Biochemical Studies Of Neurons And Other Cell Types

血管活性肠肽（VIP）具有神经营养和促进生长的作用，是通过胶质源性物质介导的。本节的一个主要目标是确定这些vip调节发育的介质并研究它们的作用机制。先前的研究表明，VIP释放的保护蛋白之一是飞分子作用蛋白：活性依赖性神经营养因子（ADNF）。本节的一个持续的优先事项是在蛋白质和遗传水平上表征ADNF，并开发基于源自ADNF的肽的治疗剂。由于ADNF丰度低、ph敏感性不稳定、高聚集性、水溶性差和分子复杂性，研究ADNF尤其具有挑战性。以前，少量的ADNF是用四种色谱方法进行生物化学分离的。这种方法虽然对确定ADNF概念至关重要，但耗时且在分离过程中导致产品大量损失。基于针对adnf衍生肽的抗血清，一种亲和柱在过去一年中被开发出来，能够从vip刺激的星形胶质细胞培养的条件培养基中一步分离纯化的adnf样蛋白。这种替代程序显著增加了ADNF蛋白在结构和药理学研究中的可用性。虽然最初被认为是一个单一的14 kDa蛋白，但进一步的结构分析表明，ADNF是一个至少由三种成分组成的复合物。通过基质辅助激光解吸/电离（MALDI）和表面增强激光解吸/电离（SELDI）质谱/飞行时间分析获得了ADNF配合物的更多信息。这些技术确定ADNF复合物的分子量为14,590道尔顿。此外，ADNF的MALDI和SELDI光谱都显示出异常宽的峰分子范围，超过1000道尔顿，这一观察结果与ADNF是一组蛋白质或糖蛋白的结论一致，这些蛋白质或糖蛋白稳定地结合形成一个功能单元。进一步支持这一分子模型的是用N-CHO毛细管电泳分离ADNF的三个组分峰。从N-CHO柱分离的三个峰表现出独特的单调剂量反应，其效价差异很大。这些峰的生物活性被一组不同的抗ADNF肽产生的抗血清所抑制。基于其相对稳定性，ADNF复合物的组分2被选择用于进一步的MALDI研究，其分子质量为13,757道尔顿。因此，ADNF的MADLI谱的宽度可能归因于存在多个大小相似且紧密相关的成分。这些研究构成了我们ADNF工作模型的基础，并表明多基因产物或跨国修饰可能解释其结构。通过继续寻找ADNF成分的生物活性肽片段并努力获得更多针对色氨酸肽的抗体，进一步研究了ADNF的性质。这些研究表明，在发现的九种色氨酸消化肽中，有四种被发现对河豚毒素处理的大脑皮层培养物产生的凋亡性死亡具有神经保护作用。通过对神经元细胞计数和羧基荧光素双醋酸酯的测定，这四种多肽对神经保护作用有效。肽的大小从10-30个氨基酸不等。在现有的数据库中搜索后，这九种肽都是独一无二的，与任何已知的蛋白质都没有明显的同源性。此外，这些肽序列的反翻译没有发现任何数据库中核苷酸序列的显著特征或相似性。今年产生了三种新的抗体，并研究了它们与ADNF可能的相互作用。当用于治疗大脑皮层培养物时，与对照组相比，这三种抗体均使神经元存活率降低了20-35%。免疫前血清对神经元存活没有影响。此外，这三种新的抗血清都阻断了生化纯化ADNF的促生存活性，支持了抗血清与ADNF复合物重要成分相互作用的假设。这些新的抗血清将用于进一步尝试表达克隆ADNF复合物的成分。一种抗ADNF肽用于评估新生儿脑切片中ADNF的免疫反应性。这些研究表明，adnf样蛋白最密集的定位是脑干，特别是在三叉神经节、网状结构和臂旁核/面核的细胞中。在纤维中，adnf样蛋白在后脑中含量最高，在尾状壳核、嗅结节中含量显著，在大脑皮层和海马中含量较少。在相同条件下用免疫前血清进行的对照研究未显示免疫反应性。先前的研究表明，由ADNF和药理学相关蛋白（活性依赖性神经保护蛋白，ADNP）衍生的短肽在飞摩尔浓度下具有神经保护作用。在用谷氨酸、β -淀粉样肽、铁、过氧化氢和gp120 （HIV包膜蛋白）等临床相关毒素处理的培养物中观察到这些肽的保护作用。ADNF-9 （ADNF激动剂）和NAP （ADNP激动剂）的药理学已进一步探索。令人惊讶的是，ADNF-9和NAP在d -异构体和l -异构体中都具有充分的活性。这一观察结果最初是在对河豚毒素在大脑皮层培养物中产生的凋亡性死亡的研究中得出的。重要的是，D-ADNF-9和D-NAP联合治疗显著增加了肽表现出有效神经保护作用的浓度范围。d -异构体肽的研究扩展到预防胎儿酒精综合征模型中的胚胎死亡。这些研究表明，腹腔和口服给药D-ADNF-9都能有效预防产前酒精治疗引起的胎儿死亡。这些研究表明，这些胶质蛋白的肽通过非手性机制表现出保护特性。此外，这些肽的口服活性形式的发现增加了它们作为药物开发先导化合物的潜力。VIP的促生长作用在胚胎发生后早期表现明显。在这个发育阶段，VIP受体定位于神经管。体外培养的神经管被用作研究vip介导的基因表达调控和神经营养因子释放的模型系统。GEArray系统用于监测控制和vip刺激神经管中的信号转导通路和细胞因子。VIP处理后，gr1、BAX、p21、p57等cDNA表达量均上调。VIP治疗后，GCSF、IL-10、TGF-B1、tnf - α、tnf - β、p53等细胞因子下调。这一系列由VIP介导的变化表明，在这个发育的关键时期，VIP调控的细胞因子和通路基因的复杂性。PACAP-38是VIP肽家族的一种肽，在培养中发现对胚胎9.5天小鼠胚胎的生长有抑制作用。因此，VIP对生长的刺激作用并不是这类肽的一般特征。

Vasoactive intestinal peptide (VIP) has neurotrophic and growth-promoting actions that are mediated through glia-derived substances. A major goal of this section is to identify these mediators of VIP-regulated development and study their mechanism of action. Previous studies have indicated that one of the protective proteins released by VIP is a femtomolar-acting protein: activity dependent neurotrophic factor (ADNF). A continuing priority of this section is to characterize ADNF at the protein and genetic level and to develop therapeutic agents based on peptides derived from ADNF. The investigation of ADNF has been particularly challenging due to its low abundance, pH-sensitive instability, high aggregational properties, poor aqueous solubility and molecular complexity. Previously, small amounts of the ADNF were isolated biochemically with the use of four chromatographic procedures. This methodology, although essential in defining the ADNF concept, was time consuming and resulted in significant loss of product during isolation. Based on antiserum made against