In vitro cultures of primary cortical neurons are widely used to investigate neuronal function.However,it has yet to be fully investigated whether there are significant differences in development and function between ...In vitro cultures of primary cortical neurons are widely used to investigate neuronal function.However,it has yet to be fully investigated whether there are significant differences in development and function between cultured rodent and primate cortical neurons,and whether these differences influence the utilization of cultured cortical neurons to model pathological conditions.Using in vitro culture techniques combined with immunofluorescence and electrophysiological methods,our study found that the development and maturation of primary cerebral cortical neurons from cynomolgus monkeys were slower than those from mice.We used a microelectrode array technique to compare the electrophysiological differences in cortical neurons,and found that primary cortical neurons from the mouse brain began to show electrical activity earlier than those from the cynomolgus monkey.Although cultured monkey cortical neurons developed slowly in vitro,they exhibited typical pathological features-revealed by immunofluorescent staining-when infected with adeno-associated viral vectors expressing mutant huntingtin(HTT),the Huntington’s disease protein.A quantitative analysis of the cultured monkey cortical neurons also confirmed that mutant HTT significantly reduced the length of neurites.Therefore,compared with the primary cortical neurons of mice,cultured monkey cortical neurons have longer developmental and survival times and greater sustained physiological activity,such as electrophysiological activity.Our findings also suggest that primary cynomolgus monkey neurons cultured in vitro can simulate a cell model of human neurodegenerative disease,and may be useful for investigating time-dependent neuronal death as well as treatment via neuronal regeneration.All mouse experiments and protocols were approved by the Animal Care and Use Committee of Jinan University of China(IACUC Approval No.20200512-04)on May 12,2020.All monkey experiments were approved by the IACUC protocol(IACUC Approval No.LDACU 20190820-01)on August 23,2019 for animal management and use.展开更多
Twenty-two lactic acid bacteria （LAB） strains isolated from Argentinean goat dairy products were evaluated for its biochemical properties and esterase activities relevant to flavor development. Streptococcus thermop...Twenty-two lactic acid bacteria （LAB） strains isolated from Argentinean goat dairy products were evaluated for its biochemical properties and esterase activities relevant to flavor development. Streptococcus thermophilus （UNSE314）, Lactobacillus （L.） delbrueckii subsp, bulgaricus （UNSE309）, L. rhamnosus （UNSE308）, L. plantarum （UNSE287, UNSE316, UNSE317） and Pediococcus pentosaceus （UNSE315） strains presented high acidifying activity. All strains tested metabolized citrate and produced diacetyl-acetoin in goat milk. Based on these results, 10 strains with the best performance in diverse technological properties were selected to determine esterolytic activity. In all evaluated strains, esterase specific activity （ESA） was detected on ct-naphthyl （ct-NA） acetate and 13-naphthyl （[3-NA） acetate, propionate, eaprylate and ct-NA butyrate. No activity was detected on [3-NA laurate. The highest values were detected when using a-NA instead of fI-NA derivatives as substrate. In Pediocoecus strains, wide variability in ESA were observed, which were species- and strain-specific. These results allow us to select strains with biochemical properties and esterase activities to design starter and adjunct cultures that contribute to flavor development during cheese ripening, thus preserving the typical organoleptic characteristics of Argentinean goat cheeses.展开更多
AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (D...AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.展开更多
AIM: To investigate the role of TR3 in induction of apoptosis in gastric cancer cells. METHODS: Human gastric cancer cell line, MGC80-3, was used. Expression of TR3 mRNA and its protein was detected by Northern blot a...AIM: To investigate the role of TR3 in induction of apoptosis in gastric cancer cells. METHODS: Human gastric cancer cell line, MGC80-3, was used. Expression of TR3 mRNA and its protein was detected by Northern blot and Western blot. Localization of TR3 protein was showed by immunofluorescence analysis under laser-scanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among 1000 cells randomly. Stable transfection assay was carried out by Lipofectamine. RESULTS: Treatment of MGC80-3 cells with TPA and VP-16 resulted in apoptosis, accompanied by the repression of Bcl-2 protein in a time-dependent manner. At the same time, TPA and VP-16 also up-regulated expression level of TR3 mRNA in MGC80-3 cells that expressed TR3 mRNA. When antisense-TR3 expression vector was transfected into the cells, expression of TR3 protein was repressed. In this case, TPA and VP-16 did not induce apoptosis. In addition, TPA and VP-16-induced apoptosis involved in translocation of TR3. In MGC80-3 cells, TR3 localized concentrative in nucleus, after treatment of cells with TPA and VP-16, TR3 translocated from nucleus to cytosol obviously. However, when this nuclear translocation was blocked by LMB, apoptosis was not occurred in MGC80-3 cells even in the presence of TPA and VP-16. CONCLUSION: Induction of apoptosis by TPA and VP-16 is through induction of TR3 expression and translocation of TR3 from nucleus to cytosol, which may be a novel signal pathway for TR3, and represent the new biological function of TR3 to exert its effect on apoptosis in gastric cancer cells.展开更多
