BIOCHEMICAL STUDIES OF NEURONS AND OTHER CELL TYPES

血管活性肠肽（VIP）的神经营养和生长刺激作用是通过分泌的胶质源性物质间接介导的。我们的重点是表征这些vip介质并研究它们的作用机制。活动依赖性神经营养因子（ADNF）是vip神经保护作用的主要胶质介质。先前的研究表明，ADNF在飞摩尔浓度下具有神经保护作用，并且与热休克蛋白60 （hsp60）具有结构相似性。然而，我们最近的研究表明，在与河豚毒素共同处理的大脑皮层培养物中，在很宽的浓度范围内测试的ADNF制剂显示出两个促进生存活性的峰值。这些活动的ec50值相差10万倍。虽然5种色谱技术均未发现ADNF的异质性，但通过毛细管电泳（CE）分析纯化的ADNF显示出两个峰，每个峰都显示出促进生存的活性，其中只有一个峰被抗hsp60血清阻断。胰蛋白酶消化ADNF后获得氨基酸序列数据。总共测序了15.8 kDa，没有一个肽与任何已知序列匹配。当与大脑皮层培养物孵育时，针对其中一种消化肽产生的抗血清产生神经元细胞死亡，并阻断ADNF的促生存活性和CE分离的非hsp60峰。这些数据表明ADNF比以前认识到的更复杂，可能由两个活性成分组成，一个与hsp60共享一个表位，另一个是一个独特的蛋白质/亚基。为了进一步探索vip相关蛋白的保护作用，在胎儿酒精综合征模型中，使用与ADNF相关的肽和从相关蛋白（活性依赖性神经保护蛋白）中获得的另一肽来预防胚胎死亡和生长限制。此外，多肽的保护作用即使在给予酒精一小时后仍然有效。VIP在神经管闭合时对小鼠胚胎生长有显著影响。为了进一步研究VIP刺激生长的介质，我们将胚胎9.5天小鼠的离体神经管与VIP和条件培养基（CM）一起孵育。经CE分析，CM中检测到35个峰。在这个复杂的混合物中，鉴定出两种细胞因子：白细胞介素-1和白细胞介素-6。这些细胞因子已被证明在发育中的中枢神经系统中具有增殖和神经营养作用。- VIP， PACAP，星形细胞，细胞因子，应激蛋白，神经保护，神经管，胎儿酒精综合征，营养因子

The neurotrophic and growth-stimulating actions of vasoactive intestinal peptide (VIP) are mediated indirectly through secreted, glia-derived substances. Our focus is to characterize these VIP-mediators and study their mechanism of action. A principal glial mediator of VIPs neuroprotective actions is activity dependent neurotrophic factor (ADNF). Previous studies indicated that ADNF exhibited neuroprotection at femtomolar concentrations and had a structural similarity to heat shock protein 60 (hsp60). However, our recent studies indicate that ADNF preparations tested over a very broad range of concentrations exhibited two peaks of survival-promoting activity when tested in cerebral cortical cultures co-treated with tetrodotoxin. The EC50s of these activities differed by 100,000-fold. Although no ADNF heterogeneity was evident with five chromatographic techniques, analysis of purified ADNF by capillary electrophoresis (CE) demonstrated two peaks, each exhibiting survival-promoting activity, only one of which was blocked by antiserum to hsp60. Amino acid sequence data was obtained after trypsin digestion of ADNF. A total of 15.8 kDa was sequenced, with none of the peptides matching any known sequence. Antisera generated against one of the digest peptides produced neuronal cell death when incubated with cerebral cortical cultures and blocked the survival-promoting activity of ADNF and the non-hsp60 peak isolated by CE. These data suggest that ADNF is more complex than previously recognized and may consist of two active components, one sharing an epitope with hsp60, the other being a unique protein/subunit. To further explore the protective actions of VIP-related proteins, a peptide associated with ADNF and another peptide obtained from a related protein (activity dependent neuroprotective protein) were used to prevent embryonic death and growth restriction in a model of fetal alcohol syndrome. Furthermore, the protective actions of the peptides were effective even one hour after the administration of alcohol. VIP has a dramatic effect on mouse embryonic growth at the time of neural tube closure. In further investigations of the mediators of VIP-stimulated growth, isolated neural tube from embryonic day 9.5 mice were incubated with VIP and the conditioned medium (CM) studied. As analyzed by CE, 35 peaks were detected in CM. In this complex mixture, two cytokines were identified: interleukin-1 and interleukin-6. These cytokines have been shown to have proliferative and neurotrophic roles in the developing CNS. - VIP, PACAP, astrocytes, cytokines, stress proteins, neuroprotection, neural tube, fetal alcohol syndrome, trophic factors