ADNF-derived peptides, an affinity column has been developed during the past year that is capable of isolating purified ADNF-like protein in a single step from conditioned medium obtained from VIP-stimulated astrocyte cultures. This alternative procedure has significantly increased the availability of ADNF protein for structural and pharmacological studies. Although initially thought to be a single 14 kDa protein, further structural analysis has indicated that ADNF exists as a complex of at least three components. Additional information on the ADNF complex has been obtained with MALDI (Matrix Assisted Laser Desorption/Ionization) and SELDI (Surface Enhanced Laser Desorption/Ionization) mass spectrometric/time of flight analyses. These techniques determined that the molecular weight of the ADNF complex was 14,590 Daltons. Furthermore, both the MALDI and SELDI spectra of ADNF exhibited an unusually broad peak molecular range in excess of 1000 Daltons, an observation consistent with the conclusion that ADNF is an array of proteins or glycoproteins that are stably associated to form a functional unit. Further support for this molecular model is the separation of three component peaks of ADNF by N-CHO capillary electrophoresis. The three peaks isolated from the N-CHO column exhibit unique monotonic dose responses differing widely in potency. The biological activity of these peaks is inhibited by a distinct group of the antisera generated against ADNF peptides. Based on its relative stability, component 2 of the ADNF complex was chosen for additional MALDI studies which demonstrated a molecular mass of 13,757 Daltons. Thus, the breadth of the MADLI spectrum for ADNF may be attributed to the existence of multiple components that are similar in size and tightly associated. These studies form the basis of our working model of ADNF and suggest the possibility that multiple gene products or post-transnational modifications may explain its structure. The nature of ADNF was further investigated with a continued search for biologically active peptide fragments of ADNF components and with an effort to obtain more antibodies directed against tryptic peptides. These studies revealed that of the nine tryptic digest peptides discovered, four were found to exhibit neuroprotection from apoptotic death produced in cerebral cortical cultures treated with tetrodotoxin. As measured by both neuronal cell counts and carboxy fluorescein diacetate, potent and efficacious neuroprotection were observed after treatment with these four peptides. The size of the peptides ranged from 10-30 amino acids. After searches in available data bases, all nine of these peptides appear unique with no apparent homology to any known protein. In addition, back translation of these peptide sequences did not reveal significant identities or similarities in nucleotide sequences within any database. Three new antibodies were generated this year and investigated for their possible interaction with ADNF. When used to treat cerebral cortical cultures, all three antibodies decreased neuronal survival by 20-35% in comparison to controls. The pre-immune sera had no effect on neuronal survival. In addition, all three new antisera blocked the survival-promoting activity of the biochemically purified ADNF, supporting the hypothesis