试验考察宰前添加天冬氨酸镁(MgAsp)与维生素D3(VD3)对试猪血液理化指标、猪肉品质以及μ-Calpain和Calpastatin基因mRNA含量的影响。选体重约90 kg DLY杂交猪48头,随机分到4个处理:对照、加MgAsp、加VD3和加MgAsp与VD3。试验期为9 d,...试验考察宰前添加天冬氨酸镁(MgAsp)与维生素D3(VD3)对试猪血液理化指标、猪肉品质以及μ-Calpain和Calpastatin基因mRNA含量的影响。选体重约90 kg DLY杂交猪48头,随机分到4个处理:对照、加MgAsp、加VD3和加MgAsp与VD3。试验期为9 d,试验结束后选取24头进行屠宰。结果表明:宰前短期添加MgAsp和VD3对育肥猪胴体品质无影响;与对照相比,添加MgAsp提高血清镁含量(P<0.05),添加VD3提高血清磷(P<0.05)和钙的含量,MgAsp和VD3联合添加可进一步提高血清镁含量(P<0.01);添加MgAsp降低臀肌L值(P<0.01),并有提高肌肉pH值和降低滴水损失的趋势,提高了μ-Calpain与Calpastatin mRNA的比值,改善了嫩度;添加VD3降低了臀肌的L24h值(P<0.05),有提高pH和嫩度的趋势;MgAsp和VD3联合添加除对剪切力略有改善而外,对其他肉质指标并没有进一步改善。展开更多
AIM: To detect how BRCA-associated protein 1(BAP1) regulates cell migration in uveal melanoma(UM) cells. METHODS: Wound healing and transwell assays were performed to detect UM cell migration abilities. Protein chip, ...AIM: To detect how BRCA-associated protein 1(BAP1) regulates cell migration in uveal melanoma(UM) cells. METHODS: Wound healing and transwell assays were performed to detect UM cell migration abilities. Protein chip, immunoprecipitations and surface plasmon resonance analyses were applied to identify BAP1 protein partners. Western blot and calpain activity assays were used to test the expression and function of calpastatin(CAST). RESULTS: CAST protein was confirmed as a new BAP1 protein partner, and loss of BAP1 reduced the expression and function of CAST in UM cells. The overexpression of CAST rescued the cell migration phenotype caused by BAP1 loss.CONCLUSION: BAP1 interacts with CAST in UM cells, and CAST and its subsequent calpain pathway may mediate BAP1-related cell migration regulation.展开更多
Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down".The factors include ATP,calpastatin and H fraction(a high molecular fraction of bovine ...Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down".The factors include ATP,calpastatin and H fraction(a high molecular fraction of bovine cardiac cytoplasm).Methods Single Ca2+ channel activities were recorded with patch clamp technique in guinea-pig cardiac myocytes.Run-down was induced by the inside-out patch formation.Calpastatin(CS),calmodulin(CaM)and three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2,CT-1(amino acids number 1509-1791),CT-2(1777-2003)and CT-3(1944-2169)were produced as GST fusion proteins.Results(1)CaM + ATP or CS + ATP restored the channels after run-down;however,the CaM or CS's effects became smaller with the longer run-down time.(2)After run down,CaM-dependent protein kinase(CaMKII)produced Ca2+ channel activity to only 2-10% of the basal activity,however,in the presence of CaMKII,the time-dependent nature of the CaM effect was abolished.(3)In pull-down assay,CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase.(4)CaMKII was detected in the H fraction of bovine cardiac cytoplasm.Conclusions The results show that CS,CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2+ channels,and that there might be cross talks among the four factors(CS,CaM,CaMKII and the undefined cytoplasmic factor).展开更多
Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins, The association of Single Nucleotide Polymorphism （SNP） in the calpastatin gene with meat tenderness...Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins, The association of Single Nucleotide Polymorphism （SNP） in the calpastatin gene with meat tenderness is an important topic in meat production. Therefore efficient procedure to investigate the SNP is necessary. The objectives of this study were to detect the SNP of calpastatin gene at domain L marker （G/C transversion） of the Kamphaengsaen beef breed （KPS cattle; n = 26） by the Amplification Refractory Mutation System （ARMS） compared with the Restriction Fragment Length Polymorphism （RFLP） methods and to determine the genotypes of the KPS cattle at that marker. Genomic DNA of calpastatin gene extracted from blood of the KPS cattle was detected with ARMS and RFLP methods. The ARMS system has utilized two primer pairs to amplify the two different alleles of a polymorphism in single PCR reaction to detected single base mutation. In this method, the alleles-specific primers had a mismatch at 3＇ terminal base and a second deliberate mismatch at position -2 from 