We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly ...We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.展开更多
OBJECTIVE: To investigate the response of multiple myeloma (MM) cells to arsenic trioxide (As2O3) and their possible mechanisms. METHODS: Two MM-derived cell lines RPMI8226 and U266 cells were used as in vitro models....OBJECTIVE: To investigate the response of multiple myeloma (MM) cells to arsenic trioxide (As2O3) and their possible mechanisms. METHODS: Two MM-derived cell lines RPMI8226 and U266 cells were used as in vitro models. Cell apoptosis was assessed by morphology, flow cytometry, and DNA gel electrophoresis. Mitochondrial transmembrane potentials (delta psi m) were evaluated by measuring cellular Rhodamine 123 staining intensity. Protein expression was analyzed using Western blot. RESULTS: Zero point one to 0.5 mumol/L As2O3 inhibited cell proliferation and 2.0 mumol/L As2O3 induced cell apoptosis, while 1.0 mumol/L As2O3 inhibited proliferation with a weak degree of apoptosis induction in RPMI8226 and U266 cell lines. As2O3-induced apoptosis was accompanied by mitochondrial transmembrane potentials (delta psi m) collapse and caspase-3 activation in the presence of intact membrane. Glutathione depleter buthionine sulfoximine enhanced, while disulfide bond-reducing agent dithiothreitol partially antagonized As2O3-induced delta psi m collapse and apoptosis in MM cells. All-trans retinoic acid (ATRA) could also induce apoptosis in RPMI8226 cells, but it did not show any cooperative effects with As2O3. CONCLUSION: As2O3 exerts apoptosis-inducing and growth-inhibiting effects on MM cells, and mitochondrium is a pivotal and common target of As2O3 for apoptosis induction.展开更多
基金This work was supported by the National Natural Science Foundation of China,No.81922026(to SY)the National Key Research and Development Program of China Stem Cell and Translational Research,No.2017YFA0105104(to SY)+3 种基金Key Field Research and Development Program of Guangdong Province,No.2018B030337001(to XJL)Guangdong Key Laboratory of Non-human Primate Models of Brain Diseases,No.2020B121201006(to XJL)Guangzhou Key Research Program on Brain Science,No.202007030008(to SY)the Fundamental Research Funds for the Central Universities,No.21619104(to SY).
文摘In vitro cultures of primary cortical neurons are widely used to investigate neuronal function.However,it has yet to be fully investigated whether there are significant differences in development and function between cultured rodent and primate cortical neurons,and whether these differences influence the utilization of cultured cortical neurons to model pathological conditions.Using in vitro culture techniques combined with immunofluorescence and electrophysiological methods,our study found that the development and maturation of primary cerebral cortical neurons from cynomolgus monkeys were slower than those from mice.We used a microelectrode array technique to compare the electrophysiological differences in cortical neurons,and found that primary cortical neurons from the mouse brain began to show electrical activity earlier than those from the cynomolgus monkey.Although cultured monkey cortical neurons developed slowly in vitro,they exhibited typical pathological features-revealed by immunofluorescent staining-when infected with adeno-associated viral vectors expressing mutant huntingtin(HTT),the Huntington’s disease protein.A quantitative analysis of the cultured monkey cortical neurons also confirmed that mutant HTT significantly reduced the length of neurites.Therefore,compared with the primary cortical neurons of mice,cultured monkey cortical neurons have longer developmental and survival times and greater sustained physiological activity,such as electrophysiological activity.Our findings also suggest that primary cynomolgus monkey neurons cultured in vitro can simulate a cell model of human neurodegenerative disease,and may be useful for investigating time-dependent neuronal death as well as treatment via neuronal regeneration.All mouse experiments and protocols were approved by the Animal Care and Use Committee of Jinan University of China(IACUC Approval No.20200512-04)on May 12,2020.All monkey experiments were approved by the IACUC protocol(IACUC Approval No.LDACU 20190820-01)on August 23,2019 for animal management and use.
文摘Twenty-two lactic acid bacteria （LAB） strains isolated from Argentinean goat dairy products were evaluated for its biochemical properties and esterase activities relevant to flavor development. Streptococcus thermophilus （UNSE314）, Lactobacillus （L.） delbrueckii subsp, bulgaricus （UNSE309）, L. rhamnosus （UNSE308）, L. plantarum （UNSE287, UNSE316, UNSE317） and Pediococcus pentosaceus （UNSE315） strains presented high acidifying activity. All strains tested metabolized citrate and produced diacetyl-acetoin in goat milk. Based on these results, 10 strains with the best performance in diverse technological properties were selected to determine esterolytic activity. In all evaluated strains, esterase specific activity （ESA） was detected on ct-naphthyl （ct-NA） acetate and 13-naphthyl （[3-NA） acetate, propionate, eaprylate and ct-NA butyrate. No activity was detected on [3-NA laurate. The highest values were detected when using a-NA instead of fI-NA derivatives as substrate. In Pediocoecus strains, wide variability in ESA were observed, which were species- and strain-specific. These results allow us to select strains with biochemical properties and esterase activities to design starter and adjunct cultures that contribute to flavor development during cheese ripening, thus preserving the typical organoleptic characteristics of Argentinean goat cheeses.
