A neuronal death model: overexpression of neuronal intermediate filament protein peripherin in PC12 cells

<p>Abstract</p> <p>Background</p> <p>Abnormal accumulation of neuronal intermediate filament (IF) is a pathological indicator of some neurodegenerative disorders. However, the underlying neuropathological mechanisms of neuronal IF accumulation remain unclear. A stable clone established from PC12 cells overexpressing a GFP-Peripherin fusion protein (pEGFP-Peripherin) was constructed for determining the pathway involved in neurodegeneration by biochemical, cell biology, and electronic microscopy approaches. In addition, pharmacological approaches to preventing neuronal death were also examined.</p> <p>Results</p> <p>Results of this study showed that TUNEL positive reaction could be detected in pEGFP-Peripherin cells. Swollen mitochondria and endoplasmic reticulum (ER) were seen by electron microscopy in pEGFP-Peripherin cells on day 8 of nerve growth factor (NGF) treatment. Peripherin overexpression not only led to the formation of neuronal IF aggregate but also causes aberrant neuronal IF phosphorylation and mislocation. Western blots showed that calpain, caspase-12, caspase-9, and caspase-3 activity was upregulated. Furthermore, treatment with calpain inhibitor significantly inhibited cell death.</p> <p>Conclusions</p> <p>These results suggested that the cytoplasmic neuronal IF aggregate caused by peripherin overexpression may induce aberrant neuronal IF phosphorylation and mislocation subsequently trapped and indirectly damaged mitochondria and ER. We suggested that the activation of calpain, caspase-12, caspase-9, and caspase-3 were correlated to the dysfunction of the ER and mitochondria in our pEGFP-Peripherin cell model. The present study suggested that pEGFP-Peripherin cell clones could be a neuronal death model for future studies in neuronal IFs aggregate associated neurodegeneration.</p

Technology Anxiety and Implicit Learning Ability Affect Technology Leadership to Promote the use of Information Technology at Elementary Schools

Abstract“Oversold & underused” is a criticism by Cuban (2001) of the investment of information technology (IT) in the classroom. Recently, Taiwan's educational administration has provided considerable financial support to IT in elementary schools, but few reports have provided evidence of its successful use. The present study aims to identify the personal factors that affect principals’ beliefs about the promotion of IT in their schools. 331 data were collected and analyzed with AMOS 19.0. The results of this study indicated that greater technology anxiety was negatively associated with perceived ease of using (PEU) IT, whereas implicit learning ability was positively correlated with perceived usefulness of IT. Technology leadership increased significantly with PEU and perceived usefulness (PU), it is also associated with the intention to overcome difficulties in promoting information technology in schools. The implications of this study may contribute to the reduction of principals’ technology anxiety, increasing their implicit learning ability and therefore fostering the future implementation of IT in schools, changing the myth of technology as “oversold & underused”

Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase

AbstractPlant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PPi) as an energy source to generate a H+ gradient potential for the secondary transport of ions and metabolites across the vacuole membrane. In this study, functional roles of arginine residues in mung bean V-PPase were determined by site-directed mutagenesis. Alignment of amino-acid sequence of K+-dependent V-PPases from several organisms showed that 11 of all 15 arginine residues were highly conserved. Arginine residues were individually substituted by alanine residues to produce R→A-substituted V-PPases, which were then heterologously expressed in yeast. The characteristics of mutant variants were subsequently scrutinized. As a result, most R→A-substituted V-PPases exhibited similar enzymatic activities to the wild-type with exception that R242A, R523A, and R609A mutants markedly lost their abilities of PPi hydrolysis and associated H+-translocation. Moreover, mutation on these three arginines altered the optimal pH and significantly reduced K+-stimulation for enzymatic activities, implying a conformational change or a modification in enzymatic reaction upon substitution. In particular, R242A performed striking resistance to specific arginine-modifiers, 2,3-butanedione and phenylglyoxal, revealing that Arg242 is most likely the primary target residue for these two reagents. The mutation at Arg242 also removed F− inhibition that is presumably derived from the interfering in the formation of substrate complex Mg2+–PPi. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg242 which is embedded in a more hydrophobic environment