3＇ terminus. While the RFLP method detected a polymorphism using PCR-base technique follow by RsaI restriction enzyme. Amplification of the ARMS method revealed that the results were not different from the conventional method of RFLP. Analysis of genotypes revealed that the KPS cattle inherited the CC, CG and GG genotypes at domain L marker. These were reliable when verified by nucleotide sequence analysis of PCR products. The animals were genotyped and determined for tenderness phenotype with this marker that predicted variation of an intronic polymorphism at domain L of the calpastatin gene. Therefore, the ARMS method was simple, efficient technique, and suitable for detecting SNP at domain L marker of the calpastatin gene.展开更多
Objective Numerous studies have indicated that excitatory amino acid toxicity,such as glutamate toxicity,is involved in glaucoma.In addition,excessive glutamate can lead to an intracellular calcium overload,resulting ...Objective Numerous studies have indicated that excitatory amino acid toxicity,such as glutamate toxicity,is involved in glaucoma.In addition,excessive glutamate can lead to an intracellular calcium overload,resulting in regulated necrosis.Our previous studies have found that the calpastatin(CAST)-calpain pathway plays an important role in retinal neuron-regulated necrosis after glutamate injury.Although inhibition of the calpain pathway can decrease regulated necrosis,necrotic cells remain.It has been suggested that there are other molecules that participate in retinal neuron-regulated necrosis.CAST is an important regulator of dynamin-related protein 1(Drp1)-mediated mitochondrial defects.Thus,the aim of this study was to determine whether the CAST-Drp1 pathway may be an underlying signaling axis in neuron-regulated necrosis.Methods Using cultured retinal neurons and in an in-vivo glaucoma model induced by glutamate overload,members of the CAST-Drp1 pathway were assessed by immunofluorescence,Western blotting,Phos-tagTM SDS-PAGE,and co-immunoprecipitation assays.Moreover,the black and white box test was performed on the rats.Results We found that more retinal neuron-regulated necrosis and Drp1 activation as well as lower CAST levels were present in the glutamate-induced glaucoma model.Rats with glutamate-induced glaucoma exhibited impaired visual function.We also observed retinal neuron-regulated necrosis and Drp1 activity decreased,and impaired vision recovered after CAST active peptide application,indicating that the CAST-Drp1 pathway plays a critical role in retinal neuron-regulated necrosis and visual function.Conclusion The results of this study indicate that the CAST-Drp1 pathway protects against retinal neuron-regulated necrosis,which may expand the therapeutic targets for the treatment of neurodegenerative disorders involving dysfunction of glutamate metabolism,such as glaucoma.展开更多
文摘试验考察宰前添加天冬氨酸镁(MgAsp)与维生素D3(VD3)对试猪血液理化指标、猪肉品质以及μ-Calpain和Calpastatin基因mRNA含量的影响。选体重约90 kg DLY杂交猪48头,随机分到4个处理:对照、加MgAsp、加VD3和加MgAsp与VD3。试验期为9 d,试验结束后选取24头进行屠宰。结果表明:宰前短期添加MgAsp和VD3对育肥猪胴体品质无影响;与对照相比,添加MgAsp提高血清镁含量(P<0.05),添加VD3提高血清磷(P<0.05)和钙的含量,MgAsp和VD3联合添加可进一步提高血清镁含量(P<0.01);添加MgAsp降低臀肌L值(P<0.01),并有提高肌肉pH值和降低滴水损失的趋势,提高了μ-Calpain与Calpastatin mRNA的比值,改善了嫩度;添加VD3降低了臀肌的L24h值(P<0.05),有提高pH和嫩度的趋势;MgAsp和VD3联合添加除对剪切力略有改善而外,对其他肉质指标并没有进一步改善。
基金Supported by the Science and Technology Commission of Shanghai (No.14411961800)
文摘AIM: To detect how BRCA-associated protein 1(BAP1) regulates cell migration in uveal melanoma(UM) cells. METHODS: Wound healing and transwell assays were performed to detect UM cell migration abilities. Protein chip, immunoprecipitations and surface plasmon resonance analyses were applied to identify BAP1 protein partners. Western blot and calpain activity assays were used to test the expression and function of calpastatin(CAST). RESULTS: CAST protein was confirmed as a new BAP1 protein partner, and loss of BAP1 reduced the expression and function of CAST in UM cells. The overexpression of CAST rescued the cell migration phenotype caused by BAP1 loss.CONCLUSION: BAP1 interacts with CAST in UM cells, and CAST and its subsequent calpain pathway may mediate BAP1-related cell migration regulation.
