Construction of chimera protein by using artificial restriction DNA cutter.
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We have already developed artificial restriction DNA cutter (ARCUT), which can hydrolyze double-stranded DNA site-selectively, by using Ce(IV)/EDTA in combination with two pseudo-complementary peptide nucleic acids (pcPNAs). Here, ARCUT was used to prepare a chimera protein. The gene for WW-domain containing oxidoreductase (WWOX) was clipped off by ARCUT just before its stop codon, and ligated with the gene for enhanced green fluorescent protein (EGFP). Conventional PCR for insertion of restriction enzyme site is never required. (+info)
Gene manipulation of fluorescent protein through site-selective hydrolysis by Ce(IV)/EDTA.
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Previously we reported that gap-site, formed in substrate DNA by using two oligonucleotide additives, was selectively hydrolyzed by Ce(IV)/EDTA. Herein this site-selective scission was used for gene manipulation, and green fluorescent protein (GFP) was converted to blue fluorescent protein (BFP). The sense strand of GFP was cleaved at predetermined site by using this system, and its upstream fragment was connected with the downstream of BFP gene by T4 DNA ligase. In this manipulation, three amino acid residues in GFP (C at the position 65, Y at 66, and T at 167) were converted to S, H, and I, respectively. The sequencing experiment confirmed that desired recombinant DNA was prepared, and the recombinant DNA was successfully expressed to emit blue fluorescence. (+info)
Highly active artificial restriction enzyme composed of Ce(IV)/EDTA and PNA bearing phosphate group--relationship between the promotion by phosphate and the structure of invasion complex.
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Recently, we developed artificial restriction DNA cutter (ARCUT) composed of pseudo-complementary peptide nucleic acid (pcPNA) and Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). Here we promoted the site-selective hydrolysis by attaching phosphate groups to the pcPNAs. The promotion by the phosphates increased with decreasing length of the gap-like site. Furthermore, the scission was successful even when phosphate groups were introduced to 0 base-gap system. (+info)
Site-selective hydrolysis of huge DNA by artificial restriction DNA cutter.
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Genomic DNAs of lambda phage (48.5 kbp) and E. coli (4.6 Mbp) were successfully cleaved at the target site by artificial restriction DNA cutter (ARCUT), which we recently developed by combining Ce(IV)/EDTA and PNA (peptide nucleic acid). PNA satisfactorily invaded the target site in such huge DNAs and activate there for Ce(IV)/EDTA-induced scission. (+info)
Component analysis of dental porcelain for assisting dental identification.
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The fluorescence of porcelain crowns recovered from the mouth of an unknown murder victim, and several control porcelain samples, were examined by fluorescent examination lamps. The fluorescence from two of the control samples was quite similar to that from the porcelain crowns recovered from the victim. To increase the objectivity of the results by quantitative analysis, the composition of each porcelain crown and control sample was also evaluated by wave dispersion X-ray microanalyser. The elements detected from the porcelain crowns of the victim matched those of two of the porcelain samples. Later, the antemortem dental records and radiographs of the victim were obtained through a dentist, who had recognized the name of the porcelain manufacturer in a postmortem dental information request placed on the Japanese Dental Association web page. Although component analysis of dental porcelain may be an effective means of assisting dental identification, a more rapid and non-destructive analysis for detecting the elements is required. The energy dispersive X-ray fluorescence (EDXRF) spectrometer was used for a pilot study of identification of porcelain composition. (+info)
Cardioprotective effects of cerium oxide nanoparticles in a transgenic murine model of cardiomyopathy.
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OBJECTIVE: Cerium oxide (CeO2) nanoparticles have been shown to protect cells in culture from lethal stress, but no protection in vivo has been reported. Cardiac-specific expression of monocyte chemoattractant protein (MCP)-1 in mice causes ischemic cardiomyopathy associated with activation of endoplasmic reticulum (ER) stress. The aim of this study was to assess the effects of CeO2 nanoparticles on cardiac function and remodeling as well as ER stress response in this murine model of cardiomyopathy. METHODS: MCP-1 transgenic mice (MCP mice) and wild-type controls were administered intravenously 15 nmol of CeO2 nanoparticles or vehicle only twice a week for 2 weeks. Cardiac function, myocardial histology, nitrotyrosine formation, expression of cytokines, and ER stress-associated genes were evaluated. RESULTS: Treatment with CeO2 nanoparticles markedly inhibited progressive left ventricular dysfunction and dilatation in MCP mice and caused a significant decrease in serum levels of MCP-1, C-reactive protein, and total nitrated proteins. The infiltration of monocytes/macrophages, accumulation of 3-nitrotyrosine, apoptotic cell death, and expression of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 in the myocardium were markedly inhibited by CeO2 nanoparticles. Expression of the key ER stress-associated genes, including glucose-regulated protein 78 (Grp78), protein disulfide isomerase (PDI), and heat shock proteins (HSP25, HSP40, HSP70), were also suppressed by CeO2 nanoparticles. CONCLUSIONS: CeO2 nanoparticles protect against the progression of cardiac dysfunction and remodeling by attenuation of myocardial oxidative stress, ER stress, and inflammatory processes probably through their autoregenerative antioxidant properties. (+info)
Auto-catalytic ceria nanoparticles offer neuroprotection to adult rat spinal cord neurons.
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This paper describes the evaluation of the auto-catalytic anti-oxidant behavior and biocompatibility of cerium oxide nanoparticles for applications in spinal cord repair and other diseases of the central nervous system. The application of a single dose of nano-ceria at a nano-molar concentration is biocompatible, regenerative and provides a significant neuroprotective effect on adult rat spinal cord neurons. Retention of neuronal function is demonstrated from electrophysiological recordings and the possibility of its application to prevent ischemic insult is suggested from an oxidative injury assay. A mechanism is proposed to explain the auto-catalytic properties of these nanoparticles. (+info)
An efficient and general approach to beta-functionalized ketones.
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[structure: see text]. The oxidation of selected anions (N3-, SCN-, I-, and Br-) by ceric ammonium nitrate (CAN) in the presence of substituted cyclopropyl alcohols provides a novel approach to beta-functionalized ketones. The protocol has a number of advantages including short reaction times, ease of reagent handling, and mild, neutral reaction conditions. Overall, this method provides an alternative pathway to important starting materials and intermediates in organic synthesis. (+info)