RASSF1C modulates the expression of a stem cell renewal gene, PIWIL1

ABSTRACT: BACKGROUND: RASSF1A and RASSF1C are two major isoforms encoded by the Ras association domain family 1 (RASSF1) gene through alternative promoter selection and mRNA splicing. RASSF1A is a well established tumor suppressor gene. Unlike RASSF1A, RASSF1C appears to have growth promoting actions in lung cancer. In this article, we report on the identification of novel RASSF1C target genes in non small cell lung cancer (NSCLC). METHODS: Over-expression and siRNA techniques were used to alter RASSF1C expression in human lung cancer cells, and Affymetrix-microarray study was conducted using NCI-H1299 cells over-expressing RASSF1C to identify RASSF1C target genes. RESULTS: The microarray study intriguingly shows that RASSF1C modulates the expression of a number of genes that are involved in cancer development, cell growth and proliferation, cell death, and cell cycle. We have validated the expression of some target genes using qRT-PCR. We demonstrate that RASSF1C over-expression increases, and silencing of RASSF1C decreases, the expression of PIWIL1 gene in NSCLC cells using qRT-PCR, immunostaining, and Western blot analysis. We also show that RASSF1C over-expression induces phosphorylation of ERK1/2 in lung cancer cells, and inhibition of the MEK-ERK1/2 pathway suppresses the expression of PIWIL1 gene expression, suggesting that RASSF1C may exert its activities on some target genes such as PIWIL1 through the activation of the MEK-ERK1/2 pathway. Also, PIWIL1 expression is elevated in lung cancer cell lines compared to normal lung epithelial cells. CONCLUSIONS: Taken together, our findings provide significant data to propose a model for investigating the role of RASSF1C/PIWIL1 proteins in initiation and progression of lung cancer.     

Properties of 2-locus genealogies and linkage disequilibrium in temporally structured samples

Archeogenetics has been revolutionary, revealing insights into demographic history and recent positive selection. However, most studies to date have ignored the nonrandom association of genetic variants at different loci (i.e. linkage disequilibrium). This may be in part because basic properties of linkage disequilibrium in samples from different times are still not well understood. Here, we derive several results for summary statistics of haplotypic variation under a model with time-stratified sampling: (1) The correlation between the number of pairwise differences observed between time-staggered samples (πΔt) in models with and without strict population continuity; (2) The product of the linkage disequilibrium coefficient, D, between ancient and modern samples, which is a measure of haplotypic similarity between modern and ancient samples; and (3) The expected switch rate in the Li and Stephens haplotype copying model. The latter has implications for genotype imputation and phasing in ancient samples with modern reference panels. Overall, these results provide a characterization of how haplotype patterns are affected by sample age, recombination rates, and population sizes. We expect these results will help guide the interpretation and analysis of haplotype data from ancient and modern samples.     

Hydrogen peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells

Axl, a receptor tyrosine kinase, is involved in cell survival, proliferation, and migration. We have shown that Axl expression increases in the neointima of balloon-injured rat carotids. Because oxidative stress is known to play a major role in remodeling of injured vessels, we hypothesized that H(2)O(2) might activate Axl by promoting autophosphorylation. H(2)O(2) rapidly stimulated Axl tyrosine phosphorylation in rat vascular smooth muscle cells within 1 min that was maximal at 5 min (6-fold). The response to H(2)O(2) was concentration-dependent with EC(50) of approximately 500 microm. Axl phosphorylation was partly dependent on production of its endogenous ligand, growth arrest gene 6 (Gas6), because Axl-Fc, a fragment of Axl extracellular domain that neutralizes Gas6, inhibited H(2)O(2)-induced Axl phosphorylation by 50%. Axl phosphorylation by H(2)O(2) was also attenuated by warfarin, which inhibits Gas6 activity by preventing post-translational modification. In intact vessels Axl was phosphorylated by H(2)O(2), and Axl phosphorylation was inhibited by warfarin treatment in balloon-injured carotids. Akt, a downstream target of Axl, was phosphorylated by H(2)O(2)in Axl(+/+) mouse aorta but significantly inhibited in Axl(-/-) aorta. Intimal proliferation was decreased significantly in a cuff injury model in Axl(-/-) mice compared with Axl(+/+) mice. In summary, Axl is an important signaling mediator for oxidative stress in cultured vascular smooth muscle cells and intact vessels and may represent an important therapeutic target for vascular remodeling and response to injury.     

Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis

The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.     

世界古老果树榅桲在中国的归化及其种群动态——以湖南省褒忠山种群为例

外来植物归化因其对本地生态系统有潜在的入侵风险而受到生态学家的广泛关注。群落和种群结构反映植物的环境适应性、种群地位、稳定性和发展趋势,可为判定外来植物归化提供可靠依据。榅桲(Cydonia oblonga)为在全球广泛引种栽培达4000多年的古老果树,引入中国已有2500多年,但关于其野外逸生种群的生存状态未见报道。以目前中国唯一逸生地——湖南省褒忠山的榅桲种群为对象,在其所在群落物种组成和群落结构分析的基础上,采用时空替代法,运用静态生命表、生存曲线和时间序列预测模型对榅桲种群动态进行了定量分析。结果表明：(1)群落内共有维管束植物20科28属31种,其中木本植物16种,草本植物15种;区系以温带成分为主,占群落内非世界分布总属数的75%;(2)群落具有明显层次结构,可分灌木层和草本层,灌木层个体集中于0—1.5 m高度级,占总个体数的81.81%;(3)种群龄级结构呈近倒“J”型,存活曲线趋近于Deevey-Ⅱ型,具有稳定的更新潜力;(4)生存分析表明榅桲种群具有前期优势、中期减少、后期稳定的特点;时间序列预测表明该种群个体数量未来能够持续增长,但幼苗及小树个体基本保持稳定,其种...     

A Role for Saccharomyces cerevisiae Tpa1 Protein in Direct Alkylation Repair

Alkylating agents induce cytotoxic DNA base adducts. In this work, we provide evidence to suggest, for the first time, that Saccharomyces cerevisiae Tpa1 protein is involved in DNA alkylation repair. Little is known about Tpa1 as a repair protein beyond the initial observation from a high-throughput analysis indicating that deletion of TPA1 causes methyl methane sulfonate sensitivity in S. cerevisiae. Using purified Tpa1, we demonstrate that Tpa1 repairs both single- and double-stranded methylated DNA. Tpa1 is a member of the Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that mutation of the amino acid residues involved in cofactor binding abolishes the Tpa1 DNA repair activity. Deletion of TPA1 along with the base excision repair pathway DNA glycosylase MAG1 renders the tpa1Δmag1Δ double mutant highly susceptible to methylation-induced toxicity. We further demonstrate that the trans-lesion synthesis DNA polymerase Polζ (REV3) plays a key role in tolerating DNA methyl-base lesions and that tpa1Δmag1revΔ3 triple mutant is extremely susceptible to methylation-induced toxicity. Our results indicate a synergism between the base excision repair pathway and direct alkylation repair by Tpa1 in S. cerevisiae. We conclude that Tpa1 is a hitherto unidentified DNA repair protein in yeast and that it plays a crucial role in reverting alkylated DNA base lesions and cytotoxicity.     

Pesticide Methoxychlor Promotes the Epigenetic Transgenerational Inheritance of Adult-Onset Disease through the Female Germline

