Objective The protein interacting with C kinase 1(PICK1)plays a critical role in vesicle trafficking,and its deficiency in sperm cells results in abnormal vesicle trafficking from Golgi to acrosome,which eventually di...Objective The protein interacting with C kinase 1(PICK1)plays a critical role in vesicle trafficking,and its deficiency in sperm cells results in abnormal vesicle trafficking from Golgi to acrosome,which eventually disrupts acrosome formation and leads to male infertility.Methods An azoospermia sample was filtered,and the laboratory detection and clinical phenotype indicated typical azoospermia in the patient.We sequenced all of the exons in the PICK1 gene and found that there was a novel homozygous variant in the PICK1 gene,c.364delA(p.Lys122SerfsX8),and this protein structure truncating variant seriously affected the biological function.Then we constructed a PICK1 knockout mouse model using clustered regularly interspaced short palindromic repeat cutting technology(CRISPRc).Results The sperm from PICK1 knockout mice showed acrosome and nucleus abnormalities,as well as dysfunctional mitochondrial sheath formation.Both the total sperm and motility sperm counts were decreased in the PICK1 knockout mice compared to wild-type mice.Moreover,the mitochondrial dysfunction was verified in the mice.These defects in the male PICK1 knockout mice may have eventually led to complete infertility.Conclusion The c.364delA novel variant in the PICK1 gene associated with clinical infertility,and pathogenic variants in the PICK1 may cause azoospermia or asthenospermia by impairing mitochondrial function in both mice and humans.展开更多
Objective-To investigate the effects of heat shock transcription factor 1) gene on the constitutivety expressed αB-CrystaUin (aBC) in mice myocardium. Methods-The expression levels of constitutive aBC in HSF1 knockou...Objective-To investigate the effects of heat shock transcription factor 1) gene on the constitutivety expressed αB-CrystaUin (aBC) in mice myocardium. Methods-The expression levels of constitutive aBC in HSF1 knockout (hsf1 - /- ) and HSFl wild type (As/1 + /+) mice myocardium were evaluated by western blot and immunohistochemistry. Results : The αBC levels in hsfl -/- and hsfl +/+ were 68. 42±4. 16, 100. 00±7. 58, respectively (P<0. 05, cytoso-lic fraction) , and 20. 53±1. 01, 37. 55±1. 91, respectively (P<0. 05, pellet fraction). The aBC signals decreased significantly in hsfl -/- myocardium when compared with those in hsfl +/+ myocardium stained with fluorescence immunohistochemistry. Conclusion-HSF1 is an important, but not the only factor, which mediates the constitutively expressed aBC.展开更多
Objective ： To investigate the mechanism of phenotypic knockout of CXCR4 on T-cell leukemia cell line Molt-4 via SDF-1α/54/KDEL intrakine technology, which the mutant SDF-1α/54, human stromal cell-derived Faceor-1 ...Objective ： To investigate the mechanism of phenotypic knockout of CXCR4 on T-cell leukemia cell line Molt-4 via SDF-1α/54/KDEL intrakine technology, which the mutant SDF-1α/54, human stromal cell-derived Faceor-1 （SDF-1α） was deleted its C- terminal α-helix and attached with a endoplasimc reticulum retention signal 4-peptide- KDEL encoding gene, so that retain the newly synthesized receptor CXCR4 within the Molt-4 cells endoplasmic reticulum. Methods： The recombinant vector pEGFP-C3/SDF- 1α/54/KDEL were transfected into Cos-7 cells by liposome, SDF-1α/54/KDEL fusion protein was confirmed with western blot. The recombinant plasmids were transfected transiently into Molt-4 by electroporation. Results：Western blot confirmed SDF-1α/54/KDEL expression in Cos-7. A dramatic downregulation of CXCR4 expression on Molt-4 was demonstrated by flow cytometric （FCM） analysis. Conelusion：SDF-1α/54/KDEL and SDF- 1αKDEL have no significant deviation for phenotypic knockout of CXCR4. These suggest that the phenotypic knockout effects of SDF-1α/54 against CXCR4 are not influenced by deleting of SDF-1α helix in the C-terminal.展开更多
基金supported by grants from Zhejiang Provincial Natural Science Foundation of China(No.LQ21H200007)National Natural Science Foundation of China(No.82202605,No.81772664 and No.82172363)+1 种基金Zhejiang Provincial People’s Hospital Excellent Scientific Research Start-up Fundation of China(No.ZRY2019C008)Hangzhou Medical College Fundamental Scientific Research Project of China(No.KYQN202116).