基金National Natural Science Foundation of China,No.39770300,30070873the Overseas Chinese Affairs Office of the State Council Foundation,No.98-33
文摘AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.
基金the National Outstanding Youth Science foundation of China (B type,39825502)the National Natural Science Foundation of China (39880015,30170477)the Natural Science Foundation of Fujian Province (C0110004).
文摘AIM: To investigate the role of TR3 in induction of apoptosis in gastric cancer cells. METHODS: Human gastric cancer cell line, MGC80-3, was used. Expression of TR3 mRNA and its protein was detected by Northern blot and Western blot. Localization of TR3 protein was showed by immunofluorescence analysis under laser-scanning confocal microscope. Apoptotic morphology was observed by DAPI fluorescence staining, and apoptotic index was counted among 1000 cells randomly. Stable transfection assay was carried out by Lipofectamine. RESULTS: Treatment of MGC80-3 cells with TPA and VP-16 resulted in apoptosis, accompanied by the repression of Bcl-2 protein in a time-dependent manner. At the same time, TPA and VP-16 also up-regulated expression level of TR3 mRNA in MGC80-3 cells that expressed TR3 mRNA. When antisense-TR3 expression vector was transfected into the cells, expression of TR3 protein was repressed. In this case, TPA and VP-16 did not induce apoptosis. In addition, TPA and VP-16-induced apoptosis involved in translocation of TR3. In MGC80-3 cells, TR3 localized concentrative in nucleus, after treatment of cells with TPA and VP-16, TR3 translocated from nucleus to cytosol obviously. However, when this nuclear translocation was blocked by LMB, apoptosis was not occurred in MGC80-3 cells even in the presence of TPA and VP-16. CONCLUSION: Induction of apoptosis by TPA and VP-16 is through induction of TR3 expression and translocation of TR3 from nucleus to cytosol, which may be a novel signal pathway for TR3, and represent the new biological function of TR3 to exert its effect on apoptosis in gastric cancer cells.
基金grants fromthe Chinese Academy of Sciences (No. KJ951-BI608), the National Natural Sciences FOundation ofChina (No. 39625007 and
文摘We reported in this manuscript that TGF-beta1 induces apoptosis in AML12 murine hepatocytes, which is associated with the activation of p38 MAPK signaling pathway. SB202190, a specific inhibitor of p38 MAPK, strongly inhibited the TGF-beta1-induced apoptosis and PAI-1 promoter activity. Treatment of cells with TGF-beta1 activates p38. Furthermore, over-expression of dominant negative mutant p38 also reduced the TGF-beta1-induced apoptosis. The data indicate that the activation of p38 is involved in TGF-beta1-mediated gene expression and apoptosis.
基金theNationalNaturalScienceFoundationofChina (No 39970 312andNo 39730 2 70 ) NationalOutstandingYoungScientificFoundationofC
文摘OBJECTIVE: To investigate the response of multiple myeloma (MM) cells to arsenic trioxide (As2O3) and their possible mechanisms. METHODS: Two MM-derived cell lines RPMI8226 and U266 cells were used as in vitro models. Cell apoptosis was assessed by morphology, flow cytometry, and DNA gel electrophoresis. Mitochondrial transmembrane potentials (delta psi m) were evaluated by measuring cellular Rhodamine 123 staining intensity. Protein expression was analyzed using Western blot. RESULTS: Zero point one to 0.5 mumol/L As2O3 inhibited cell proliferation and 2.0 mumol/L As2O3 induced cell apoptosis, while 1.0 mumol/L As2O3 inhibited proliferation with a weak degree of apoptosis induction in RPMI8226 and U266 cell lines. As2O3-induced apoptosis was accompanied by mitochondrial transmembrane potentials (delta psi m) collapse and caspase-3 activation in the presence of intact membrane. Glutathione depleter buthionine sulfoximine enhanced, while disulfide bond-reducing agent dithiothreitol partially antagonized As2O3-induced delta psi m collapse and apoptosis in MM cells. All-trans retinoic acid (ATRA) could also induce apoptosis in RPMI8226 cells, but it did not show any cooperative effects with As2O3. CONCLUSION: As2O3 exerts apoptosis-inducing and growth-inhibiting effects on MM cells, and mitochondrium is a pivotal and common target of As2O3 for apoptosis induction.
