Molecular biological research in the differentiation of osteoclast

1.中国仓鼠卵巢细胞表达人组织蛋白酶E单体突变体的生化性质为了解二聚体形成的生理意义，采用定点突变的方法构建了人组织蛋白酶E单体突变体，并在中国仓鼠卵巢细胞中表达。对突变蛋白的免疫定位表明，单体CE主要与内质网和非溶酶体内吞细胞器有关。该单体蛋白主要以46 kDa的Pro-CE形式产生，细胞内含有高甘露糖苷型低聚糖链。相反，二聚体Pro-CE在pH为7的条件下几乎不被激活。单体形式比这些二聚体形式对pH 7和温度变化更不稳定。这些结果表明CE的二聚化不一定是表达活性、正确的细胞内定位和…的正确折叠所必需的更多的碳水化合物修饰，但它可能是必不可少的结构稳定的分子结构在体内。组织蛋白酶D在调节骨吸收的成骨细胞中的免疫细胞化学显示组织蛋白酶D是一种溶酶体天冬氨酸蛋白酶，用免疫金标法对其免疫细胞化学定位进行了研究。当成骨细胞的突起与破骨前细胞部分接触时，在靠近破骨前细胞的整个胞浆突起的溶酶体样结构(溶酶体/内体)内可见组织蛋白酶D阳性颗粒。然而，在活跃的破骨细胞中，成骨细胞中组织蛋白酶D标记的溶酶体样结构散布在细胞质中，阳性结构不集中在过程中。这些结果表明，组织蛋白酶D通过成骨细胞与破骨细胞之间的相互作用参与破骨细胞性骨吸收的过程。较少

1. Biochemical properties of the monomeric mutant of human cathepsin E expressed in chinese hamster ovary cellsTo understand the physiological siginificance of the dimer formation, the monomeric mutant of human Cathepsin E (CE) was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Immunolocalization of the mutant protein at both the light and electron microscopic levels revealed the monomeric CE to be associated predominantly with the endoplasmic reticulum and the non-lysosomal endocytic organelles. The monomeric protein was generated primarily as the 46-kDa pro-CE with a high-mannosetype oligosaccharide chain in the cells. In contrast, the dimeric pro-CE was scarcely activated by treatment at pH 7.The monomeric form was more unstable to pH7 and temperature changes than these dimeric forms. These results indicate that the dimerization of the CE is not necessarily required for proper folding to express activity, correct intracellular localization and … More carbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo.2. Immunocytochemical demonstration of cathepsin D in the osteoblastic cells modulating bone resorptionImmunocytochemical localization of cathepsin D,a lysosomal aspartic proteinase, was investigated using immunogold methods. When the process in the osteoblastic cells partially contacted with the preosteoclasts, cathepsin D-positive particles were found within lysosome-like structures (lysosomes/endosomes) in the process of whole cytoplasm which is located close to the preosteclasts. However, in the case of active osteoclasts, cathepsin D-labeling lysosome-like structures in the osteoblastic cells were scattered within the cytoplasm.The positive structures were not concentrated within the process. This localization pattern was clearly different from that of the osteoblastic cells located in contact with preosteoclasts.These results suggest that cathepsin D is involved in the proceeding of osteoclastic bone resorption through the interaction between osteoblastic cells and osteoclasts. Less