Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide, metabolism of fatty acids, photorespiration, and the bio...Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide, metabolism of fatty acids, photorespiration, and the biosyn- thesis of plant hormones. Plant peroxisomes have been traditionally classified into three major subtypes, and in-depth mass spectrometry （MS）-based proteomics has been per- formed to explore the proteome of the two major subtypes present in green leaves and etiolated seedlings. Here, we carried out a comprehensive proteome analysis of perox- isomes from Arabidopsis leaves given a 48-h dark treatment. Our goal was to determine the proteome of the third major subtype of plant peroxisomes from senescent leaves, and further catalog the plant peroxisomal proteome. We identified a total of 111 peroxisomal proteins and verified the peroxisomal localization for six new proteins with potential roles in fatty acid metabolism and stress response by in vivo targeting analysis. Metabolic pathways compartmentalized in the three major subtypes of peroxisomes were also compared, which revealed a higher number of proteins involved in the detoxification of reactive oxygen species in peroxisomes from senescent leaves. Our study takes an important step towards mapping the full function of plant peroxisomes.展开更多
Both female and male gametophytes harbor companion cells and gametes. MET1, a DNA methyltransferase, is down-regulated in companion cells. However, how MET1 is differentially regulated in gametophytes remains unexplor...Both female and male gametophytes harbor companion cells and gametes. MET1, a DNA methyltransferase, is down-regulated in companion cells. However, how MET1 is differentially regulated in gametophytes remains unexplored. ARID1, a transcription factor that is specifically depleted in sperm cells, is occupied by MET1- dependent CG methylation. Here, we show that MET1 confines ARID1 to the vegetative cell of male gametes, but ARID1 conversely represses MET1 in the central cell of female gametes. Compared to the vegetative celllocalization in wild type pollen, ARID1 expands to sperm cells in the met1 mutant. To understand whether MET1- dependent ARID1 inhibition exists during female gametogenesis, we first show that ARID1 is expressed in the megaspore mother cell （MMC）, ARID1 but not MET1 is detectable in the central cell at maturity. Interestingly, compared to the absence of MET1 in the central cell and the egg cell of wild type ovules, MET1 significantly accumulates in these two cells in aridl ovules. Lastly, we show that both ARIDI and METI are required for the cell specification of MMC. Collectively, our results uncover a reciprocal dependence between ARIDI and METI, and provide a clue to further understand how the specification of MMC is likely regulated by DNA methylation.展开更多
Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affe...Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.展开更多
In order to investigate the pathogenesis of Alzheimer disease （AD） and study the enzymatic progress of amyloid precursor protein （APP）, the fluorescent eukaryotic expression plasmid of C99 was constructed containi...In order to investigate the pathogenesis of Alzheimer disease （AD） and study the enzymatic progress of amyloid precursor protein （APP）, the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP （which was named C99 containing APP717 mutation）, together with the fragment encoding yellow fluorescence protein （which was named YFP） were amplified by PCR. The two fragments （YFP and C99） were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β （Aβ） was detected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, membrane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the deposition in the cell space and the cell shape was rough. It was suggested that Aβ could be generated within the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to further study the cleavage mechanism of APP and to explore the pathogenesis of AD.展开更多
Polyethylene glycol (PEG)-mediated DNA transformation for transient gene expression in protoplasts and Agrobacterium tumefaciens-mediated transformation in lower epidermis of leaves are readily available in several pl...Polyethylene glycol (PEG)-mediated DNA transformation for transient gene expression in protoplasts and Agrobacterium tumefaciens-mediated transformation in lower epidermis of leaves are readily available in several plant species. In the study, these two versatile tools were used in rapeseed. A simple and efficient method was established for isolating protoplasts from rapeseed cotyledons and leaves, and found that cotyledons might be better than true leaves. Transient expression analysis showed that yellow fluorescent protein (YFP) and luciferase (LUC) could be expressed in rapeseed protoplasts. Moreover,GUS histochemical assays indicated that Agrobacterium-mediated DNA transient expression was achievable only in lower epidermis of rapeseed cotyledons and expression signal was the highest on the 5th day after injection with the bacterial suspension (OD600=0.8).These methods might provide valuable tools for rapid functional gene analysis in rapeseed.展开更多
