Studies of a synthetic substrate in canine globoid cell leukodystrophy

Gal et al. ((1977) Clin. Chim. Acta 77, 53–59) reported the use of a new synthetic substrate, 2-hexadecanoylamino-4-nitrophenyl-β-D-galactopyranoside for the diagnosis of human globoid cell leukodystrophy. Assay of β-galactosidase in brain homogenates from normal, carrier, and globoid cell leukodystrophy-affected dogs utilizing this new substrate demonstrated overlapping activities. Instead of reflecting specific $\text{D}\text{-galactosyl}\text{-N-}\text{acylsphingosine}$ galactohydrolase (EC 3.2.1.46), the 2-hexadecanoylamino-4-nitrophenyl-β-D-galactopyranoside β-galactosidase activity in canine brain is highly correlated with nonspecific 4-methylumbelliferyl β-galactosidase. Optimization of the 2-hexadecanoylamino-4-nitrophenyl-β-D-galactopyranoside assay system for canine brain and the use of varying concentrations of taurocholate or taurodeoxycholate in the assay mixture did not alter the lack of specificity. These results indicate a significant difference in the nature of the underlying defect in galactosylceramide β-galactosidase in canine globoid cell leukodystrophy compared to human globoid cell leukodystrophy.     

A humanin analog decreases oxidative stress and preserves mitochondrial integrity in cardiac myoblasts

A potent analog (HNG) of the endogenous peptide humanin protects against myocardial ischemia–reperfusion (MI–R) injury in vivo, decreasing infarct size and improving cardiac function. Since oxidative stress contributes to the damage from MI–R we tested the hypotheses that: (1) HNG offers cardioprotection through activation of antioxidant defense mechanisms leading to preservation of mitochondrial structure and that, (2) the activity of either of a pair of non-receptor tyrosine kinases, c-Abl and Arg is required for this protection. Rat cardiac myoblasts (H₉C₂ cells) were exposed to nanomolar concentrations of HNG and to hydrogen peroxide (H₂O₂). Cells treated with HNG in the presence of H₂O₂ demonstrated reduced intracellular reactive oxygen species (ROS), preserved mitochondrial membrane potential, ATP levels and mitochondrial structure. HNG induced activation of catalase and glutathione peroxidase (GPx) within 5 min and decreased the ratio of oxidized to reduced glutathione within 30 min. siRNA knockdown of both Abl and Arg, but neither alone, abolished the HNG-mediated reduction of ROS in myoblasts exposed to H₂O₂. These findings demonstrate an HNG-mediated, Abl- and Arg-dependent, rapid and sustained activation of critical cellular defense systems and attenuation of oxidative stress, providing mechanistic insights into the observed HNG-mediated cardioprotection in vivo.     

新型神经保护肽[Gly14]-Humanin的表达，纯化和活性鉴定

Humanin（HN）是近年来在人体内发现的内源性多肽,能够保护神经细胞免于各种阿尔采默病病相关因素诱导的凋亡,HN肽链第14位氨基酸被Gly取代的突变体[Gly14]-Humanin（HNG）,神经保护活性比HN高近1 000倍。但由于HNG天然获取的途径有限,限制了对其功能的进一步研究。本课题组构建了HNG的表达质粒pET32a/HNG,并将其转化大肠杆菌BL21 trxB (DE3),诱导表达后HNG以可溶性融合蛋白的形式出现并用镍离子亲和层析纯化,纯化的融合蛋白用肠激酶切割后经反相层析得到纯化的HNG多肽（23 mg每1 L细菌培养液）,重组HNG多肽的分子量经电喷雾电离质谱（ESI-MS）检测为2876.5道尔顿,其N末端8个氨基酸残基的序列经Edman降解法测定为A-M-M-A-P-R-G-F,均与理论值一致。神经细胞保护实验表明,重组HNG多肽的神经保护作用与天然的HNG相近