Cell Biological Approach to the Elucidation of Intercellular Messenger Molecules

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The localizations and changes of mRNAs of the precursors and synthesizing enzymes for peptides, amines, and steroids were semiquantitatively investigated by using an in situ hybridization histochemistry. In addition, their translated proteins from mRNAs were analyzed on tissue sections with the aid of a modified immunocytochemistry in conjunction with tritiated antibodies. In the first year of this project, we tried to focus on the development and establishment of technical matters, including the standardization of parameters for the quantification of mRNAs and their proteins on the sections. The mRNAs and proteins used in this project were the precursors of vasopressin, oxytocin, CRF, enkephalin, CGRP, NPY and MAP2, and enzymes of tyrosine hydroxylase, tryptophan hydroxylase and choline acetyltransferase, and receptor proteins of serotonin, estrogen, progesterone, testosterone and glucocorticoids. Probes for these materials were designed and made by computer-assisted DNA synthesizer t … More o minimize the homologous sequence to their similar mRNAs.In the second year we carried out experiments which include physiological and pharmacological manipulations such as salt loading, dehydration, ovariectomy, castration, steroid treatment, colchicine injiction and reserpine treatment. Under such conditions, each mRNA in neurons of the hypothalamic nuclei and in endocrine cells of adrenal gland was investigated on its changes. At the same time immunocytochemical changes were examined on the manipulated tissue in comparison with the control. It seems to us that these messengers and proteins are good indicator for the intracellular mechanisms of the gene expression, production, transport, and release of bioactive molecules. Furthermore posterior pituitaries, spinal cords, midbrain regions and skins were implanted into the third ventricle of the brain or anterior eye chamber to elucidate the specificity of cellular target recognition. One of our results showed specific reinnervation pattern of several kinds of neurons to the original target areas. Less