The hypothalamic Arg-Phe-amide-related peptides, gonadotropin-inhibitory hormone and orthologous mammalian peptides of Arg-Phe-amide, may be important regulators of the hypothalamus-pituitary-gonadal reproductive axis...The hypothalamic Arg-Phe-amide-related peptides, gonadotropin-inhibitory hormone and orthologous mammalian peptides of Arg-Phe-amide, may be important regulators of the hypothalamus-pituitary-gonadal reproductive axis. These peptides may modulate the effects of kisspeptins because they are presently recognized as the most potent activators of the hypothalamus-pituitary-gonadal axis. However, their effects on gonadotropin-releasing hormone neurons have not been investigated. In the current study, the GT1-7 cell line-expressing gonadotropin-releasing hormone was used as a model to explore the effects of Arg-Phe- amide-related peptides on kisspeptin activation. Intracellular calcium concentration was quantified using the calcium-sensitive dye, fura-2 acetoxymethyl ester. Gonadotropin-releasing hormone released into the medium was detected via enzyme-linked immunosorbent assay. Results showed that 100 nmol/L kisspeptin-10 significantly increased gonadotropin-releasing hormone levels （at 120 minutes of exposure） and intracellular calcium concentrations. Co-treatment of kisspeptin with 1 μmol/L gonadotropin-inhibitory hormone or 1 μmol/L Arg-Phe-amide-related peptide-1 significantly attenuated levels of kisspeptin-induced gonadotropin-releasing hormone but did not affect kisspeptin-induced elevations of intracellular calcium concentration. Overall, the results suggest that gonadotropin-inhibitory hormone and Arg-Phe-amide-related peptide-1 may have inhibitory effects on kisspeptin-activated gonadotropin-releasing hormone neurons independent of the calcium signaling pathway.展开更多
自组装的纳米纤维肽作为一种新型材料，被广泛应用于细胞的三维培养以及组织缺损的修复。本研究在传统自组装多肽RADA16—1（RAD16）的基础上，为成骨细胞体外培养构建了一种支架材料，即把细胞黏附基序arginine—glycine—aspartic（RG...自组装的纳米纤维肽作为一种新型材料，被广泛应用于细胞的三维培养以及组织缺损的修复。本研究在传统自组装多肽RADA16—1（RAD16）的基础上，为成骨细胞体外培养构建了一种支架材料，即把细胞黏附基序arginine—glycine—aspartic（RGD）连接到RAD16上，再与纯RAD16按比例混合，构成本实验的研究材料Hybrid。用原子力显微镜（atomic force microscope，AFM）观察材料的微观结构。通过相差显微镜、MTT、组织化学染色等工具或方法观察成骨细胞（MC3T3-E1）在材料中的生长、增殖和分化情况。结果显示，Hybrid可以形成良好的纳米纤维结构。与RAD16相比，这种改进的纳米材料可以促进细胞的增殖，且碱性磷酸酶（alkaline phosphatase，ALP）的表达呈强阳性，有明显的钙结节形成。实验表明该材料在骨组织工程方面有潜在的应用价值。展开更多
目的:对重组Arg^(34)-胰高血糖素样肽-1(Arg^(34)-Glucagon-like Peptide-1[7-37],Arg^(34)-GLP-1[7-37])生产制备过程中的潜在杂质进行分离纯化和结构确证。方法:将发酵后的料液进行高效液相色谱分析检测,利用离子交换色谱和制备液相...目的:对重组Arg^(34)-胰高血糖素样肽-1(Arg^(34)-Glucagon-like Peptide-1[7-37],Arg^(34)-GLP-1[7-37])生产制备过程中的潜在杂质进行分离纯化和结构确证。方法:将发酵后的料液进行高效液相色谱分析检测,利用离子交换色谱和制备液相纯化得到Arg^(34)-GLP-1[7-37]纯品和杂质组分,使用纯品进行高温、酸碱等不同条件下的破坏实验,来探知杂质产生机理,利用MALDI-TOF质谱、蛋白质全序列测序等方式推断杂质的分子量与结构。结果:经阴离子交换色谱及反相制备纯化后,Arg^(34)-GLP-1[7-37]的纯度可达97.9%。分离出3种主要杂质并将其纯化至91.4%,99.2%和96.7%。基质辅助激光解吸电离飞行时间质谱(Matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF)测试其[M+H]+分子量分别为1033.5446,2744.2397和3112.4475,通过破坏实验及Arg^(34)-GLP-1[7-37]的氨基酸序列来推测杂质1、杂质3与杂质9可能序列分别为E-F-I-A-W-L-V-R,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-R。将纯化前后的成分进行比对,表明上述3个主要杂质是由发酵过程产生,通过极端条件处理这些杂质,其含量会显著增加。本研究解析的3个杂质结构及机理探究均为首次发表。结论:由于重组Arg^(34)-GLP-1[7-37]在高温、过酸过碱及氧化环境中不稳定,为防止纯化过程中主产物降解,应注意避免这些条件。本研究为胰高血糖素样肽-1(glucagon-like peptide1,GLP-1)及其类似物的研究指出了一些应避免的误区。展开更多
