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1.
Biol Trace Elem Res ; 142(1): 117-26, 2011 Jul.
Article in English | MEDLINE | ID: mdl-20556539

ABSTRACT

A high concentration of vanadium (V) has toxic effects on human and animals and is one of environmental pollutants. In the present study, we have conducted a fundamental study using cultured Vero cells from monkey kidney for the future environmental monitoring. Orthovanadate (VAN), one of V compounds, of 10(-10) and 10(-8) M did not affect the cell growth although the higher concentration of above 10(-6) M VAN inhibited the cell growth accompanied with the decrease in cell numbers and morphological changes. Given that the washing method with ice-cold Li is also effective for determination of the cellular Na content, we used this method for the determination of the V content of the Vero cells. The V distributions in Vero cell; in the 10(-3) M VAN solution, extracellular and intracellular were obtained as 1:0.564:0.036 and 1:0.662:0.098 at 60 and 120 min after the treatment of VAN. The intracellular V content was 10% of the applied concentration of VAN. Consequently, it was suggested that V concentration of 10(-7) and 10(-6) M in the tissue and environment, respectively, might become the threshold concentration; a criterion of the environmental contamination when we carry out environmental monitoring.


Subject(s)
Environmental Monitoring/methods , Environmental Pollutants/toxicity , Toxicity Tests , Vanadium/toxicity , Animals , Cell Enlargement , Cell Proliferation , Chlorocebus aethiops , Lithium/metabolism , Sodium/metabolism , Vanadium/metabolism , Vero Cells
2.
Neurosci Lett ; 446(1): 11-5, 2008 Nov 28.
Article in English | MEDLINE | ID: mdl-18809465

ABSTRACT

The neuroprotective function of prion protein (PrP) was revealed first by the fact that reintroduction of the mouse prion protein gene (Prnp) into a mouse Prnp(-/-) neuronal cell line, HpL3-4, could prevent apoptosis induced by serum deprivation. In the present study, the anti-apoptotic activities of mouse, hamster, and bovine PrP were compared by expressing mouse PrP (MoPrP), hamster PrP (HaPrP), and bovine PrP (BoPrP) in HpL3-4 cells, respectively. Morphological analysis and DNA fragmentation assays demonstrated that HpL3-4 cells expressing HaPrP, BoPrP, and empty vector (EM) showed the typical features of apoptosis with DNA laddering and apoptotic bodies after serum deprivation, whereas HpL3-4 cells expressing MoPrP showed decreased levels of apoptosis in comparison. The levels of histone-associated DNA fragments (mono- and oligonucleosomes) in the cytosol fractions of the cells correlated with the levels of DNA laddering. These results indicate a species-specific anti-apoptotic function of PrP exists, suggesting that the interaction of the mouse PrP with mouse host factors is required for its anti-apoptotic activity.


Subject(s)
Apoptosis/physiology , Neurons/metabolism , Prions/physiology , Animals , Apoptosis/drug effects , Apoptosis/genetics , Blotting, Western , Cattle , Cell Line , Cricetinae , Culture Media, Serum-Free , DNA Fragmentation/drug effects , Mice , Mice, Knockout , Neurons/cytology , Polymerase Chain Reaction , Prions/genetics , Prions/metabolism , Species Specificity , Statistics as Topic , Transfection/methods
3.
Protein Pept Lett ; 15(3): 250-4, 2008.
Article in English | MEDLINE | ID: mdl-18336352

ABSTRACT

Cytochrome c oxidases of prion protein (PrP) gene-deficient (Prnp(-/-)) and Prnp(+/+) mice were examined in vivo and in vitro. Non-invasive near-infrared spectra revealed that oxidation of copper and heme a+a(3) in cytochrome c oxidase of Prnp(-/-) mice was similar to that in Prnp(+/+) mice. Biochemical analysis of mitochondrial fractions also supported the results. PrP might not be involved in regulation of cytochrome c oxidase.


