Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy(cryo-EM).However,the strong s...Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy(cryo-EM).However,the strong signal of liposomes under cryo-EM imaging conditions often interferes with the structural determination of the embedded membrane proteins.Here,we propose a liposome signal subtraction method based on single-particle two-dimensional(2D)classification average images,aimed at enhancing the reconstruction resolution of membrane proteins.We analyzed the signal distribution characteristics of liposomes and proteins within the 2D classification average images of protein–liposome complexes in the frequency domain.Based on this analysis,we designed a method to subtract the liposome signals from the original particle images.After the subtraction,the accuracy of single-particle three-dimensional(3D)alignment was improved,enhancing the resolution of the final 3D reconstruction.We demonstrated this method using a PIEZO1-proteoliposome dataset by improving the resolution of the PIEZO1 protein.展开更多
[Objective] The aim of the study is to clone and analyze the gene encoding 14-3-3 protein from banana. [Method] Combined with PCR amplification, RACE (rapid amplification of cDNA ends) technique was employed to clone ...[Objective] The aim of the study is to clone and analyze the gene encoding 14-3-3 protein from banana. [Method] Combined with PCR amplification, RACE (rapid amplification of cDNA ends) technique was employed to clone 14-3-3 gene from banana; then the amplified sequence was sequenced and homologically analyzed. [Result] A new cDNA homologous with 14-3-3 protein genes were obtained by RT-PCR and RACE ( rapid amplification of cDNA ends ) approaches. The full length of this cDNA was 866 bp encoding 197 amino acids. Alignment of deduced amino acid sequence with those from other plants revealed that the cDNA shared high homology with 14-3-3 protein genes from other plants, and was designated as Musa acuminata 14-3-3 gene (Ma-14-3-3d). Phylogenetic analysis reveals that Ma-14-3-3d has closer genetic relationship with those from monocotyledon species than those from other species. [Conclusion] Ma-14-3-3d belongs to the same lineage of 14-3-3 from monocotyledon.展开更多
Oryza sativa L. ssp. japonica and indica exhibit different sensitivity to photoinhibition and they show different stability of their core proteins D1 in the chloroplast photosystem Ⅱ. Using in situ hybridization, psb...Oryza sativa L. ssp. japonica and indica exhibit different sensitivity to photoinhibition and they show different stability of their core proteins D1 in the chloroplast photosystem Ⅱ. Using in situ hybridization, psbA, the gene encoding D1 protein of O. sativa ssp. japonica cv. 9516, and that of O. sativa ssp. indica cv. Shanyou 63 was cloned. As revealed by homology comparison of their sequences, the sequences are identical in the regions of promoter and 5′-UTR; differences are found in individual bases in the coding region all of which, being in the third position of respective codons, however, do not affect the amino acids coded finally; a difference is noted in the length of the oligo-U sequence in the region of 3′-UTR. It is thus apparent that, rather than a result of any difference in the amino acid sequences, the differences in the sensitivity to photoinhibition of D1 proteins between japonica and indica rice may be related to the upstream factors that regulate expression of psbA or to differences of photoprotective mechanisms.展开更多
Objective In the present study, we investigated the antioxidant and anti‐aging effects of Silybum marianum protein hydrolysate（SMPH） in D‐galactose‐treated mice. Methods D‐galactose（500 mg/kg body weight） was ...Objective In the present study, we investigated the antioxidant and anti‐aging effects of Silybum marianum protein hydrolysate（SMPH） in D‐galactose‐treated mice. Methods D‐galactose（500 mg/kg body weight） was intraperitoneally injected daily for 7 weeks to accelerate aging, and SMPH（400, 800, 1,200 mg/kg body weight, respectively） was simultaneously administered orally. The antioxidant and anti‐aging effects of SMPH in the liver and brain were measured by biochemical assays. Transmission electron microscopy（TEM） was performed to study the ultrastructure of liver mitochondria. Results SMPH decreased triglyceride and cholesterol levels in the D‐galactose‐treated mice. It significantly elevated the activities of superoxide dismutase（SOD） and glutathione peroxidase（GSH‐Px）, and total antioxidant capacity（T‐AOC）, which were suppressed by D‐galactose. Monoamine oxidase（MAO） and malondialdehyde（MDA） levels as well as the concentrations of caspase‐3 and 8‐OHd G in the liver and brain were significantly reduced by SMPH. Moreover, it increased Bcl‐2 levels in the liver and brain. Furthermore, SMPH significantly attenuated D‐galactose‐induced liver mitochondrial dysfunction by improving the activities of Na＋‐K＋‐ATPase and Ca2＋‐Mg2＋‐ATPase as well as mitochondrial membrane potential（ΔΨm） and fluidity. TEM showed that the degree of liver mitochondrial damage was significantly decreased by SMPH. Conclusion The results indicated that SMPH protects against D‐galactose‐induced accelerated aging in mice through its antioxidant and anti‐aging activities.展开更多
