Regulation of cyclin D1 RNA stability by SNIP1

Cyclin D1 expression represents one of the key mitogen-regulated events during the G1 phase of the cell cycle, whereas Cyclin D1 overexpression is frequently associated with human malignancy. Here, we describe a novel mechanism regulating Cyclin D1 levels. We find that SNIP1, previously identified as a regulator of Cyclin D1 expression, does not, as previously thought, primarily function as a transcriptional coactivator for this gene. Rather, SNIP1 plays a critical role in cotranscriptional or posttranscriptional Cyclin D1 mRNA stability. Moreover, we show that the majority of nucleoplasmic SNIP1 is present within a previously undescribed complex containing SkIP, THRAP3, BCLAF1, and Pinin, all proteins with reported roles in RNA processing and transcriptional regulation. We find that this complex, which we have termed the SNIP1/SkIP–associated RNA-processing complex, is coordinately recruited to both the 3' end of the Cyclin D1 gene and Cyclin D1 RNA. Significantly, SNIP1 is required for the further recruitment of the RNA processing factor U2AF65 to both the Cyclin D1 gene and RNA. This study shows a novel mechanism regulating Cyclin D1 expression and offers new insight into the role of SNIP1 and associated proteins as regulators of proliferation and cancer.

Variations in Apolipoprotein E Frequency With Age in a Pooled Analysis of a Large Group of Older People

Variation in the apolipoprotein E gene (APOE) has been reported to be associated with longevity in humans. The authors assessed the allelic distribution of APOE isoforms e2, e3, and e4 among 10,623 participants from 15 case-control and cohort studies of age-related macular degeneration (AMD) in populations of European ancestry (study dates ranged from 1990 to 2009). The authors included only the 10,623 control subjects from these studies who were classified as having no evidence of AMD, since variation within the APOE gene has previously been associated with AMD. In an analysis stratified by study center, gender, and smoking status, there was a decreasing frequency of the APOE e4 isoform with increasing age (?2 for trend = 14.9 (1 df); P = 0.0001), with a concomitant increase in the e3 isoform (?2 for trend = 11.3 (1 df); P = 0.001). The association with age was strongest in e4 homozygotes; the frequency of e4 homozygosity decreased from 2.7% for participants aged 60 years or less to 0.8% for those over age 85 years, while the proportion of participants with the e3/e4 genotype decreased from 26.8% to 17.5% across the same age range. Gender had no significant effect on the isoform frequencies. This study provides strong support for an association of the APOE gene with human longevity.

Endocrine disrupting effects of zearalenone, alpha- and beta-zearalenol at the level of nuclear receptor binding and steroidogenesis.

The mycotoxin zearalenone (ZEN) is a secondary metabolite of fungi which is produced by certain species of the genus Fusarium and can occur in cereals and other plant products. Reporter gene assays incorporating natural steroid receptors and the H295R steroidogenesis assay have been implemented to assess the endocrine disrupting activity of ZEN and its metabolites -zearalenol (-ZOL) and -zearalenol (-ZOL). -ZOL exhibited the strongest estrogenic potency (EC50 0.022 ± 0.001 nM), slightly less potent than 17- estradiol (EC50 0.015 ± 0.002 nM). ZEN was ~70 times less potent than -ZOL and twice as potent as -ZOL. Binding of progesterone to the progestagen receptor was shown to be synergistically increased in the presence of ZEN, -ZOL or -ZOL. ZEN, -ZOL or -ZOL increased production of progesterone, estradiol, testosterone and cortisol hormones in the H295R steroidogenesis assay, with peak productions at 10 M. At 100 M, cell viability decreased and levels of hormones were significantly reduced except for progesterone. -ZOL increased estradiol concentrations more than -ZOL or ZEN, with a maximum effect at 10 M, with -ZOL (562 ± 59 pg/ml) > -ZOL (494 ± 60 pg/ml) > ZEN (375 ± 43 pg/ml). The results indicate that ZEN and its metabolites can act as potential endocrine disruptors at the level of nuclear receptor signalling and by altering hormone production.

Consultation charges in Ireland deter a large proportion of patients from seeing the GP: results of a cross-sectional survey

To estimate the effect of a consultation charge on the health-seeking behaviour of patients.

Endocrine disrupting effects of ochratoxin A at the level of nuclear receptor activation and steroidogenesis

Ochratoxin A (OTA) is a mycotoxin and extrolite of fungi which has been reported in a range of foods. This study uses mammalian reporter gene assays (RGAs) with natural steroid receptors and the H295R steroidogenesis assay to assess the endocrine disrupting activity of OTA.

