Biomedical subjects

Y Morita-Fujimura

Publications and source records attributed to Y Morita-Fujimura.

23 records · Page 2Linked to original sources

PubMed1999

Early decrease of XRCC1, a DNA base excision repair protein, may contribute to DNA fragmentation after transient focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: DNA damage and the DNA repair mechanism are known to be involved in ischemia/reperfusion injury in the brain. The x-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase beta. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after transient focal cerebral ischemia (FCI). METHODS: Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal blockade of the middle cerebral artery. XRCC1 protein expression was analyzed by immunohistochemistry and Western blot analysis. DNA damage was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL). The spatial relationship between XRCC1 expression and DNA damage was examined by double staining with XRCC1 and TUNEL after FCI. RESULTS: Immunohistochemistry showed the nuclear expression of XRCC1 in all regions of the control brains and that it was predominant in the hippocampus. The XRCC1 level was markedly reduced in the caudate putamen at 10 minutes, further decreased in the entire middle cerebral artery territory at 1 hour, and remained reduced until 4 and 24 hours after FCI. Western blot analysis of the normal control brain showed a characteristic band of 70 kDa, which decreased after FCI. A significant amount of DNA fragmentation was detected by DNA gel electrophoresis 24 hours but not 4 hours after FCI. Double staining showed that the neurons that lost XRCC1 immunoreactivity became TUNEL positive. CONCLUSIONS: These results suggest that the early decrease of XRCC1 and the failure of the DNA repair mechanism may contribute, at least in part, to DNA fragmentation after FCI.

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PubMed1999

Reduction of apurinic/apyrimidinic endonuclease expression after transient global cerebral ischemia in rats: implication of the failure of DNA repair in neuronal apoptosis.

BACKGROUND AND PURPOSE: To clarify the relationship between apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, and delayed neuronal cell death associated with apoptosis, we examined the expression of APE/Ref-1 before and after transient global ischemia in rats. METHODS: Global ischemia was induced by bilateral common carotid artery occlusion and hypotension. Expression of the APE/Ref-1 protein was evaluated by Western blot and immunohistochemical analyses. Apoptosis after global ischemia was observed by DNA electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. RESULTS: Immunohistochemistry showed the nuclear expression of APE/Ref-1 in the control brains. Nuclear immunoreactivity of APE/Ref-1 was significantly decreased 2 days after 10 minutes of ischemia in the hippocampal CA1 subregion. Western blot analysis of a sample from the normal brains showed a characteristic 37-kDa band, which was reduced in the hippocampal CA1 subregion after ischemia. A significant amount of DNA fragmentation was observed at 3 days but not at 1 day after ischemia. Double staining with APE/Ref-1 and TUNEL clearly showed that the neurons that lost APE/Ref-1 immunoreactivity became TUNEL positive. CONCLUSIONS: Our data provide evidence that APE/Ref-1 decreased in hippocampal CA1 neurons after transient global ischemia and that this reduction precedes DNA fragmentation, which is destined to cause apoptosis. Our results suggest the possibility that a decrease of APE/Ref-1 activity and the failure of DNA repair may underlie the mechanism of apoptosis after transient focal ischemia.

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PubMed1999

Exacerbation of delayed cell injury after transient global ischemia in mutant mice with CuZn superoxide dismutase deficiency.

BACKGROUND AND PURPOSE: We have demonstrated that copper-zinc superoxide dismutase (CuZn-SOD), a cytosolic isoenzyme of SODs, has a protective role in the pathogenesis of superoxide radical-mediated brain injury. Using mice bearing a disruption of the CuZn-SOD gene (Sod1), the present study was designed to clarify the role of superoxide anion in the pathogenesis of selective vulnerability after transient global ischemia. METHODS: Sod1 knockout homozygous mutant mice (Sod1 -/-) with a complete absence of endogenous CuZn-SOD activity, heterozygous mutant mice (Sod1 +/-) with a 50% decrease in the activity, and littermate wild-type mice (male, 35 to 45 g) were subjected to global ischemia. Since the plasticity of the posterior communicating artery (PcomA) has been reported to influence the outcome of hippocampal injury, we assessed the relation between the plasticity of PcomAs and the decrease of regional cerebral blood flow in global ischemia. RESULTS: The fluorescence intensity of hydroethidine oxidation, a measurement of ethidium fluorescence for superoxide radicals, was increased in mutant mice 1 day after both 5 and 10 minutes of global ischemia, compared with wild-type mice. Hippocampal injury in the PcomA hypoplastic brains showed significant exacerbation in mutant mice compared with wild-type littermates 3 days after 5 minutes of global ischemia, although a marked difference was not observed at 1 day. CONCLUSIONS: These data suggest that superoxide radicals play an important role in the pathogenesis of delayed injury in the vulnerable hippocampal CA1 subregion after transient global ischemia.

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PubMed1998

Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats.

Recent in vitro cell-free studies have shown that cytochrome c release from mitochondria is a critical step in the apoptotic process. The present study examined the expression of cytochrome c protein after transient focal cerebral ischemia in rats, in which apoptosis was assumed to contribute to the expansion of the ischemic lesion. In situ labeling of DNA breaks in frozen sections after 90 minutes of middle cerebral artery (MCA) occlusion showed a significant number of striatal and cortical neurons, which were maximized at 24 hours after ischemia, exhibiting chromatin condensation, nuclear segmentation, and apoptotic bodies. Cytosolic localization of cytochrome c was detected immunohistochemically in the ischemic area as early as 4 hours after 90 minutes of MCA occlusion. Western blot analysis of the cytosolic fraction revealed a strong single 15-kDa band, characteristic of cytochrome c, only in the samples from the ischemic hemisphere. Western blot analysis of the mitochondrial fraction showed a significant amount of mitochondrial cytochrome c in nonischemic brain, which was decreased in ischemic brain 24 hours after ischemia. These results provide the first evidence that cytochrome c is being released from mitochondria to the cytosol after transient focal ischemia. Although further evaluation is necessary to elucidate its correlation with DNA fragmentation, our results suggest the possibility that cytochrome c release may play a role in DNA-damaged neuronal cell death after transient focal cerebral ischemia in rats.

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PubMed1996

Reduced microtubule-nucleation activity of tau after dephosphorylation.

Based on video-enhanced differential interference contrast (DIC) microscopy, we developed a small-scale method which is capable of measuring both the lengths and the number densities of microtubules (MTs) assembled in vitro. With this method, effect of dephosphorylation on the activity of bovine brain tau protein to promote the assembly of tubulin at physiological concentration (15 microM) was quantitatively analyzed. The MT number density was selectively reduced when tau isolated directly in the presence of phosphatase inhibitors (N-tau) was dephosphorylated in vitro (DP-tau), without significant changes in the mean MT length or the binding affinity toward preformed MTs. Tau obtained from brain MTs (MT-tau) also exhibited lower nucleation activity in spite of its high MT-binding affinity. The results indicate that nucleation, elongation and MT-binding are distinct aspects of tau function which are differentially affected by the phosphorylation state of tau.

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