BACKGROUND Massive hepatocyte death is the core event in acute liver failure(ALF).Gasdermin D(GSDMD)-mediated pyroptosis is a type of highly inflammatory cell death.However,the role of hepatocyte pyroptosis and its me...BACKGROUND Massive hepatocyte death is the core event in acute liver failure(ALF).Gasdermin D(GSDMD)-mediated pyroptosis is a type of highly inflammatory cell death.However,the role of hepatocyte pyroptosis and its mechanisms of expanding inflammatory responses in ALF are unclear.AIM To investigate the role and mechanisms of GSDMD-mediated hepatocyte pyroptosis through in vitro and in vivo experiments.METHODS The expression of pyroptosis pathway-associated proteins in liver tissues from ALF patients and a hepatocyte injury model was examined by Western blot.GSDMD short hairpin RNA(shRNA)was used to investigate the effects of downregulation of GSDMD on monocyte chemotactic protein 1(MCP1)and its receptor CC chemokine receptor-2(CCR2)in vitro.For in vivo experiments,we used GSDMD knockout mice to investigate the role and mechanism of GSDMD in a D-galactose/lipopolysaccharide(D-Galn/LPS)-induced ALF mouse model.RESULTS The levels of pyroptosis pathway-associated proteins in liver tissue from ALF patients and a hepatocyte injury model increased significantly.The level of GSDMD-N protein increased most obviously(P<0.001).In vitro,downregulation of GSDMD by shRNA decreased the cell inhibition rate and the levels of MCP1/CCR2 proteins(P<0.01).In vivo,GSDMD knockout dramatically eliminated inflammatory damage in the liver and improved the survival of DGaln/LPS-induced ALF mice(P<0.001).Unlike the mechanism of immune cell pyroptosis that involves releasing interleukin(IL)-1βand IL-18,GSDMDmediated hepatocyte pyroptosis recruited macrophages via MCP1/CCR2 to aggravate hepatocyte death.However,this pathological process was inhibited after knocking down GSDMD.CONCLUSION GSDMD-mediated hepatocyte pyroptosis plays an important role in the pathogenesis of ALF,recruiting macrophages to release inflammatory mediators by upregulating MCP1/CCR2 and leading to expansion of the inflammatory responses.GSDMD knockout can reduce hepatocyte death and inflammatory responses,thus alleviating ALF.展开更多
AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial (HCE) cells Exposed to aspergillus fumigatus (AF) antigens. METHODS: HCE cells cultured 47 in vitro with AF a...AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial (HCE) cells Exposed to aspergillus fumigatus (AF) antigens. METHODS: HCE cells cultured 47 in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The Expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-kappa B and relative downstream cytokines such as TNF-alpha, IL-1 beta, IL-8 and IL-10 in supernatant fluid were measured by ELISA. RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-kappa B was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-alpha, IL-1 beta, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-alpha and IL-1 beta reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner. ' CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-kappa B pathway. SP-D possibly mediates the recognition to AF mycelium.展开更多
基金Project supported by the National Natural Science Foundation of China (Grant Nos.32241023 and 92254306)the Fund from the Tsinghua–Peking Joint Center for Life SciencesBeijing Frontier Research Center for Biological Structure。
文摘Reconstituting membrane proteins in liposomes and determining their structure is a common method for determining membrane protein structures using single-particle cryo-electron microscopy(cryo-EM).However,the strong signal of liposomes under cryo-EM imaging conditions often interferes with the structural determination of the embedded membrane proteins.Here,we propose a liposome signal subtraction method based on single-particle two-dimensional(2D)classification average images,aimed at enhancing the reconstruction resolution of membrane proteins.We analyzed the signal distribution characteristics of liposomes and proteins within the 2D classification average images of protein–liposome complexes in the frequency domain.Based on this analysis,we designed a method to subtract the liposome signals from the original particle images.After the subtraction,the accuracy of single-particle three-dimensional(3D)alignment was improved,enhancing the resolution of the final 3D reconstruction.We demonstrated this method using a PIEZO1-proteoliposome dataset by improving the resolution of the PIEZO1 protein.
文摘[Objective] The aim of the study is to clone and analyze the gene encoding 14-3-3 protein from banana. [Method] Combined with PCR amplification, RACE (rapid amplification of cDNA ends) technique was employed to clone 14-3-3 gene from banana; then the amplified sequence was sequenced and homologically analyzed. [Result] A new cDNA homologous with 14-3-3 protein genes were obtained by RT-PCR and RACE ( rapid amplification of cDNA ends ) approaches. The full length of this cDNA was 866 bp encoding 197 amino acids. Alignment of deduced amino acid sequence with those from other plants revealed that the cDNA shared high homology with 14-3-3 protein genes from other plants, and was designated as Musa acuminata 14-3-3 gene (Ma-14-3-3d). Phylogenetic analysis reveals that Ma-14-3-3d has closer genetic relationship with those from monocotyledon species than those from other species. [Conclusion] Ma-14-3-3d belongs to the same lineage of 14-3-3 from monocotyledon.