Summary Vacuolar trafficking routes and their regu- lators have recently drawn lots of attention in plant cell biology. A recent study reported the discovery of a plant-specific vacuolar trafficking route, i.e., a dir...Summary Vacuolar trafficking routes and their regu- lators have recently drawn lots of attention in plant cell biology. A recent study reported the discovery of a plant-specific vacuolar trafficking route, i.e., a direct ER- to-vacuole route, through analysis of VHA-a3 subcellular targeting, a key component for the tonoplast V- ATPases. Our recent findings showed that VHA-a3 targets to the tonoplast through a Rab5-mediated but Rab7-independent pathway, shedding new lights on the unconventional vacuolar trafficking route in plant cells.展开更多
Microsurgical suturing is the gold standard of nerve coaptation. Although literature on the usefulness of fibrin glue as an alternative is becoming increasingly available, it remains contradictory. Furthermore, no dat...Microsurgical suturing is the gold standard of nerve coaptation. Although literature on the usefulness of fibrin glue as an alternative is becoming increasingly available, it remains contradictory. Furthermore, no data exist on how both repair methods might influence the morphological aspects（arborization; branching） of early peripheral nerve regeneration. We used the sciatic nerve transplantation model in thy-1 yellow fluorescent protein mice（YFP; n = 10）. Pieces of nerve（1cm） were grafted from YFP-negative mice（n = 10） into those expressing YFP. We performed microsuture coaptations on one side and used fibrin glue for repair on the contralateral side. Seven days after grafting, the regeneration distance, the percentage of regenerating and arborizing axons, the number of branches per axon, the coaptation failure rate, the gap size at the repair site and the time needed for surgical repair were all investigated. Fibrin glue repair resulted in regenerating axons travelling further into the distal nerve. It also increased the percentage of arborizing axons. No coaptation failure was detected. Gap sizes were comparable in both groups. Fibrin glue significantly reduced surgical repair time. The increase in regeneration distance, even after the short period of time, is in line with the results of others that showed faster axonal regeneration after fibrin glue repair. The increase in arborizing axons could be another explanation for better functional and electrophysiological results after fibrin glue repair. Fibrin glue nerve coaptation seems to be a promising alternative to microsuture repair.展开更多
A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activa...A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(K d=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.展开更多
Ubiquitin-proteasome system(UPS)plays an important role in neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD).The discovery of UPS activators for anti-ne...Ubiquitin-proteasome system(UPS)plays an important role in neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD).The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important.In this study,we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD.At first,stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells,together with G418 screening.The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1.By employing the high-content fluorescence imaging system,together with stable YFP-CL1 HT22 cells,the UPS activators were successfully screened from our established TCM library.The representative images were captured and analyzed,and quantification of the YFP fluorescence intensity was performed by flow cytometry.Then,the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP(APP),pRK5-EGFP-Tau P301L(Tau P301L),or pRK5-EGFP-Tau(Tau)transiently transfected HT22 cells using fluorescence imaging,flow cytometry,and Western blot.In conclusion,our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the highthroughput screening of the UPS activators.Three compounds,namely salvianolic acid A(SAA),salvianolic acid B(SAB),and ellagic acid(EA),were identified to significantly decrease YFP fluorescence intensity,which suggested that these three compounds are UPS activators.The identified UPS activators were demonstrated to clear AD-related proteins,including APP,Tau,and Tau P301L.Therefore,these findings provide a novel insight into the discovery and development of anti-AD drugs.展开更多
Increasing evidence indicates that sialic acid plays an important role during nerve regeneration. Sialic acids can be modified in vitro as well as in vivo using metabolic oligosaccharide engineering of the N-awl side ...Increasing evidence indicates that sialic acid plays an important role during nerve regeneration. Sialic acids can be modified in vitro as well as in vivo using metabolic oligosaccharide engineering of the N-awl side chain. N-Propionylmannosamine （ManNProp） increases neurite outgrowth and accelerates the reestablishment of functional synapses in vitro. We investigated the influence of systemic ManNProp application using a specific in vivo mouse model. Using mice expressing axonal fluorescent proteins, we quantified the extension of regenerating axons, the number of regenerating axons, the number of arborising axons and the number of branches per axon 5 days after injury. Sciatic nerves from non-expressing mice were grafted into those expressing yellow fluorescent protein. We began a twice-daily intraperitoneal application of either peracetylated ManNProp （200 mg/kg） or saline solution 5 days before injury, and continued it until nerve harvest （5 days after transection）. ManNProp significantly increased the mean distance of axonal regeneration （2.49 mm vs. 1.53 mm; P 〈 0.005） and the number of arborizing axons （21% vs. 16%; P = 0.008） 5 days after sciatic nerve grafting. ManNProp did not affect the number of regenerating axons or the number of branches per arborizing axon. The biochemical glycoengineering of the N-acyl side chain of sialic acid might be a promising approach for improving peripheral nerve regeneration.展开更多
A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of...A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.展开更多
基金supported by grants from the National Science Foundation to J.H.(MCB 0618335MCB 1330441)and L.J.O.(MCB 0618279)
文摘Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide, metabolism of fatty acids, photorespiration, and the biosyn- thesis of plant hormones. Plant peroxisomes have been traditionally classified into three major subtypes, and in-depth mass spectrometry （MS）-based proteomics has been per- formed to explore the proteome of the two major subtypes present in green leaves and etiolated seedlings. Here, we carried out a comprehensive proteome analysis of perox- isomes from Arabidopsis leaves given a 48-h dark treatment. Our goal was to determine the proteome of the third major subtype of plant peroxisomes from senescent leaves, and further catalog the plant peroxisomal proteome. We identified a total of 111 peroxisomal proteins and verified the peroxisomal localization for six new proteins with potential roles in fatty acid metabolism and stress response by in vivo targeting analysis. Metabolic pathways compartmentalized in the three major subtypes of peroxisomes were also compared, which revealed a higher number of proteins involved in the detoxification of reactive oxygen species in peroxisomes from senescent leaves. Our study takes an important step towards mapping the full function of plant peroxisomes.
基金supported by grants of the National Natural Science Foundation of China(31422029,31470281,31671261)the Recruitment Program of Global Experts(China)
文摘Both female and male gametophytes harbor companion cells and gametes. MET1, a DNA methyltransferase, is down-regulated in companion cells. However, how MET1 is differentially regulated in gametophytes remains unexplored. ARID1, a transcription factor that is specifically depleted in sperm cells, is occupied by MET1- dependent CG methylation. Here, we show that MET1 confines ARID1 to the vegetative cell of male gametes, but ARID1 conversely represses MET1 in the central cell of female gametes. Compared to the vegetative celllocalization in wild type pollen, ARID1 expands to sperm cells in the met1 mutant. To understand whether MET1- dependent ARID1 inhibition exists during female gametogenesis, we first show that ARID1 is expressed in the megaspore mother cell （MMC）, ARID1 but not MET1 is detectable in the central cell at maturity. Interestingly, compared to the absence of MET1 in the central cell and the egg cell of wild type ovules, MET1 significantly accumulates in these two cells in aridl ovules. Lastly, we show that both ARIDI and METI are required for the cell specification of MMC. Collectively, our results uncover a reciprocal dependence between ARIDI and METI, and provide a clue to further understand how the specification of MMC is likely regulated by DNA methylation.
基金financed by the National Natural Science Foundation of China (30900972, 31572111)the Special Found for Agro-scientific Research in the Public Interest, China (201203076-06)the Graduate Innovative Projects of Hunan Province, China (CX2013B290)
文摘Agrobacterium-mediated transient expression assays are a convenient alternative to stable expression because they are simple,easy to perform,and achieve gene expression rapidly.This study investigated the factors affecting transient gene expression efficiency in citrus by observing the cryo-sectioning of leaf samples under a laser confocal microscope.These factors included the composition of the infiltration buffer,the Agrobacterium cell density,the leaf development stage,the incubation temperature,and plant genotype.The highest transient expression level of yellow fluorescent protein（YFP）was detected in Mexican lime（Citrus aurantifolia）on the third day after the intermediate-aged leaves were infiltrated with the improved infiltration buffer 1（15 mmol L^-1 2-（N-morpholino）ethanesulfonic acid,10 mmol L^-1 MgCl2,and 200μmol L^-1acetosyringone）,which had an optical density of 0.8 and was incubated at 22°C.Additionally,this transient expression assay was applied to other citrus genotypes.Of note,trifoliate orange（Poncirus trifoliata）and kumquat（Fortunella obovate）had higher expression efficiency than other six genotypes of the Citrus genus.Our study provides research basis for the selection of optimization strategies in transient gene expression and improves the method for available genome investigation in citrus.