Subject(s)
Electron Transport Complex IV/metabolism , Prions/genetics , Animals , Copper/metabolism , Heme/metabolism , Mice , Mice, Transgenic , Mitochondria/enzymology
4.
Int J Mol Med ; 20(2): 169-76, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17611634

ABSTRACT

Several lines of prion protein gene (Prnp)-knockout mice such as ZrchI, ZrchII, Npu, Ngsk and Rcm0 have been generated. Of these, ZrchII, Ngsk and Rcm0 exhibit late-onset ataxia due to ectopic expression of Doppel (Dpl); a result of damage to the splicing acceptor of Prnp exon 3. Recently, we developed another line of Prnp-/- mice (Rikn), which was generated by gene targeting with more nucleotides by replacing intron 2 with the pgk-neo gene (cf. Ngsk Prnp-/- mice) and showed not only ataxia but also a lower olfactory sensitivity than the other Prnp-/- mouse line ZrchI at over 60 weeks of age. The histopathology of the elderly Rikn Prnp-/- mice showed mitral cell loss concomitantly observed with gliosis of astrocytes. Western blot analysis showed that Dpl was detected in the cerebrum, cerebellum and olfactory bulb of Rikn Prnp-/- mice, where aberrant histopathology was observed. Thus, mitral cell loss and gliosis induced by ectopic Dpl expression were probably associated with the late-onset olfactory deficits in Rikn Prnp-/- mice.


Subject(s)
Olfaction Disorders/genetics , Olfactory Bulb/pathology , Prions/genetics , Age of Onset , Aging/physiology , Animals , Brain/metabolism , Brain/pathology , Caspase 3/metabolism , Cell Count , DNA, Single-Stranded/metabolism , GPI-Linked Proteins , Gene Expression Regulation , Gliosis/genetics , Gliosis/pathology , Mice , Mice, Knockout , Olfaction Disorders/pathology , Prion Proteins , Prions/metabolism , Tissue Distribution
5.
Biochem Biophys Res Commun ; 358(2): 469-74, 2007 Jun 29.
Article in English | MEDLINE | ID: mdl-17498663

ABSTRACT

Splenocytes of wild-type (Prnp(+/+)) and prion protein gene-deficient (Prnp(-/-)) mice were treated with various activation stimuli such as T cell mitogen concanavalin A (ConA), phorbol 12-myristate 13-acetate (PMA)+ionomycin (Io), or B cell mitogen lipopolysaccharide (LPS). Cellular prion protein (PrP(C)) expression was enhanced following ConA stimulation, but not PMA+Io or LPS in Prnp(+/+) splenocytes. Rikn Prnp(-/-) splenocytes elicited lower cell proliferations than Prnp(+/+) or Zrch I Prnp(-/-) splenocytes after LPS stimulation and showed sporadic nerve cells in the cerebral cortex and deeper structure. Around the degenerated nerve cells, mild vacuolation in the neuropil was observed. This neural alteration correlated well to the suppressed response of B cells in the spleen. The finding that discrete lesions within the central nervous systems induced marked modulation of immune function probably indicates the existence of a delicately balanced neural-endocrine network by PrP(C) and PrPLP/Doppel.


Subject(s)
Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Lymphocytes/metabolism , Lymphocytes/pathology , Mitogens/pharmacology , Prions/metabolism , Animals , Cerebral Cortex/drug effects , GPI-Linked Proteins , Lymphocytes/drug effects , Mice , Mice, Knockout , Prions/genetics
6.
Biochem Biophys Res Commun ; 333(2): 448-54, 2005 Jul 29.
Article in English | MEDLINE | ID: mdl-15950943