文摘Oryza sativa L. ssp. japonica and indica exhibit different sensitivity to photoinhibition and they show different stability of their core proteins D1 in the chloroplast photosystem Ⅱ. Using in situ hybridization, psbA, the gene encoding D1 protein of O. sativa ssp. japonica cv. 9516, and that of O. sativa ssp. indica cv. Shanyou 63 was cloned. As revealed by homology comparison of their sequences, the sequences are identical in the regions of promoter and 5′-UTR; differences are found in individual bases in the coding region all of which, being in the third position of respective codons, however, do not affect the amino acids coded finally; a difference is noted in the length of the oligo-U sequence in the region of 3′-UTR. It is thus apparent that, rather than a result of any difference in the amino acid sequences, the differences in the sensitivity to photoinhibition of D1 proteins between japonica and indica rice may be related to the upstream factors that regulate expression of psbA or to differences of photoprotective mechanisms.
基金supported by University natural science foundation of Jiangsu Province(16KJB550001)Postdoctoral research funding project of Jiangsu Province(1601058A)key research and development plan of Zhenjiang city(NY2016020)
文摘Objective In the present study, we investigated the antioxidant and anti‐aging effects of Silybum marianum protein hydrolysate（SMPH） in D‐galactose‐treated mice. Methods D‐galactose（500 mg/kg body weight） was intraperitoneally injected daily for 7 weeks to accelerate aging, and SMPH（400, 800, 1,200 mg/kg body weight, respectively） was simultaneously administered orally. The antioxidant and anti‐aging effects of SMPH in the liver and brain were measured by biochemical assays. Transmission electron microscopy（TEM） was performed to study the ultrastructure of liver mitochondria. Results SMPH decreased triglyceride and cholesterol levels in the D‐galactose‐treated mice. It significantly elevated the activities of superoxide dismutase（SOD） and glutathione peroxidase（GSH‐Px）, and total antioxidant capacity（T‐AOC）, which were suppressed by D‐galactose. Monoamine oxidase（MAO） and malondialdehyde（MDA） levels as well as the concentrations of caspase‐3 and 8‐OHd G in the liver and brain were significantly reduced by SMPH. Moreover, it increased Bcl‐2 levels in the liver and brain. Furthermore, SMPH significantly attenuated D‐galactose‐induced liver mitochondrial dysfunction by improving the activities of Na＋‐K＋‐ATPase and Ca2＋‐Mg2＋‐ATPase as well as mitochondrial membrane potential（ΔΨm） and fluidity. TEM showed that the degree of liver mitochondrial damage was significantly decreased by SMPH. Conclusion The results indicated that SMPH protects against D‐galactose‐induced accelerated aging in mice through its antioxidant and anti‐aging activities.
基金Supported by the National Natural Science Foundation of China,No.81570543 and No.81560104
文摘BACKGROUND Massive hepatocyte death is the core event in acute liver failure(ALF).Gasdermin D(GSDMD)-mediated pyroptosis is a type of highly inflammatory cell death.However,the role of hepatocyte pyroptosis and its mechanisms of expanding inflammatory responses in ALF are unclear.AIM To investigate the role and mechanisms of GSDMD-mediated hepatocyte pyroptosis through in vitro and in vivo experiments.METHODS The expression of pyroptosis pathway-associated proteins in liver tissues from ALF patients and a hepatocyte injury model was examined by Western blot.GSDMD short hairpin RNA(shRNA)was used to investigate the effects of downregulation of GSDMD on monocyte chemotactic protein 1(MCP1)and its receptor CC chemokine receptor-2(CCR2)in vitro.For in vivo experiments,we used GSDMD knockout mice to investigate the role and mechanism of GSDMD in a D-galactose/lipopolysaccharide(D-Galn/LPS)-induced ALF mouse model.RESULTS The levels of pyroptosis pathway-associated proteins in liver tissue from ALF patients and a hepatocyte injury model increased significantly.The level of GSDMD-N protein increased most obviously(P<0.001).In vitro,downregulation of GSDMD by shRNA decreased the cell inhibition rate and the levels of MCP1/CCR2 proteins(P<0.01).In vivo,GSDMD knockout dramatically eliminated inflammatory damage in the liver and improved the survival of DGaln/LPS-induced ALF mice(P<0.001).Unlike the mechanism of immune cell pyroptosis that involves releasing interleukin(IL)-1βand IL-18,GSDMDmediated hepatocyte pyroptosis recruited macrophages via MCP1/CCR2 to aggravate hepatocyte death.However,this pathological process was inhibited after knocking down GSDMD.CONCLUSION GSDMD-mediated hepatocyte pyroptosis plays an important role in the pathogenesis of ALF,recruiting macrophages to release inflammatory mediators by upregulating MCP1/CCR2 and leading to expansion of the inflammatory responses.GSDMD knockout can reduce hepatocyte death and inflammatory responses,thus alleviating ALF.
基金National Natural Science Foundation of China(No.81170825)
文摘AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial (HCE) cells Exposed to aspergillus fumigatus (AF) antigens. METHODS: HCE cells cultured 47 in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The Expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-kappa B and relative downstream cytokines such as TNF-alpha, IL-1 beta, IL-8 and IL-10 in supernatant fluid were measured by ELISA. RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-kappa B was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-alpha, IL-1 beta, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-alpha and IL-1 beta reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner. ' CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-kappa B pathway. SP-D possibly mediates the recognition to AF mycelium.
