A role of a novel brain-derived proteinase inhibitor in the apoptosis in neurons

免疫组织化学研究表明，从牛脑中纯化和克隆的丝氨酸蛋白酶抑制剂B-43存在于牛脑中的中到大的神经元中，特别是在脑干和脊髓中。B-43免疫反应阳性神经元包括脑桥和延髓的运动核、脊髓前部的运动神经元、大脑皮质的锥体细胞、海马CA 3区的锥体细胞和纹状体的大神经元。β-43也定位于GFAP阳性星形胶质细胞。用[35 S]-甲硫氨酸标记的重组B-43与凝血酶、胰蛋白酶、神经生长因子和激肽释放酶形成SDS稳定的复合物。GST-B-43融合蛋白浓度依赖性地抑制凝血酶、胰蛋白酶和α-糜蛋白酶的酰胺水解活性，但不抑制弹性蛋白酶的酰胺水解活性。这些结果表明凝血酶和胰蛋白酶样丝氨酸蛋白酶是B-43的首选靶蛋白酶。克隆了牛B-43的小鼠同源物（P1-6）。北方印迹分析显示，与成人大脑其他区域相比，脑干和间脑中Pl-6 mRNA的表达水平较高，全脑中Pl-6 mRNA的表达逐渐增加，直至出生后11天，成人大脑中Pl-6 mRNA的表达再次下降。原位杂交结果显示，该mRNA定位于生后海马锥体细胞层，尤其是CA 3区和大脑皮质V层。在脑干中，面神经核和三叉神经运动核为Pl-6 mRNA强阳性表达，双侧颈总动脉结扎引起的脑缺血导致Pl-6 mRNA表达增加，同时伴有海马锥体细胞变性。这些结果表明PI-6/B-43参与了神经元的成熟和变性及再生过程。

Immunohistochemical studies revealed that a serine proteinase inhibitor B-43 purified and cloned from bovine brain was present in the neurons medium to large in size in the bovine brain, especially in the brain stem and the spinal cord. The B-43-immunoreactive neurons contained motor nuclei in the pons and medulla oblongata, motor neurons in the anterior spinal cord, pyramidal cells in the cerebral cortex, pyramidal cells in the hippocampus CA 3 regions and large neurons in the striatum. beta-43 was also localized in GFAP-positive astrocytes. Recombinant B-43 labeled with [35S]-methionine formed SDS-stable complexes with thrombin, trypsin, nerve growth factor and kallikrein. GST-B-43 fusion protein concentration-dependently inhibited the amidolytic activity of thrombin, trypsin and alpha-chymotrypsin but not elastase, These results suggested that thrombin and trypsin-like serine proteinase were the preferred target proteinases of B-43. The mouse homolog (P1-6) of bovine B-43 was cloned. Northern blot analysis showed a high level of expression of Pl-S mRNA in brain stem and diencephalon as compared with other regions in the adult brain, The expression of Pl-6 mRNA in the Whole brain was increased gradually until 11 days after birth and was decreased again in the adult brain. In situ hybridization analysis revealed that the mRNA was localized in pyramidal cell layer of the post-natal hippocampus, especially in CA 3 region, and layer V of the cerebral cortex. In the brain stem, the facial nucleus and the motor nucleus of trigeminal nerve were strongly positive for Pl-6 mRNA.Brain ischemia induced by bilateral ligation of the common carotid artery led to an increase in Pl-6 mRNA expression, accompanied by the degeneration of hippocampal pyramidal cells. These results suggested the involvement of PI-6/B-43 in the maturation of neurons and degenerative and regenerative processes.