that antisera interact with important components of the ADNF complex. These new antisera will be used in further attempts to expression clone the components of the ADNF complex. One anti-ADNF peptide was used to assess ADNF immunoreactivity in sections of the neonatal brain. These studies revealed that the most dense localization of ADNF-like protein was the brain stem, particularly in cells of the trigeminal ganglion, reticular formation and parabrachial/facial nuclei. In fibers, the greatest abundance of ADNF-like protein was found in the hindbrain, with significant amounts localized in the caudate putamen, olfactory tubercle, and to a lesser extent in the cerebral cortex and hippocampus. Control studies conducted under the same conditions with pre-immune serum did not reveal immunoreactivity. Previous studies have shown that short peptides derived from ADNF and a pharmacologically related protein (activity dependent neuroprotective protein, ADNP) exhibit neuroprotection at femtomolar concentrations. The protective action of these peptides is observed in cultures treated with clinically relevant toxins including glutamate, beta amyloid peptide, iron, hydrogen peroxide and gp120, the HIV envelope protein. The pharmacology of ADNF-9 (agonist to ADNF) and NAP (agonist to ADNP) has been further explored. Surprisingly, ADNF-9 and NAP were found to be fully active in the D-isomer form as well as in the L-isomer form. This observation was initially made in studies of apoptotic death produced by tetrodotoxin in cerebral cortical cultures. Importantly, treatment with the combination of D-ADNF-9 and D-NAP significantly increased the range of concentrations at which the peptides exhibited effective neuroprotection. The studies with D-isomer peptides were extended to the prevention of embryonic death in a model of fetal alcohol syndrome. These studies indicated that both intraperitoneally and orally administered D-ADNF-9 was efficacious in preventing fetal death produced by prenatal alcohol treatment. These studies indicate that the peptides from these glial proteins exhibit protective properties through non-chiral mechanisms. Furthermore, the discovery of orally active forms of these peptides increases their potential as lead compounds for drug development. The growth-promoting action of VIP is evident in the early postimplantation period of embryogenesis. VIP receptors are localized to the neural tube at this stage of development. Explanted neural tubes have been used as a model system to study VIP-mediated regulation of gene expression and neurotrophin release. The GEArray system was utilized to monitor signal transduction pathways and cytokines in control and VIP-stimulated neural tubes. The following cDNA's were up regulated after VIP treatment: GRO1, BAX, p21 and p57. A number of cytokines were down regulated after VIP treatment: GCSF, IL-10, TGF-B1, TNF-alpha, TNF-beta and p53. This constellation of changes mediated by VIP demonstrate the complexity of cytokine and pathway genes regulated by VIP at this critical period of development. PACAP-38, a peptide that belongs to the VIP family of peptides, was found to inhibit the growth of embryonic day 9.5 mouse embryos in culture. Thus the stimulatory action VIP on growth is not a general feature of this group of peptides.