Environmental compounds including fungicides, plastics, pesticides, dioxin and hydrocarbons can promote the epigenetic transgenerational inheritance of adult-onset disease in future generation progeny following ancestral exposure during the critical period of fetal gonadal sex determination. This study examined the actions of the pesticide methoxychlor to promote the epigenetic transgenerational inheritance of adult-onset disease and associated differential DNA methylation regions (i.e. epimutations) in sperm. Gestating F0 generation female rats were transiently exposed to methoxychlor during fetal gonadal development (gestation days 8 to 14) and then adult-onset disease was evaluated in adult F1 and F3 (great-grand offspring) generation progeny for control (vehicle exposed) and methoxychlor lineage offspring. There were increases in the incidence of kidney disease, ovary disease, and obesity in the methoxychlor lineage animals. In females and males the incidence of disease increased in both the F1 and the F3 generations and the incidence of multiple disease increased in the F3 generation. There was increased disease incidence in F4 generation reverse outcross (female) offspring indicating disease transmission was primarily transmitted through the female germline. Analysis of the F3 generation sperm epigenome of the methoxychlor lineage males identified differentially DNA methylated regions (DMR) termed epimutations in a genome-wide gene promoters analysis. These epimutations were found to be methoxychlor exposure specific in comparison with other exposure specific sperm epimutation signatures. Observations indicate that the pesticide methoxychlor has the potential to promote the epigenetic transgenerational inheritance of disease and the sperm epimutations appear to provide exposure specific epigenetic biomarkers for transgenerational disease and ancestral environmental exposures.     

Direct Pro-Inflammatory Effects of Prorenin on Microglia

Neuroinflammation has been implicated in hypertension, and microglia have been proposed to play an important role in the progression of this disease. Here, we have studied whether microglia are activated within cardiovascular regulatory area(s) of the brain during hypertension, especially in high blood pressure that is associated with chronic activation of the renin-angiotensin-system. In addition, we determined whether prorenin, an essential component of the renin-angiotensin-system, exerts direct pro-inflammatory effects on these microglia. Our data indicate that two rodent models which display neurogenic hypertension and over activation of the renin-angiotensin-system in the brain (sRA mice and spontaneously hypertensive rats) exhibit microglial activation, and increased levels of pro-inflammatory cytokines, in the paraventricular nucleus of the hypothalamus, an area crucial for regulation of sympathetic outflow. Further, the renin-angiotensin-system component prorenin elicits direct activation of hypothalamic microglia in culture and induction of pro-inflammatory mechanisms in these cells, effects that involve prorenin receptor-induced NFκB activation. In addition, the prorenin-elicited increases in cytokine expression were fully abolished by microglial inhibitor minocycline, and were potentiated by pre-treatment of cells with angiotensin II. Taken together with our previous data which indicate that pro-inflammatory processes in the paraventricular nucleus are involved in the hypertensive action of renin-angiotensin-system, the novel discovery that prorenin exerts direct stimulatory effects on microglial activation and pro-inflammatory cytokine production provides support for the idea that renin-angiotensin-system -induced neurogenic hypertension is not restricted to actions of angiotensin II alone.     

Auxin and Cytokinin Alleviate Chromium-Induced Oxidative Stress in Nostoc muscorum and Anabaena sp. by Modulating Ascorbate–Glutathione Cycle

Journal of Plant Growth Regulation - Numerous advances have been made to mitigate the repercussion of toxic metals on photosynthetic prokaryotes, i.e. cyanobacteria. One strategy is exogenous...     

Cholinergic Receptor Alterations in the Brain Stem of Spinal Cord Injured Rats

Cholinergic receptors in upper motor neurons of brain stem control locomotion and coordination. Present study unravels cholinergic alterations in brain stem during spinal cord injury to understand signalling pathway changes which may be associated with spinal cord injury mediated motor deficits. We evaluated cholinergic function in brain stem by studying the expression of choline acetyl transferase and acetylcholine esterase. We quantified metabotropic muscarinic cholinergic receptors by receptor assays for total muscarinic, muscarinic M1 and M3 receptor subunits, gene expression studies using Real Time PCR and confocal imaging using FITC tagged secondary antibodies. The gene expression of ionotropic nicotinic cholinergic receptors and confocal imaging were also studied. The results from our study showed metabolic disturbance in cholinergic pathway as choline acetyl transferase is down regulated and acetylcholine esterase is up regulated in spinal cord injury group. The significant decrease in muscarinic receptors showed by decreased receptor number along with down regulated gene expression and confocal imaging accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic acetylcholine receptors and confocal imaging. The motor coordination was analysed by Grid walk test which showed an increased foot slips in spinal cord injured rats. The significant reduction in brain stem cholinergic function might have intensified the motor dysfunction and locomotor disabilities.