Dual Role of α-Secretase Cleavage in the Regulation of γ-Secretase Activity for Amyloid Production

Processing of the amyloid precursor protein (APP) by β- and γ-secretases generates pathogenic β-amyloid (Aβ) peptides associated with Alzheimer disease (AD), whereas cleavage of APP by α-secretases precludes Aβ formation. Little is known about the role of α-secretase cleavage in γ-secretase regulation. Here, we show that α-secretase-cleaved APP C-terminal product (αCTF) functions as an inhibitor of γ-secretase. We demonstrate that the substrate inhibitory domain (ASID) within αCTF, which is bisected by the α-secretase cleavage site, contributes to this negative regulation because deleting or masking this domain turns αCTF into a better substrate for γ-secretase. Moreover, α-secretase cleavage can potentiate the inhibitory effect of ASID. Inhibition of γ-secretase activity by αCTF is observed in both in vitro and cellular systems. This work reveals an unforeseen role for α-secretase in generating an endogenous γ-secretase inhibitor that down-regulates the production of Aβ. Deregulation of this feedback mechanism may contribute to the pathogenesis of AD.     

Nitrofen induces apoptosis independently of retinaldehyde dehydrogenase (RALDH) inhibition

BACKGROUND: Nitrofen is a diphenyl ether that induces congenital diaphragmatic hernia (CDH) in rodents. Its mechanism of action has been hypothesized as inhibition of the retinaldehyde dehydrogenase (RALDH) enzymes with consequent reduced retinoic acid signaling. METHODS: To determine if nitrofen inhibits RALDH enzymes, a reporter gene construct containing a retinoic acid response‐element (RARE) was transfected into HEK‐293 cells and treated with varying concentrations of nitrofen in the presence of retinaldehyde (retinal). Cell death was characterized by caspace‐cleavage microplate assays and terminal deoxynucleotidyl transferase dUTP nick end‐labeling (TUNEL) assays. Ex vivo analyses of cell viability were characterized in fetal rat lung explants using Live/Dead staining. Cell proliferation and apoptosis were assessed using fluorescent immunohistochemistry with phosphorylated histone and activated caspase antibodies on explant tissues. Nile red staining was used to identify intracellular lipid droplets. RESULTS: Nitrofen‐induced dose‐dependent declines in RARE‐reporter gene expression. However, similar reductions were observed in control‐reporter constructs suggesting that nitrofen compromised cell viability. These observed declines in cell viability resulted from increased cell death and were confirmed using two independent assays. Ex vivo analyses showed that mesenchymal cells were particularly susceptible to nitrofen‐induced apoptosis while epithelial cell proliferation was dramatically reduced in fetal rat lung explants. Nitrofen treatment of these explants also showed profound lipid redistribution, primarily to phagocytes. CONCLUSIONS: The observed declines in nitrofen‐associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition. Birth Defects Res (Part B) 89:223–232, 2010. © 2010 Wiley‐Liss, Inc.     

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer

Background

Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As⁺³ and Cd⁺² sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants.

Results

It was shown that both As⁺³ and Cd⁺² exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As⁺³ or Cd⁺². Localization studies showed that ENO2 in the MCF-10A cells transformed by As⁺³ or Cd⁺² had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1.

Conclusion

The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As⁺³ or Cd⁺². The findings also suggest a possible link between As⁺³ and Cd⁺² exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As⁺³ and Cd⁺².     

Science in Risk Assessment and Policy (SciRAP): An Online Resource for Evaluating and Reporting In Vivo (Eco)Toxicity Studies

(Eco)toxicity studies conducted according to internationally standardized test guidelines are often considered reliable by default and preferred as key evidence in regulatory risk assessment. At the same time regulatory agencies emphasize the use of all relevant (eco)toxicity data in the risk assessment process, including non-standard studies. However, there is a need to facilitate the use of such studies in regulatory risk assessment. Therefore, we propose a framework that facilitates a systematic and transparent evaluation of the reliability and relevance of (eco)toxicity in vivo studies for health and environmental risk assessment. The framework includes specific criteria to guide study evaluation, as well as a color-coding tool developed to aid the application of these criteria. In addition we provide guidance intended for researchers on how to report non-standard studies to ensure that they meet regulatory requirements. The intention of the evaluating and reporting criteria is to increase the usability of all relevant data that may fill information gaps in chemical risk assessments. The framework is publically available online, free of charge, at the Science in Risk Assessment and Policy (SciRAP) website: www.scirap.org. The aim of this article is to present the framework and resources available at the SciRAP website.     

Synthesis of a folate functionalized PEGylated poly(propylene imine) dendrimer as prospective targeted drug delivery system

Based on fourth generation diaminobutane poly(propylene imine) dendrimer, a novel targeted drug nanocarrier was prepared, bearing protective PEG chains and a folate targeting ligand. As a control a PEGylated derivative without folate was also synthesized. The encapsulation and release properties of these PEGylated derivatives were investigated employing etoposide, an anticancer hydrophobic drug. Enhanced solubility of etoposide was achieved inside the dendrimeric scaffold which was subsequently released in a controlled manner. These properties coupled with specificity towards the folate receptor and the low toxicity render folate functionalized PEGylated poly(propylene imine) dendrimer promising candidate for targeted drug delivery.     

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

Leptotene/zygotene chromosome movement via the SUN/KASH protein bridge in Caenorhabditis elegans

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2-dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates.