Markers of mRNA stabilization and degradation, and RNAi within astrocytoma GW bodies

GW bodies (GWBs) are unique cytoplasmic structures that contain the mRNA binding protein GW182 and other proteins involved in mRNA processing pathways. The rationale for this study arose from clinical studies indicating that 33% of patients with GWB autoantibodies have a motor/sensory neuropathy and/or ataxia. The novelty of this study is the identification of GWBs in astrocytes and astrocytoma cells within cell bodies and cytoplasmic projections. Astrocytoma GWBs exhibit complex heterogeneity with combinations of LSm4 and XRN1 as well as Ago2 and Dicer, key proteins involved in mRNA degradation and RNA interference, respectively. GWB subsets contained the mRNA transport and stabilization proteins SYNCRIP, hnRNPA1, and FMRP, not previously described as part of the GWB complex. Immunoprecipitation of astrocytoma GWBs suggested that Dicer, hDcp, LSm4, XRN1, SYNCRIP, and FMRP form a multiprotein complex. GWBs are likely involved in a number of regulatory mRNA pathways in astrocytes and astrocytoma cells.     

Cloning and pharmacological characterization of the dog P2X7 receptor

Background and purpose:

Human and rodent P2X7 receptors exhibit differences in their sensitivity to antagonists. In this study we have cloned and characterized the dog P2X7 receptor to determine if its antagonist sensitivity more closely resembles the human or rodent orthologues.

Experimental approach:

A cDNA encoding the dog P2X7 receptor was isolated from a dog heart cDNA library, expressed in U-2 OS cells using the BacMam viral expression system and characterized in electrophysiological, ethidium accumulation and radioligand binding studies. Native P2X7 receptors were examined by measuring ATP-stimulated interleukin-1β release in dog and human whole blood.

Key results:

The dog P2X7 receptor was 595 amino acids long and exhibited high homology (>70%) to the human and rodent orthologues although it contained an additional threonine at position 284 and an amino acid deletion at position 538. ATP possessed low millimolar potency at dog P2X7 receptors. 2′-&3′-O-(4benzoylbenzoyl) ATP had slightly higher potency but was a partial agonist. Dog P2X7 receptors possessed relatively high affinity for a number of selective antagonists of the human P2X7 receptor although there were some differences in potency between the species. Compound affinities in human and dog blood exhibited a similar rank order of potency as observed in studies on the recombinant receptor although absolute potency was considerably lower.

Conclusions and implications:

Dog recombinant and native P2X7 receptors display a number of pharmacological similarities to the human P2X7 receptor. Thus, dog may be a suitable species for assessing target-related toxicity of antagonists intended for evaluation in the clinic.     

Constitutive activity of cannabinoid-2 (CB2) receptors plays an essential role in the protean agonism of (+)AM1241 and L768242

Background and purpose:

Cannabinoid-2 (CB₂) receptor-selective agonists have shown anti-nociceptive activity in models of neuropathic and inflammatory pain, and the two agonists most widely used, (+/−)AM1241 [(2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl-methanone] and L768242 [(2,3-dichloro-phenyl)-[5-methoxy-2-methyl-3-(2-morpholin-4-yl-ethyl)-indol-1-yl]-methanone] ({"type":"entrez-nucleotide","attrs":{"text":"GW405833","term_id":"288331434","term_text":"GW405833"}}GW405833), have been suggested to be protean agonists. Here we investigated the role of the constitutive activity of CB₂ receptors in (+)AM1241 and L768242 protean agonism.

Experimental approach:

Pharmacological profiles of CB₂ receptor ligands were evaluated in Chinese hamster ovary cells expressing recombinant human (hCB₂) or rat (rCB₂) receptors, by measuring modulation of cAMP. To assess the influence of constitutive activity on pharmacological profile, constitutive activity was abolished by pretreatment with AM630 [(6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl) methanone)], followed by extensive washing.

Key results:

In cell lines expressing either hCB₂ or rCB₂ receptors, (+)AM1241 did not reverse forskolin stimulation of cAMP levels. Conversely, L768242 was an inverse agonist at both hCB₂ and rCB₂ receptors. Abolition of constitutive activity disclosed (+)AM1241 and L768242 agonist activity, while activity of CP55940 [5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxy-propyl)-cyclohexyl]-phenol] was unaffected and AM630 became a neutral antagonist. In presence of constitutively active CB₂ receptors, (+)AM1241 antagonized CP55940, but when constitutive activity was abolished, it acted as a partial agonist with additive or antagonistic behaviour, depending on concentration.