基金supported by the grants from the Japan Society for the Promotion of Science and the National Natural Science Foundation of China(No.30670761,No.30671726)
文摘Objective To explore the mechanism that cytoplasmic factors could recover L-type Ca2+ channel activity after "run-down".The factors include ATP,calpastatin and H fraction(a high molecular fraction of bovine cardiac cytoplasm).Methods Single Ca2+ channel activities were recorded with patch clamp technique in guinea-pig cardiac myocytes.Run-down was induced by the inside-out patch formation.Calpastatin(CS),calmodulin(CaM)and three GST-fusion fragment peptides derived from the C-terminal tail of guinea-pig Cav1.2,CT-1(amino acids number 1509-1791),CT-2(1777-2003)and CT-3(1944-2169)were produced as GST fusion proteins.Results(1)CaM + ATP or CS + ATP restored the channels after run-down;however,the CaM or CS's effects became smaller with the longer run-down time.(2)After run down,CaM-dependent protein kinase(CaMKII)produced Ca2+ channel activity to only 2-10% of the basal activity,however,in the presence of CaMKII,the time-dependent nature of the CaM effect was abolished.(3)In pull-down assay,CT-1 treated with CaMKII showed a higher affinity for CaM than that treated with phosphatase.(4)CaMKII was detected in the H fraction of bovine cardiac cytoplasm.Conclusions The results show that CS,CaM and CaMKII are all involved in the maintenance of the basal activity of L-type Ca2+ channels,and that there might be cross talks among the four factors(CS,CaM,CaMKII and the undefined cytoplasmic factor).
文摘Calpastatin is an endogenous inhibitor of calpain which is responsible for the breakdown of myofibrillar proteins, The association of Single Nucleotide Polymorphism （SNP） in the calpastatin gene with meat tenderness is an important topic in meat production. Therefore efficient procedure to investigate the SNP is necessary. The objectives of this study were to detect the SNP of calpastatin gene at domain L marker （G/C transversion） of the Kamphaengsaen beef breed （KPS cattle; n = 26） by the Amplification Refractory Mutation System （ARMS） compared with the Restriction Fragment Length Polymorphism （RFLP） methods and to determine the genotypes of the KPS cattle at that marker. Genomic DNA of calpastatin gene extracted from blood of the KPS cattle was detected with ARMS and RFLP methods. The ARMS system has utilized two primer pairs to amplify the two different alleles of a polymorphism in single PCR reaction to detected single base mutation. In this method, the alleles-specific primers had a mismatch at 3＇ terminal base and a second deliberate mismatch at position -2 from 3＇ terminus. While the RFLP method detected a polymorphism using PCR-base technique follow by RsaI restriction enzyme. Amplification of the ARMS method revealed that the results were not different from the conventional method of RFLP. Analysis of genotypes revealed that the KPS cattle inherited the CC, CG and GG genotypes at domain L marker. These were reliable when verified by nucleotide sequence analysis of PCR products. The animals were genotyped and determined for tenderness phenotype with this marker that predicted variation of an intronic polymorphism at domain L of the calpastatin gene. Therefore, the ARMS method was simple, efficient technique, and suitable for detecting SNP at domain L marker of the calpastatin gene.
基金supported by the National Natural Science Foundation of China(No.82101126)the Natural Science Foundation of Hunan Province(No.2021JJ40873).
文摘Objective Numerous studies have indicated that excitatory amino acid toxicity,such as glutamate toxicity,is involved in glaucoma.In addition,excessive glutamate can lead to an intracellular calcium overload,resulting in regulated necrosis.Our previous studies have found that the calpastatin(CAST)-calpain pathway plays an important role in retinal neuron-regulated necrosis after glutamate injury.Although inhibition of the calpain pathway can decrease regulated necrosis,necrotic cells remain.It has been suggested that there are other molecules that participate in retinal neuron-regulated necrosis.CAST is an important regulator of dynamin-related protein 1(Drp1)-mediated mitochondrial defects.Thus,the aim of this study was to determine whether the CAST-Drp1 pathway may be an underlying signaling axis in neuron-regulated necrosis.Methods Using cultured retinal neurons and in an in-vivo glaucoma model induced by glutamate overload,members of the CAST-Drp1 pathway were assessed by immunofluorescence,Western blotting,Phos-tagTM SDS-PAGE,and co-immunoprecipitation assays.Moreover,the black and white box test was performed on the rats.Results We found that more retinal neuron-regulated necrosis and Drp1 activation as well as lower CAST levels were present in the glutamate-induced glaucoma model.Rats with glutamate-induced glaucoma exhibited impaired visual function.We also observed retinal neuron-regulated necrosis and Drp1 activity decreased,and impaired vision recovered after CAST active peptide application,indicating that the CAST-Drp1 pathway plays a critical role in retinal neuron-regulated necrosis and visual function.Conclusion The results of this study indicate that the CAST-Drp1 pathway protects against retinal neuron-regulated necrosis,which may expand the therapeutic targets for the treatment of neurodegenerative disorders involving dysfunction of glutamate metabolism,such as glaucoma.