基金This project was supported by the Key Project of Chinese Ministry of Education (No 10420)
文摘In order to investigate the pathogenesis of Alzheimer disease （AD） and study the enzymatic progress of amyloid precursor protein （APP）, the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP （which was named C99 containing APP717 mutation）, together with the fragment encoding yellow fluorescence protein （which was named YFP） were amplified by PCR. The two fragments （YFP and C99） were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β （Aβ） was detected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, membrane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the deposition in the cell space and the cell shape was rough. It was suggested that Aβ could be generated within the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to further study the cleavage mechanism of APP and to explore the pathogenesis of AD.
基金supported by the National KeyBasic Research Program of China (2015CB150200)the Natural Science Foundation of Hubei Province (2016CFB286)
文摘Polyethylene glycol (PEG)-mediated DNA transformation for transient gene expression in protoplasts and Agrobacterium tumefaciens-mediated transformation in lower epidermis of leaves are readily available in several plant species. In the study, these two versatile tools were used in rapeseed. A simple and efficient method was established for isolating protoplasts from rapeseed cotyledons and leaves, and found that cotyledons might be better than true leaves. Transient expression analysis showed that yellow fluorescent protein (YFP) and luciferase (LUC) could be expressed in rapeseed protoplasts. Moreover,GUS histochemical assays indicated that Agrobacterium-mediated DNA transient expression was achievable only in lower epidermis of rapeseed cotyledons and expression signal was the highest on the 5th day after injection with the bacterial suspension (OD600=0.8).These methods might provide valuable tools for rapid functional gene analysis in rapeseed.
基金supported by Major Research Plan(2013CB945102) from the Ministry of Science,Technology of ChinaNational Natural Science Foundation of China(31261160490 to Y.Z.)Natural Science Foundation of Shandong Province(ZR2014CM027 to S.L.)
文摘Summary Vacuolar trafficking routes and their regu- lators have recently drawn lots of attention in plant cell biology. A recent study reported the discovery of a plant-specific vacuolar trafficking route, i.e., a direct ER- to-vacuole route, through analysis of VHA-a3 subcellular targeting, a key component for the tonoplast V- ATPases. Our recent findings showed that VHA-a3 targets to the tonoplast through a Rab5-mediated but Rab7-independent pathway, shedding new lights on the unconventional vacuolar trafficking route in plant cells.
基金supported by funding from the Charité–Universittsmedizin Berlin
文摘Microsurgical suturing is the gold standard of nerve coaptation. Although literature on the usefulness of fibrin glue as an alternative is becoming increasingly available, it remains contradictory. Furthermore, no data exist on how both repair methods might influence the morphological aspects（arborization; branching） of early peripheral nerve regeneration. We used the sciatic nerve transplantation model in thy-1 yellow fluorescent protein mice（YFP; n = 10）. Pieces of nerve（1cm） were grafted from YFP-negative mice（n = 10） into those expressing YFP. We performed microsuture coaptations on one side and used fibrin glue for repair on the contralateral side. Seven days after grafting, the regeneration distance, the percentage of regenerating and arborizing axons, the number of branches per axon, the coaptation failure rate, the gap size at the repair site and the time needed for surgical repair were all investigated. Fibrin glue repair resulted in regenerating axons travelling further into the distal nerve. It also increased the percentage of arborizing axons. No coaptation failure was detected. Gap sizes were comparable in both groups. Fibrin glue significantly reduced surgical repair time. The increase in regeneration distance, even after the short period of time, is in line with the results of others that showed faster axonal regeneration after fibrin glue repair. The increase in arborizing axons could be another explanation for better functional and electrophysiological results after fibrin glue repair. Fibrin glue nerve coaptation seems to be a promising alternative to microsuture repair.
基金the Start- up Fund for Returned Overseas Scholars from Northeast Normal U niversity,National ScienceFund for Distinguished Young Scholars (No. 30 32 5 0 11) ,Distinguished Young Scholars Fund of Jilin Province(No.2 0 0 30 112 ) ,Excellent Young Teachers
文摘A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(K d=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation.
