To investigate the distribution of mitochondria ONA 4 977 bp deletion, a common deletion （CD）, in normal populations of Chinese, human peripheral blood samples from sixty healthy donors were collected, and levels of...To investigate the distribution of mitochondria ONA 4 977 bp deletion, a common deletion （CD）, in normal populations of Chinese, human peripheral blood samples from sixty healthy donors were collected, and levels of the CD in genomic DNA from the samples were detected using real-time PCR. The results showed that the CD was found in 27 health donors, with its positive rate being 45% （27/60）. The CD ratio was between 0 and 0.000308%, and not affected by age and gender in sixty healthy donors. Our studies indicate that the CD ratio is low, and do not show the age-dependent accumulation and any gender difference in peripheral whole blood from the normal Chinese population.展开更多
Background In perinatal dairy cows,ketosis is a prevalent metabolic disorder that lowers milk output and per-formance.Mitochondrial dysfunction and chronic inflammation in mammary tissue are linked to elevated blood k...Background In perinatal dairy cows,ketosis is a prevalent metabolic disorder that lowers milk output and per-formance.Mitochondrial dysfunction and chronic inflammation in mammary tissue are linked to elevated blood ketone levels,particularlyβ-hydroxybutyrate(BHB).Recent research has linked cytosolic mitochondrial DNA(mtDNA)with chronic aseptic inflammation by activating the cGAS-STING pathway during metabolic disorders,while autophagy activation effectively reverses this process.However,whether it is involved in mammary gland damage during ketosis is poorly understood.Therefore,this study aimed to explore the underlying mechanisms of mtDNA-induced inflammation under BHB stress and evaluate the potential therapeutic strategy of autophagy activation in mitigating this damage.Results Our study found an increased cytoplasmic mtDNA abundance in mammary gland tissues of dairy cows with ketosis and bovine mammary epithelial cell line(MAC-T)subjected to BHB stress.Further investigations revealed the activation of the cGAS-STING pathway and inflammatory response,indicated by elevated levels of cGAS and STING,along with increased phosphorylation levels of TBK1,P65,and IκB,and higher transcript levels of pro-inflammatory factors(IL-1B,IL-6,and TNF-α)in both in vivo and in vitro experiments.Notably,STING inhibition via si-STING transfection reversed BHB-induced inflammation.Additionally,autophagy activation appeared to protect against BHB stress by facilitating the removal of cytoplasmic mtDNA and preventing cGAS-STING pathway-mediated inflammation.Conclusions The findings illustrate that elevated BHB levels lead to the release of cytoplasmic mtDNA,which in turn activates the cGAS-STING pathway and triggers an inflammatory response in the mammary glands during hyper-ketonemia.Conversely,autophagy activation has been shown to alleviate this process by promoting cytoplasmic mtDNA degradation.展开更多
Objective： To obtain high quality of mitochondrial DNA （mtDNA） and carry out extra-long PCR （XL-PCR）. Methods： Mitochondria were isolated by differential centrifugation, and membranes were disrupted using 10%SDS...Objective： To obtain high quality of mitochondrial DNA （mtDNA） and carry out extra-long PCR （XL-PCR）. Methods： Mitochondria were isolated by differential centrifugation, and membranes were disrupted using 10%SDS （pH 7.0）. mtDNA was then extracted using phenol and chloroform. Resuits： The mtDNA obtained by using our improved method can be used as effective template for XL-PCR, and total mtDNA （16 kb） can be amplified easily. Conclusion： Our improved method is effective in preparing high quality of mtDNA, which can be used as template for XL-PCR.展开更多
Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration,...Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has ad- vanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.展开更多
It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells’ mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson’s disease rats. To prepare fo...It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells’ mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson’s disease rats. To prepare for mitochondrial transplantation study in human neurodegenerative diseases, we select human fibroblasts as mitochondrial donor because that fibroblasts share many characteristics with mesenchymal stromal cells (MSCs). We isolate human primary fibroblasts and develop a mitochondrial DNA (mtDNA)-depleted mouse motor neuron NSC-34 cells (NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells). Fibroblast and NSC-34 cell’s mitochondria are co-cultured with NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells. Mitochondrial transplantation is observed by fluorescent microscopy. Gene expression is determined by polymerase chain reaction (PCR) and real time PCR (qPCR). Also, mitochondria are injected to mice bearing mammary adenocarcinoma 4T1 cells. We find results as following: 1) There are abundant mitochondria in fibroblasts (337 ± 80 mitochondria per fibroblast). 42.4% of viable mitochondria are obtained by using differential centrifugation. The isolated mitochondria actively transplant into NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells after co-culture. 2) Fibroblasts transfer mitochondria to human mammary adenocarcinoma MCF-7 cells. 3) There is no expression of HLA-I antigen in fibroblast’s mitochondria indicating they can be used for allogeneic mitochondrial transplantation without HLA antigen match. 4) PCR and qPCR show that NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells lose mitochondrially encoded cytochrome c oxidase I (MT-CO1) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and upregulate expression of glycolysis-associated genes hexokinase (HK2), glucose transporter 1 (SLC2A1) and lactate dehydrogenase A (LDHA). 5) Transplantation of NSC-34 mitochondria restores MT-CO1 and MT-ND1 and downregulates gene expression of HK2, SLC2A1 and LDHA. 6) Normal mammary epithelial mitochondria successfully enter to 4T1 cells in mice. Subcutaneous injection of mitochondria is safe for mice. In summary, mitochondrial transplantation replenishes mtDNA and rescues aerobic respiration of diseased cells with mitochondrial dysfunction. Human primary fibroblasts are potential mitochondrial donor for mitochondrial transplantation study in human neurodegenerative diseases.展开更多
基金supported by grants from the building project for the National Key Clinical Special Department of China(No.2011-17)the Medical Science and Technology Foundation of Henan Province(No.201204123)the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘To investigate the distribution of mitochondria ONA 4 977 bp deletion, a common deletion （CD）, in normal populations of Chinese, human peripheral blood samples from sixty healthy donors were collected, and levels of the CD in genomic DNA from the samples were detected using real-time PCR. The results showed that the CD was found in 27 health donors, with its positive rate being 45% （27/60）. The CD ratio was between 0 and 0.000308%, and not affected by age and gender in sixty healthy donors. Our studies indicate that the CD ratio is low, and do not show the age-dependent accumulation and any gender difference in peripheral whole blood from the normal Chinese population.