Conclusions and implications:

These results show that (+)AM1241 and L768242 are protean agonists at both hCB₂ and rCB₂ receptors. Abolition of constitutive activity reveals the agonist activity of these compounds. Thus, differences between in vivo and in vitro profiles of CB₂ receptor agonists could be due to different levels of constitutive activity in recombinant versus native CB₂ receptors.     

Impaired arsenic metabolism in children during weaning

Background

Methylation of inorganic arsenic (iAs) via one-carbon metabolism is a susceptibility factor for a range of arsenic-related health effects, but there is no data on the importance of arsenic metabolism for effects on child development.

Aim

To elucidate the development of arsenic metabolism in early childhood.

Methods

We measured iAs, methylarsonic acid (MA) and dimethylarsinic acid (DMA), the metabolites of iAs, in spot urine samples of 2400 children at 18 months of age. The children were born to women participating in a population-based longitudinal study of arsenic effects on pregnancy outcomes and child development, carried out in Matlab, a rural area in Bangladesh with a wide range of arsenic concentrations in drinking water. Arsenic metabolism was evaluated in relation to age, sex, anthropometry, socio-economic status and arsenic exposure.

Results

Arsenic concentrations in child urine (median 34 μg/L, range 2.4-940 μg/L), adjusted to average specific gravity of 1.009 g/mL, were considerably higher than that measured at 3 months of age, but lower than that in maternal urine. Child urine contained on average 12% iAs, 9.4% MA and 78% DMA, which implies a marked change in metabolite pattern since infancy. In particular, there was a marked increase in urinary %MA, which has been associated with increased risk of health effects.

Conclusion

The arsenic metabolite pattern in urine of children at 18 months of age in rural Bangladesh indicates a marked decrease in arsenic methylation efficiency during weaning.     

Evaluation of the therapeutic potential of PPARalpha agonists for X-linked adrenoleukodystrophy

Adrenoleukodystrophy protein (ABCD1), a peroxisomal membrane protein, is mutated in patients affected by X-linked adrenoleukodystrophy (X-ALD). Adrenoleukodystrophy-related protein (ABCD2) is the closest relative of ABCD1. Pharmacological induction of ABCD2 gene expression has been proposed as a novel therapy strategy for X-ALD. Fibrates induce peroxisome proliferation and Abcd2 expression in rodent liver. Here we evaluate the possibility of using peroxisome proliferator-activated receptor alpha (PPARalpha) agonists for pharmacological induction of ABCD2 expression. In the liver of PPARalpha-deficient mice, both the constitutive and the fenofibrate-inducible Abcd2 gene expression was found to be PPARalpha-dependent. In the brain, PPARalpha-deficiency has no effect on Abcd2 expression. In mice orally treated with the novel, highly selective, and potent PPARalpha agonists GW 7647, GW 6867, and tetradecylthioacetic acid, Abcd2 expression was induced in liver and adrenal glands, but not in brain and testis. None of four putative PPREs identified in the 5(')-flanking DNA and in intron 1 of the Abcd2 gene conferred fibrate response in luciferase reporter assays. Thus, although fibrate-mediated Abcd2 induction is PPARalpha-dependent, it appears to be an indirect mechanism. Within the mouse Abcd2 promoter, a putative sterol regulatory element (SRE) similar in sequence and position to the characterized SRE sequence of the human ABCD2 promoter, was identified. A PPARalpha dependent induction of the sterol regulatory-binding protein 2 (SREBP2) and a down-regulation of SREBP1c mRNA levels could be demonstrated after fenofibrate treatment of mice. Our results suggest that the PPARalpha agonist-mediated induction of Abcd2 expression seems to be indirect and possibly mediated by SREBP2.     

GW4064对果糖诱导非酒精性脂肪肝小鼠的保护作用研究

摘 要 目的： 研究GW4064对果糖诱导非酒精性脂肪肝(NAFLD)小鼠的保护作用。方法: 小鼠随机分为正常对照组、模型组和治疗组,采用30%果糖水造模,8周后,治疗组灌胃给予GW4064（30 mg·kg^-1）,2周后测定各组小鼠血清中葡萄糖、胰岛素、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）、三酰甘油（TG）和胆固醇（CHO）水平;观察肝脏病理改变。结果: 与NAFLD模型组相比,GW4064组小鼠空腹血糖及胰岛素含量降低,血清中TG、CHO 和LDL-C含量显著降低,HDL-C水平显著升高。病理结果显示,GW4064能显著降低NAFLD小鼠肝脏脂肪变性,明显减轻肝脏损伤程度。结论:采用30%果糖可以成功诱导NAFLD模型,GW4064通过降低脂质沉积和增加胰岛素敏感性来改善NAFLD。