ABSTRACT

The Prnd-encoded prion protein (PrP)-like protein, Doppel (Dpl), is a homologue of Prnp-encoded PrP, and is N-glycosylated protein with glycosylphosphatidylinositol anchor like PrP. Recently, ectopic expressions of Prnp/Prnd chimeric mRNAs have been identified as the cause of late-onset ataxia observed in several lines of Prnp-knockout mice such as ZrchII, Ngsk, Rcm0, and Rikn mice. However, it remains unclear whether the toxic effect of Dpl expression is a cell-autonomous mechanism but rather reflect a systemic process of heterogeneous cell population in the brain. In this study, the cell-autonomous role of Dpl was estimated by investigating PrP-deficient cells (HpL3-4)-the SV40 large T-antigen immortalized and Rikn Prnp(-/-) mice-derived neuronal cell line expressing Prnp/Prnd chimeric mRNAs. The reverse transcription polymerase chain reaction revealed that serum deprivation did not increase Prnp/Prnd chimeric mRNAs, which in fact was translated into a small amount of Dpl in HpL3-4 cells, whereas serum deprivation induced apoptotic cell death of HpL3-4 cells. Dpl overexpression enhanced apoptotic cell death, whereas the toxic effect of Dpl on apoptotic cell death was neutralized by PrP expression. These results indicate that Dpl elicited dose-dependently toxic effects on PrP-deficient cells without affecting on PrP-expressing cells, suggesting that the PrP-Dpl interaction can regulate cell death in a cell-autonomous manner.


Subject(s)
Amyloid/deficiency , Apoptosis/physiology , Neurons/cytology , Neurons/metabolism , Prions/metabolism , Protein Precursors/deficiency , Amyloid/metabolism , Animals , Cells, Cultured , GPI-Linked Proteins , Gene Silencing , Hippocampus/cytology , Hippocampus/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Prion Proteins , Protein Precursors/metabolism , Signal Transduction/physiology
7.
Biochem Biophys Res Commun ; 323(1): 218-22, 2004 Oct 08.
Article in English | MEDLINE | ID: mdl-15351724

ABSTRACT

The function of cellular prion protein (PrPC), which is a copper binding protein, remains unclear. To elucidate the mechanisms in which PrPC is involved in neuroprotection, we compared death signals in prion protein gene-deficient (Prnp-/-) primary cerebellar granular neurons (CGNs) to those with wild-type (WT) CGNs. When copper was exposed to these CGNs, ZrchI, and Rikn Prnp-/- CGNs were more sensitized and underwent apoptotic cell death more readily than WT CGNs. Furthermore, the level of intracellular hydrogen peroxide (H2O2) in WT CGNs increased by copper toxicity, whereas those in ZrchI and Rikn Prnp-/- CGNs did not. These results suggest that PrPC modulates the intracellular H2O2 level as a copper-binding protein to protect CGNs from apoptotic cell death possibly due to inhibiting a Fenton reaction.


Subject(s)
Apoptosis , Copper/chemistry , Hydrogen Peroxide/pharmacology , Prions/chemistry , Animals , Cell Nucleus/metabolism , Cell Survival , Cerebellum/metabolism , Copper/pharmacology , Dose-Response Relationship, Drug , Flow Cytometry , Mice , Mice, Inbred C57BL , Neurons/metabolism , Prions/genetics , Protein Binding , Time Factors
8.
J Clin Microbiol ; 42(5): 1869-74, 2004 May.
Article in English | MEDLINE | ID: mdl-15131142

ABSTRACT

Herpes B virus DNA was specifically amplified by PCR, targeting the regions that did not cross-react with herpes simplex virus (HSV). The amplified products, which were shown to be highly genetic polymorphisms among herpes B virus isolates, were identified by microplate hybridization with probes generated by PCR. The products immobilized in microplate wells were hybridized with the biotin-labeled probes derived from the SMHV strain of herpes B virus. The amplified products derived from the SMHV and E2490 strains of herpes B virus were identified by microplate hybridization. PCR products amplified from the trigeminal ganglia of seropositive cynomolgus macaques were identified as herpes B virus DNA. The utility of the PCR-microplate hybridization assay for genetic detection and identification of the polymorphic region of herpes B virus was determined.


Subject(s)
Herpesvirus 1, Cercopithecine/genetics , Herpesvirus 1, Cercopithecine/isolation & purification , Polymerase Chain Reaction/methods , Animals , Base Sequence , DNA Primers/genetics , DNA, Viral/genetics , DNA, Viral/isolation & purification , Herpesviridae Infections/virology , Humans , Macaca fascicularis , Nucleic Acid Hybridization , Polymerase Chain Reaction/statistics & numerical data , Sensitivity and Specificity , Trigeminal Ganglion/virology
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