolidines (BNO1-BNO8) were synthesized and evaluated for their affect on the synthesis of fructans by FTF using the biomolecular interaction analysis (BIAcore) system which involves the use of real-time surface plasmon resonance (SPR) technique. The tested oxazaborolidines demonstrated a significant and immediate inhibitory effect on immobilized FTF activity. This effect was reversible. Our results show that oxazaborolidines can act as enzymatic inhibitors of FTF immobilized on the surface, also at levels lower than their MIC. Part of the anti-biofilm effect of BNOs may be accounted for this enzymatic inhibition.     

Radiotherapy for Stage IIA seminoma: The Northern Israel Oncology Center Experience, 1971–2010

Aim

To evaluate treatment details, outcome, relapse rate and side-effects in Stage IIA seminoma irradiated and followed for a period of 39 years.

Background

Seminoma is a very radiosensitive disease and radiation therapy alone is able to achieve long-term disease-free survival, even in advanced Stage disease. Due to the lack of long-term prospective studies, it is of value to follow patients and try to determine the appropriate volume to be irradiated and the dose which can achieve total cure with minimal acute and chronic side-effects.

Patients and methods

A retrospective review of 24 Stage IIA seminoma patients irradiated between 1971 and 2010 was performed. All patients underwent orchiectomy and meticulous clinical, biochemical and radiological staging.

Results

Median age at diagnosis was 36 years and median follow-up was 84 months. A majority of patients received the “hockey-stick” irradiation schedule (para-aortic lymph nodes and hemi-pelvis) to a total dose of 2250–2500 cGy and a boost to radiologically involved nodes of 500–1000 cGy. Treatment was well-tolerated. Twenty-one (88%) patients are alive with no evidence of disease. Two patients died due to unknown causes, while one patient died due to head of the pancreas carcinoma, most probably radiation-induced.

Conclusions

In Stage II seminoma, radiotherapy can provide excellent results with low rates of toxicity. Reduction of total dose and size of fields without affecting the good results should be considered. Due to prolonged survival, awareness of second primary tumor is indicated.     

The role of onion-associated fungi in bulb mite infestation and damage to onion seedlings

In Israel Rhizoglyphus robini is considered to be a pest in its own right, even though the mite is usually found in association with fungal pathogens. Plant protection recommendations are therefore to treat germinating onions seedlings, clearly a crucial phase in crop production, when mites are discovered. The aim of this study was to determine the role of fungi in bulb mite infestation and damage to germinating onion seedlings. Accordingly we (1) evaluated the effect of the mite on onion seedling germination and survival without fungi, (2) compared the attraction of the mite to species and isolates of various fungi, (3) assessed the effect of a relatively non-pathogenic isolate of Fusarium oxysporum on mite fecundity, and (4) determined the effects of the mite and of F. oxysporum separately and together, on onion seedling germination and sprout development. A significant reduction of seedling survival was recorded only in the 1,000 mites/pot treatment, after 4 weeks. Mites were attracted to 6 out of 7 collected fungi isolates. Mite fecundity on onion sprouts infested with F. oxysporum was higher than on non-infested sprouts. Survival of seedlings was affected by mites, fungi, and their combination. Sprouts on Petri dishes after 5 days were significantly longer in the control and mite treatments than both fungi treatments. During the 5-day experiment more mites were always found on the fungi-infected sprouts than on the non-infected sprouts. Future research using suppressive soils to suppress soil pathogens and subsequent mite damage is proposed.