At the receptor level, OTA (within a concentration range of 0.25–2500 ng/ml) did not induce an agonistic response in an oestrogen, androgen, progestagen or glucocorticoid RGA. An antagonistic effect was observed in all of the RGAs at the highest concentration tested (2500 ng/ml). However, while there was no significant cytotoxic effect observed in the MTT (thiazolyl blue tetrazolium bromide) cell viability assay at this concentration, there was a corresponding change in cell morphology which may be related to the resulting antagonistic effect.

At the hormone production level, H295R cells were used as a steroidogenesis model and exposed to OTA (within a concentration range of 0.1–1000 ng/ml). Treatment of the cells with 1000 ng/ml OTA increased the production of estradiol (117 ± 14 ng/ml) over 3 times that of the solvent control (36 ± 9 pg/ml). Western blotting confirmed an increase in aromatase protein.

Overall the results indicate that OTA does not appear to interact with steroid receptors but has the potential to cause endocrine disruption by interfering with steroidogenesis. This is the first study identifying the effect OTA may have on production of the steroid hormone estradiol.

Discovery of a Potent Boronic Acid Derived Inhibitor of the HCV RNA-Dependent RNA Polymerase

A boronic acid moiety was found to be a critical pharmacophore for enhanced in vitro potency against wild type hepatitis C replicons and known clinical polymorphic and resistant HCV mutant replicons. The synthesis, optimization, and structure-activity relationships associated with inhibition of HCV replication in a sub-genomic replication system for a series of non-nucleoside boron-containing HCV RNA-Dependent RNA Polymerase (NS5B) inhibitors are described. A summary of the discovery of GSK5852 (3), a molecule which entered clinical trials in subjects infected with HCV in 2011, is included.

Effects of the mycotoxin patulin at the level of nuclear receptor transcriptional activity and steroidogenesis in vitro

Patulin (PAT) is a mycotoxin produced by various species of fungi, with Penicillium expansum being the most commonly occurring. Apples and apple products are the main sources of PAT contamination. This mycotoxin has been shown to induce toxic effects in animals, a few of which include reproductive toxicity and interference with the endocrine system. Here the endocrine disrupting potential of PAT has been investigated in vitro to identify disruption at the level of oestrogen, androgen, progestagen and glucocorticoid nuclear receptor transcriptional activity, and to assess interferences in estradiol, testosterone and progesterone steroid hormone production. At the receptor level, 0.5-5000ng/ml (0.0032-32μM) PAT did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs); however, nuclear transcriptional activity was affected. A >6 fold increase in the glucocorticoid receptor transcriptional activity was observed following treatment with 5000ng/ml PAT in the presence of cortisol. At the hormone production level, despite cytotoxicity being observed after treatment with 5000ng/ml PAT, estradiol levels had increased >2 fold. At 500ng/ml PAT treatment, an increase in progesterone and a decrease in testosterone production were observed. The findings of this study could be considered in assessing the health risks following exposure to PAT.

Vitamin D and mortality: meta-analysis of individual participant data from a large consortium of cohort studies from Europe and the United States

Objective: To investigate the association between serum 25-hydroxyvitamin D concentrations (25(OH)D) and mortality in a large consortium of cohort studies paying particular attention to potential age, sex, season, and country differences.

Design: Meta-analysis of individual participant data of eight prospective cohort studies from Europe and the US.

Setting: General population.

Participants: 26 018 men and women aged 50-79 years

Main outcome measures: All-cause, cardiovascular, and cancer mortality.

Results: 25(OH)D concentrations varied strongly by season (higher in summer), country (higher in US and northern Europe) and sex (higher in men), but no consistent trend with age was observed. During follow-up, 6695 study participants died, among whom 2624 died of cardiovascular diseases and 2227 died of cancer. For each cohort and analysis, 25(OH)D quintiles were defined with cohort and subgroup specific cut-off values. Comparing bottom versus top quintiles resulted in a pooled risk ratio of 1.57 (95% CI 1.36 to 1.81) for all-cause mortality. Risk ratios for cardiovascular mortality were similar in magnitude to that for all-cause mortality in subjects both with and without a history of cardiovascular disease at baseline. With respect to cancer mortality, an association was only observed among subjects with a history of cancer (risk ratio, 1.70 (1.00 to 2.88)). Analyses using all quintiles suggest curvilinear, inverse, dose-response curves for the aforementioned relationships. No strong age, sex, season, or country specific differences were detected. Heterogeneity was low in most meta-analyses.

Conclusions: Despite levels of 25(OH)D strongly varying with country, sex, and season, the association between 25(OH)D level and all-cause and cause-specific mortality was remarkably consistent. Results from a long term randomised controlled trial addressing longevity are being awaited before vitamin D supplementation can be recommended in most individuals with low 25(OH)D levels.