OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were car...OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. RESULTS: Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P展开更多
Background Parkinson’s disease(PD)is the second most common neurodegenerative disease.The pathologic characteristic feature is the loss of dopaminergic neurons in the substantia nigra(SN).However,the biochemical mech...Background Parkinson’s disease(PD)is the second most common neurodegenerative disease.The pathologic characteristic feature is the loss of dopaminergic neurons in the substantia nigra(SN).However,the biochemical mechanisms are unclear.A large number of studies have shown that oxidative damage is the primary cause of PD.Hence,antioxidants could become a suitable option to treat PD.The thioredoxin(Trx)system represents a useful,potentially disease-relevant oxidation-reduction system.Thioredoxin reductase 1(TR1)is a significant component of the Trx system.Methods The overexpression lentivirus(LV)or LV-TR1 in the TR1-A53T model of PD by the stereotactic brain,and successful overexpression of LV or LV-TR1 in the MPP^(+)-induced cellular model by LV or LV-TR1 transfection.Results We confirmed that interleukin-7 mRNA levels increased in MPP^(+)compared to that in the control and MPP^(+)-TR1 groups using quantitative polymerase chain reaction.Theγ-H_(2)AX level was increased in the Tg-A53T group compared to that in the TR1-A53T group by western blotting.The expression of Na^(+)-K^(+)-ATP was decreased in the MPP^(+)group compared to that in the control and MPP^(+)-TR1 groups by high content screening.Tg-A53T(the C57BL/6 mice transferred with mutant human a-syn);TR1-A53T(A53T mice which were injected TR1-LV 2µl in SNc on two sides with minipump).The mice were fed for 10 months.control(the N2a cells cultivated with DMEM);MPP^(+)(the N2a cells dealt with MPP^(+)(1 mM)48 h),MPP^(+)-LV(the N2a cells over-expressed LV for 24 h then dealt with MPP^(+)(1 mM)48 h).MPP^(+)-TR1(the N2a cell over-expressed TR1-LV for 24 h then dealt with MPP^(+)(1 mM)48 h).From the Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,we confirmed that the overexpression of TR1 in SN pars compacta cells decreased oxidative stress,apoptosis,DNA damage,and inflammatory response and increased NADPH,Na^(+)-K^(+)-ATP,and immune response in this PD model.Conclusions Our study shows that overexpressed TR1 can be developed as a neuroprotective agent for PD.Therefore,our findings demonstrate a new targeted protein for the treatment of PD.展开更多
文摘OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. RESULTS: Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P
基金supported by the Cuiying Science and technology Innovation Project(CY2021-MS-B07)Cuiying studentsp scientific research and cultivation program(CYXZ2020-34)Natural Science Foundation of Gansu Province(22JR5RA987)
文摘Background Parkinson’s disease(PD)is the second most common neurodegenerative disease.The pathologic characteristic feature is the loss of dopaminergic neurons in the substantia nigra(SN).However,the biochemical mechanisms are unclear.A large number of studies have shown that oxidative damage is the primary cause of PD.Hence,antioxidants could become a suitable option to treat PD.The thioredoxin(Trx)system represents a useful,potentially disease-relevant oxidation-reduction system.Thioredoxin reductase 1(TR1)is a significant component of the Trx system.Methods The overexpression lentivirus(LV)or LV-TR1 in the TR1-A53T model of PD by the stereotactic brain,and successful overexpression of LV or LV-TR1 in the MPP^(+)-induced cellular model by LV or LV-TR1 transfection.Results We confirmed that interleukin-7 mRNA levels increased in MPP^(+)compared to that in the control and MPP^(+)-TR1 groups using quantitative polymerase chain reaction.Theγ-H_(2)AX level was increased in the Tg-A53T group compared to that in the TR1-A53T group by western blotting.The expression of Na^(+)-K^(+)-ATP was decreased in the MPP^(+)group compared to that in the control and MPP^(+)-TR1 groups by high content screening.Tg-A53T(the C57BL/6 mice transferred with mutant human a-syn);TR1-A53T(A53T mice which were injected TR1-LV 2µl in SNc on two sides with minipump).The mice were fed for 10 months.control(the N2a cells cultivated with DMEM);MPP^(+)(the N2a cells dealt with MPP^(+)(1 mM)48 h),MPP^(+)-LV(the N2a cells over-expressed LV for 24 h then dealt with MPP^(+)(1 mM)48 h).MPP^(+)-TR1(the N2a cell over-expressed TR1-LV for 24 h then dealt with MPP^(+)(1 mM)48 h).From the Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,we confirmed that the overexpression of TR1 in SN pars compacta cells decreased oxidative stress,apoptosis,DNA damage,and inflammatory response and increased NADPH,Na^(+)-K^(+)-ATP,and immune response in this PD model.Conclusions Our study shows that overexpressed TR1 can be developed as a neuroprotective agent for PD.Therefore,our findings demonstrate a new targeted protein for the treatment of PD.