文摘Objective The protein interacting with C kinase 1(PICK1)plays a critical role in vesicle trafficking,and its deficiency in sperm cells results in abnormal vesicle trafficking from Golgi to acrosome,which eventually disrupts acrosome formation and leads to male infertility.Methods An azoospermia sample was filtered,and the laboratory detection and clinical phenotype indicated typical azoospermia in the patient.We sequenced all of the exons in the PICK1 gene and found that there was a novel homozygous variant in the PICK1 gene,c.364delA(p.Lys122SerfsX8),and this protein structure truncating variant seriously affected the biological function.Then we constructed a PICK1 knockout mouse model using clustered regularly interspaced short palindromic repeat cutting technology(CRISPRc).Results The sperm from PICK1 knockout mice showed acrosome and nucleus abnormalities,as well as dysfunctional mitochondrial sheath formation.Both the total sperm and motility sperm counts were decreased in the PICK1 knockout mice compared to wild-type mice.Moreover,the mitochondrial dysfunction was verified in the mice.These defects in the male PICK1 knockout mice may have eventually led to complete infertility.Conclusion The c.364delA novel variant in the PICK1 gene associated with clinical infertility,and pathogenic variants in the PICK1 may cause azoospermia or asthenospermia by impairing mitochondrial function in both mice and humans.
文摘Objective-To investigate the effects of heat shock transcription factor 1) gene on the constitutivety expressed αB-CrystaUin (aBC) in mice myocardium. Methods-The expression levels of constitutive aBC in HSF1 knockout (hsf1 - /- ) and HSFl wild type (As/1 + /+) mice myocardium were evaluated by western blot and immunohistochemistry. Results : The αBC levels in hsfl -/- and hsfl +/+ were 68. 42±4. 16, 100. 00±7. 58, respectively (P<0. 05, cytoso-lic fraction) , and 20. 53±1. 01, 37. 55±1. 91, respectively (P<0. 05, pellet fraction). The aBC signals decreased significantly in hsfl -/- myocardium when compared with those in hsfl +/+ myocardium stained with fluorescence immunohistochemistry. Conclusion-HSF1 is an important, but not the only factor, which mediates the constitutively expressed aBC.
基金Grant sponsor:National Natural Science Foundation of ChinaGrant number:30572209
文摘Objective ： To investigate the mechanism of phenotypic knockout of CXCR4 on T-cell leukemia cell line Molt-4 via SDF-1α/54/KDEL intrakine technology, which the mutant SDF-1α/54, human stromal cell-derived Faceor-1 （SDF-1α） was deleted its C- terminal α-helix and attached with a endoplasimc reticulum retention signal 4-peptide- KDEL encoding gene, so that retain the newly synthesized receptor CXCR4 within the Molt-4 cells endoplasmic reticulum. Methods： The recombinant vector pEGFP-C3/SDF- 1α/54/KDEL were transfected into Cos-7 cells by liposome, SDF-1α/54/KDEL fusion protein was confirmed with western blot. The recombinant plasmids were transfected transiently into Molt-4 by electroporation. Results：Western blot confirmed SDF-1α/54/KDEL expression in Cos-7. A dramatic downregulation of CXCR4 expression on Molt-4 was demonstrated by flow cytometric （FCM） analysis. Conelusion：SDF-1α/54/KDEL and SDF- 1αKDEL have no significant deviation for phenotypic knockout of CXCR4. These suggest that the phenotypic knockout effects of SDF-1α/54 against CXCR4 are not influenced by deleting of SDF-1α helix in the C-terminal.
