Structure and Function of New Neurotransmitters in Brains in the Silkworm Bombyx Mori

1)在蚕的情况下，在母蛹阶段从食道下神经节分泌的一种内在滞育激素作用于卵巢和产卵的胚胎发育停滞。AnyParP（从天蚕体内分离得到的麻痹肽，命名为AnyParP）与该滞育激素具有相同的作用。即使AnyParP（23个残基，C-末端是羧酸）和滞育激素（24个残基，C-末端是酰胺）之间的氨基酸序列完全不同，两种肽在胚胎滞育中也可以引发相同的功能。2）本研究采用两步层析法从家蚕蛹脑提取物中分离出一种蛋白质，并鉴定了其N端氨基酸序列。根据该氨基酸序列，合成简并寡核苷酸，并将其作为杂交探针筛选蛹脑cDNA文库。序列分析表明，603 bp的开放阅读框编码一个201个氨基酸的蛋白质，含有一个15个氨基酸的前导肽。这种蛋白质与作为脂质运载蛋白家族成员的大蜡螟Gallerin具有高度相似性。我们将这种来自神经系统的新蛋白质命名为Escherichirin。免疫组化和Western blot分析表明，P2X_1蛋白仅在家蚕中枢神经系统（CNS）表达。3）利用抗哺乳动物P2X_1受体382-399肽段的P2X_1抗体，首次在昆虫中枢神经系统中发现了P2X_1样受体的免疫活性。在家蚕5龄幼虫和蛹的脑中分布有6对免疫反应胞体，它们与发育阶段和一种特异的P2X_1蛋白有关。

1) In the case of the silkworm, an intrinsic diapause hormone secreted from subesophageal ganglion in the maternal-pupal stage, acts on ovaries and embryonic development arrests in oviposited eggs. AnyParP (a paralytic peptide isolated from the wild silkmoth, Antheraea yamamai, named as AnyParP) had the same function with this diapause hormone. Even if the sequence of amino acids between AnyParP (23 residues and C-terminal is carboxylic acid) and diapause hormone (24 residues and C-terminal is amide) is entirely different, both peptides can elicit the same function in embryonic diapause. This is the first report of a functional mimicry of the silkworm diapasue hormone by an unrelated insect peptide2) This study isolated a protein from pupal brain extract of the silkworm, Bomby mori using two chromatographic steps, and identified its N-terminal amino acid sequence. Based on the amino acid sequence, degenerate oligodeoxynucleotide was synthesized and used as a hybridization probe to screen a pupal brain cDNA library. The sequence analysis of the identified cDNA clones indicated that a 603 bp open reading frame encodes a 201-amino-acid protein containing a 15-amino-acid leader peptide. This protein has high similarity to the Galleria mellonella Gallerin as a member of the lipocalin family. We named this novel protein from the Bombyx central nervous system as Bombyrin. The immunohistochemistry and Western blot analysis indicated that Bombyrin is only expressed in the central nervous system (CNS) of Bombyx mori.3) The immunoactivity of P2X_1-like receptor was first found in the central nervous system of insects, using P2X_1 antibody against the peptide of mammalian P2X_1 receptor 382-399. Six pairs of immunoreactive somata were distributed in brains of 5th instar larvae and pupae of Bombyx mori, and they showed a relationship between developmental stages and a specific P2 X_1-like protein.