基金supported by the National Natural Science Foundation of China(32125038)the National Key R&B Program of China(2023YFD1801100)China Agriculture Research System(CARS-36).
文摘Background In perinatal dairy cows,ketosis is a prevalent metabolic disorder that lowers milk output and per-formance.Mitochondrial dysfunction and chronic inflammation in mammary tissue are linked to elevated blood ketone levels,particularlyβ-hydroxybutyrate(BHB).Recent research has linked cytosolic mitochondrial DNA(mtDNA)with chronic aseptic inflammation by activating the cGAS-STING pathway during metabolic disorders,while autophagy activation effectively reverses this process.However,whether it is involved in mammary gland damage during ketosis is poorly understood.Therefore,this study aimed to explore the underlying mechanisms of mtDNA-induced inflammation under BHB stress and evaluate the potential therapeutic strategy of autophagy activation in mitigating this damage.Results Our study found an increased cytoplasmic mtDNA abundance in mammary gland tissues of dairy cows with ketosis and bovine mammary epithelial cell line(MAC-T)subjected to BHB stress.Further investigations revealed the activation of the cGAS-STING pathway and inflammatory response,indicated by elevated levels of cGAS and STING,along with increased phosphorylation levels of TBK1,P65,and IκB,and higher transcript levels of pro-inflammatory factors(IL-1B,IL-6,and TNF-α)in both in vivo and in vitro experiments.Notably,STING inhibition via si-STING transfection reversed BHB-induced inflammation.Additionally,autophagy activation appeared to protect against BHB stress by facilitating the removal of cytoplasmic mtDNA and preventing cGAS-STING pathway-mediated inflammation.Conclusions The findings illustrate that elevated BHB levels lead to the release of cytoplasmic mtDNA,which in turn activates the cGAS-STING pathway and triggers an inflammatory response in the mammary glands during hyper-ketonemia.Conversely,autophagy activation has been shown to alleviate this process by promoting cytoplasmic mtDNA degradation.
基金Supported by the National Natural Science Foundation of China (No. C30171030, C30472175)China Postdoctoral Science Foundation (No. 2004036014)
文摘Objective： To obtain high quality of mitochondrial DNA （mtDNA） and carry out extra-long PCR （XL-PCR）. Methods： Mitochondria were isolated by differential centrifugation, and membranes were disrupted using 10%SDS （pH 7.0）. mtDNA was then extracted using phenol and chloroform. Resuits： The mtDNA obtained by using our improved method can be used as effective template for XL-PCR, and total mtDNA （16 kb） can be amplified easily. Conclusion： Our improved method is effective in preparing high quality of mtDNA, which can be used as template for XL-PCR.
基金supported by the National Institute of Aging/National Institution of Health, USA (No. AG025223 and AG024640) to YB.
文摘Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has ad- vanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.
文摘It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells’ mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson’s disease rats. To prepare for mitochondrial transplantation study in human neurodegenerative diseases, we select human fibroblasts as mitochondrial donor because that fibroblasts share many characteristics with mesenchymal stromal cells (MSCs). We isolate human primary fibroblasts and develop a mitochondrial DNA (mtDNA)-depleted mouse motor neuron NSC-34 cells (NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells). Fibroblast and NSC-34 cell’s mitochondria are co-cultured with NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells. Mitochondrial transplantation is observed by fluorescent microscopy. Gene expression is determined by polymerase chain reaction (PCR) and real time PCR (qPCR). Also, mitochondria are injected to mice bearing mammary adenocarcinoma 4T1 cells. We find results as following: 1) There are abundant mitochondria in fibroblasts (337 ± 80 mitochondria per fibroblast). 42.4% of viable mitochondria are obtained by using differential centrifugation. The isolated mitochondria actively transplant into NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells after co-culture. 2) Fibroblasts transfer mitochondria to human mammary adenocarcinoma MCF-7 cells. 3) There is no expression of HLA-I antigen in fibroblast’s mitochondria indicating they can be used for allogeneic mitochondrial transplantation without HLA antigen match. 4) PCR and qPCR show that NSC-34 <em>ρ</em><span style="white-space:nowrap;">°</span> cells lose mitochondrially encoded cytochrome c oxidase I (MT-CO1) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and upregulate expression of glycolysis-associated genes hexokinase (HK2), glucose transporter 1 (SLC2A1) and lactate dehydrogenase A (LDHA). 5) Transplantation of NSC-34 mitochondria restores MT-CO1 and MT-ND1 and downregulates gene expression of HK2, SLC2A1 and LDHA. 6) Normal mammary epithelial mitochondria successfully enter to 4T1 cells in mice. Subcutaneous injection of mitochondria is safe for mice. In summary, mitochondrial transplantation replenishes mtDNA and rescues aerobic respiration of diseased cells with mitochondrial dysfunction. Human primary fibroblasts are potential mitochondrial donor for mitochondrial transplantation study in human neurodegenerative diseases.