基金the Joint Project of Luzhou Municipal People’s Government and Southwest Medical University(No.2020LZXNYDJ37)the National Natural Science Foundation of China(Nos.81903829 and 81801398)+2 种基金the Science and Technology Planning Project of Sichuan Province(Nos.2019JDPT0010 and 2020YJ0494)the Project of Southwest Medical University(Nos.2021ZKZD015,2021ZKZD018,and 2021-ZKMS046)Sichuan University Student Innovation and Entrepreneurship Project(Nos.2019424 and 201816032066).
文摘Ubiquitin-proteasome system(UPS)plays an important role in neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD).The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important.In this study,we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD.At first,stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells,together with G418 screening.The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1.By employing the high-content fluorescence imaging system,together with stable YFP-CL1 HT22 cells,the UPS activators were successfully screened from our established TCM library.The representative images were captured and analyzed,and quantification of the YFP fluorescence intensity was performed by flow cytometry.Then,the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP(APP),pRK5-EGFP-Tau P301L(Tau P301L),or pRK5-EGFP-Tau(Tau)transiently transfected HT22 cells using fluorescence imaging,flow cytometry,and Western blot.In conclusion,our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the highthroughput screening of the UPS activators.Three compounds,namely salvianolic acid A(SAA),salvianolic acid B(SAB),and ellagic acid(EA),were identified to significantly decrease YFP fluorescence intensity,which suggested that these three compounds are UPS activators.The identified UPS activators were demonstrated to clear AD-related proteins,including APP,Tau,and Tau P301L.Therefore,these findings provide a novel insight into the discovery and development of anti-AD drugs.
文摘Increasing evidence indicates that sialic acid plays an important role during nerve regeneration. Sialic acids can be modified in vitro as well as in vivo using metabolic oligosaccharide engineering of the N-awl side chain. N-Propionylmannosamine （ManNProp） increases neurite outgrowth and accelerates the reestablishment of functional synapses in vitro. We investigated the influence of systemic ManNProp application using a specific in vivo mouse model. Using mice expressing axonal fluorescent proteins, we quantified the extension of regenerating axons, the number of regenerating axons, the number of arborising axons and the number of branches per axon 5 days after injury. Sciatic nerves from non-expressing mice were grafted into those expressing yellow fluorescent protein. We began a twice-daily intraperitoneal application of either peracetylated ManNProp （200 mg/kg） or saline solution 5 days before injury, and continued it until nerve harvest （5 days after transection）. ManNProp significantly increased the mean distance of axonal regeneration （2.49 mm vs. 1.53 mm; P 〈 0.005） and the number of arborizing axons （21% vs. 16%; P = 0.008） 5 days after sciatic nerve grafting. ManNProp did not affect the number of regenerating axons or the number of branches per arborizing axon. The biochemical glycoengineering of the N-acyl side chain of sialic acid might be a promising approach for improving peripheral nerve regeneration.
文摘A thiazolidinone CFTR inhibitor(CFTR_ inh-172 ) was synthesized by a three-step procedure with trifluromethylaniline as the starting material. The synthesized CFTR inhibitor was characterized structurally by means of 1H NMR and functionally in a CFTR-expressing cell line FRT/hCFTR/EYFP-H148Q by both fluorescent and electrophysiological methods. A large amount(100 g) of high-quality small molecule thiazolidinone CFTR chloride channel inhibitor,CFTR_ inh-172 ,can be produced with this simple three-step synthetic procedure. The structure of the final product 2-thioxo-3-(3-trifluromethylphenyl)-5-[4-carboxyphenyl- methylene]-4-thiazolidinone was confirmed by 1H NMR. The overall yield was 58% with a purity over 99% as analyzed by HPLC. The synthesized CFTR_ inh-172 specifically inhibited CFTR chloride channel function in a cell-based fluorescence assay( K _d≈1.5 μmol/L) and in a Ussing chamber-based short-circuit current assay( K _d≈0.2 μmol/L),indicating better quality than that of the commercial combinatorial compound. The synthesized inhibitor is nontoxic to cultured cells at a high concentration and to mouse at a high dose. The synthetic procedure developed here can be used to produce a large amount of the high-quality CFTR_ inh-172 suitable for antidiarrheal studies and for creation of cystic fibrosis models in large animals. The procedure can be used to synthesize radiolabled CFTR_ inh-172 for in vivo pharmacokinetics studies.
