An improved terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end labeling method for the quantification of DNA damage in tissues and cultured cells was developed. Many reports have revealed that histoc...An improved terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end labeling method for the quantification of DNA damage in tissues and cultured cells was developed. Many reports have revealed that histochemistry of DNA damage can be visualized using immunohistochemistry for the terminal deoxynucleotidyl transferase reaction in tissue sections. However, few reports have described quantification of DNA damage in tissues or cells. In this study, to estimate the degree of DNA damage, the confirmed method for histochemistry using biotinylated dUTP and deoxynucleotidyl transferase was applied to label the cleaved DNA ends caused by DNA damage in tissues or cells. After end-labeling, avidin-conjugated peroxidase was reacted. A significant correlation was observed between numbers of cleaved DNA ends and peroxidase activity after the reaction. The obtained signals for presented method showed higher than those for ordinary method, and correlate with degree of DNA damage caused by serum deprivation and chemical dose. In addition, DNA damage caused by apoptosis in cells treated with 6-hydroxydopamine or Cu and in the tissues of rats administered a diet containing no Zn could be evaluated quantitatively using the present method.展开更多
Populations from the Kii peninsula of Japan and Guam present a high incidence of amyotrophic lateral sclerosis and parkinsonism-dementia complex. It is thought that the low levels of calcium (Ca) and magnesium (Mg) in...Populations from the Kii peninsula of Japan and Guam present a high incidence of amyotrophic lateral sclerosis and parkinsonism-dementia complex. It is thought that the low levels of calcium (Ca) and magnesium (Mg) in the drinking water are involved in the pathogenesis of these diseases. The present study aimed to test the hypothesis that catalepsy, a behavioral immobility and one of the Parkinsonian symptoms, may result from functionally impaired dopaminergic neurons in low Ca and Mg (LCa/Mg) fed mice. A group of mice fed with an LCa/Mg diet for 6 weeks was compared to a control group on a standard diet. Cataleptic symptoms such as akinesia and rigidity were measured using the bar test. The antiparkinsonian drugs dopamine (DA) precursor L-3, 4-dihydroxy phenylamine (L-DOPA), the selective DA receptor D2 agonist bromocriptine and the DA releaser amantadine were tested for their effects on the induced catalepsy. Mice developped catalepsy after 3 weeks on the LCa/Mg diet. LCa/Mg diet-induced catalepsy was improved by the administration of either L-DOPA (50 - 200 mg/kg i.p.) in combination with benserazide (25 mg/kg i.p.), bromo- criptine (0.25 - 4 mg/kg i.p.) or amantadine (5 - 20 mg/kg i.p.). These results suggest that catalepsy in LCa/Mg mice might result from a hypofunction of dopaminergic neurons. Moreover, our results support the hypothesis that LCa/Mg in-take may be one etiological factor in neurodegenerative disorders including Parkinson’s disease.展开更多
文摘An improved terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end labeling method for the quantification of DNA damage in tissues and cultured cells was developed. Many reports have revealed that histochemistry of DNA damage can be visualized using immunohistochemistry for the terminal deoxynucleotidyl transferase reaction in tissue sections. However, few reports have described quantification of DNA damage in tissues or cells. In this study, to estimate the degree of DNA damage, the confirmed method for histochemistry using biotinylated dUTP and deoxynucleotidyl transferase was applied to label the cleaved DNA ends caused by DNA damage in tissues or cells. After end-labeling, avidin-conjugated peroxidase was reacted. A significant correlation was observed between numbers of cleaved DNA ends and peroxidase activity after the reaction. The obtained signals for presented method showed higher than those for ordinary method, and correlate with degree of DNA damage caused by serum deprivation and chemical dose. In addition, DNA damage caused by apoptosis in cells treated with 6-hydroxydopamine or Cu and in the tissues of rats administered a diet containing no Zn could be evaluated quantitatively using the present method.
文摘Populations from the Kii peninsula of Japan and Guam present a high incidence of amyotrophic lateral sclerosis and parkinsonism-dementia complex. It is thought that the low levels of calcium (Ca) and magnesium (Mg) in the drinking water are involved in the pathogenesis of these diseases. The present study aimed to test the hypothesis that catalepsy, a behavioral immobility and one of the Parkinsonian symptoms, may result from functionally impaired dopaminergic neurons in low Ca and Mg (LCa/Mg) fed mice. A group of mice fed with an LCa/Mg diet for 6 weeks was compared to a control group on a standard diet. Cataleptic symptoms such as akinesia and rigidity were measured using the bar test. The antiparkinsonian drugs dopamine (DA) precursor L-3, 4-dihydroxy phenylamine (L-DOPA), the selective DA receptor D2 agonist bromocriptine and the DA releaser amantadine were tested for their effects on the induced catalepsy. Mice developped catalepsy after 3 weeks on the LCa/Mg diet. LCa/Mg diet-induced catalepsy was improved by the administration of either L-DOPA (50 - 200 mg/kg i.p.) in combination with benserazide (25 mg/kg i.p.), bromo- criptine (0.25 - 4 mg/kg i.p.) or amantadine (5 - 20 mg/kg i.p.). These results suggest that catalepsy in LCa/Mg mice might result from a hypofunction of dopaminergic neurons. Moreover, our results support the hypothesis that LCa/Mg in-take may be one etiological factor in neurodegenerative disorders including Parkinson’s disease.