基金supported by a grant from TUBITAK,project No.110S381
文摘The hypothalamic Arg-Phe-amide-related peptides, gonadotropin-inhibitory hormone and orthologous mammalian peptides of Arg-Phe-amide, may be important regulators of the hypothalamus-pituitary-gonadal reproductive axis. These peptides may modulate the effects of kisspeptins because they are presently recognized as the most potent activators of the hypothalamus-pituitary-gonadal axis. However, their effects on gonadotropin-releasing hormone neurons have not been investigated. In the current study, the GT1-7 cell line-expressing gonadotropin-releasing hormone was used as a model to explore the effects of Arg-Phe- amide-related peptides on kisspeptin activation. Intracellular calcium concentration was quantified using the calcium-sensitive dye, fura-2 acetoxymethyl ester. Gonadotropin-releasing hormone released into the medium was detected via enzyme-linked immunosorbent assay. Results showed that 100 nmol/L kisspeptin-10 significantly increased gonadotropin-releasing hormone levels （at 120 minutes of exposure） and intracellular calcium concentrations. Co-treatment of kisspeptin with 1 μmol/L gonadotropin-inhibitory hormone or 1 μmol/L Arg-Phe-amide-related peptide-1 significantly attenuated levels of kisspeptin-induced gonadotropin-releasing hormone but did not affect kisspeptin-induced elevations of intracellular calcium concentration. Overall, the results suggest that gonadotropin-inhibitory hormone and Arg-Phe-amide-related peptide-1 may have inhibitory effects on kisspeptin-activated gonadotropin-releasing hormone neurons independent of the calcium signaling pathway.
文摘自组装的纳米纤维肽作为一种新型材料，被广泛应用于细胞的三维培养以及组织缺损的修复。本研究在传统自组装多肽RADA16—1（RAD16）的基础上，为成骨细胞体外培养构建了一种支架材料，即把细胞黏附基序arginine—glycine—aspartic（RGD）连接到RAD16上，再与纯RAD16按比例混合，构成本实验的研究材料Hybrid。用原子力显微镜（atomic force microscope，AFM）观察材料的微观结构。通过相差显微镜、MTT、组织化学染色等工具或方法观察成骨细胞（MC3T3-E1）在材料中的生长、增殖和分化情况。结果显示，Hybrid可以形成良好的纳米纤维结构。与RAD16相比，这种改进的纳米材料可以促进细胞的增殖，且碱性磷酸酶（alkaline phosphatase，ALP）的表达呈强阳性，有明显的钙结节形成。实验表明该材料在骨组织工程方面有潜在的应用价值。
文摘目的:对重组Arg^(34)-胰高血糖素样肽-1(Arg^(34)-Glucagon-like Peptide-1[7-37],Arg^(34)-GLP-1[7-37])生产制备过程中的潜在杂质进行分离纯化和结构确证。方法:将发酵后的料液进行高效液相色谱分析检测,利用离子交换色谱和制备液相纯化得到Arg^(34)-GLP-1[7-37]纯品和杂质组分,使用纯品进行高温、酸碱等不同条件下的破坏实验,来探知杂质产生机理,利用MALDI-TOF质谱、蛋白质全序列测序等方式推断杂质的分子量与结构。结果:经阴离子交换色谱及反相制备纯化后,Arg^(34)-GLP-1[7-37]的纯度可达97.9%。分离出3种主要杂质并将其纯化至91.4%,99.2%和96.7%。基质辅助激光解吸电离飞行时间质谱(Matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF)测试其[M+H]+分子量分别为1033.5446,2744.2397和3112.4475,通过破坏实验及Arg^(34)-GLP-1[7-37]的氨基酸序列来推测杂质1、杂质3与杂质9可能序列分别为E-F-I-A-W-L-V-R,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-R。将纯化前后的成分进行比对,表明上述3个主要杂质是由发酵过程产生,通过极端条件处理这些杂质,其含量会显著增加。本研究解析的3个杂质结构及机理探究均为首次发表。结论:由于重组Arg^(34)-GLP-1[7-37]在高温、过酸过碱及氧化环境中不稳定,为防止纯化过程中主产物降解,应注意避免这些条件。本研究为胰高血糖素样肽-1(glucagon-like peptide1,GLP-1)及其类似物的研究指出了一些应避免的误区。
