Fumiyo Takeda

Molecular cloning and characterization of a novel developmentally regulated gene, Bdm1, showing predominant expression in postnatal rat brain.

Postnatal development, such as synapse refinement, is necessary for the establishment of a mature and functional central nervous system (CNS). Using differential display analysis, we identified a novel gene, termed Bdm1, that is more abundantly expressed in the adult brain than in the embryonic brain. The full-length Bdm1 cDNA is 2718 base pairs long and contains an open reading frame of 1059 base pairs encoding a 38-kDa protein. Northern blot analysis revealed that expression of Bdm1 mRNA in the brain was weak on embryonic days and increased in the early postnatal period. Bdm1 mRNA was significantly expressed in the brain and heart, but there was no or little expression in other tissues. During the differentiation of mouse carcinoma cells P19 to neuron-like cells by retinoic acid, Bdm1 mRNA was up-regulated almost parallel to neurofilament mRNA. Expression of Bdm1 mRNA was observed appreciably in PC12 cells after neuronal differentiation but not in the nonneural cell lines examined. In situ hybridization demonstrated that Bdm1 was expressed widely in the olfactory bulb, cerebral cortex, hippocampus, cerebellum, thalamus, and medulla oblongata. Taken together, these data suggest that Bdm1 gene plays a role in the early postnatal development and function of neuronal cells.

Divalent cation, ATP-dependent [3H]Leu-enkephalin uptake by synaptic vesicle fraction isolated from bovine caudate nucleus

The mechanism of [3H]Leu-enkephalin uptake into synaptic vesicle fraction from bovine caudate nucleus was investigated. The simultaneous addition of 2 mM MgCl2, 2 mM CaCl2, and 2 mM ATP stimulated the uptake 22 times over the control, whereas the separate addition of these agents augmented the uptake 2.4 times at most. The addition of 2 mM MGCl2 plus 2 mM ATP and of 2 mM CaCl2 plus 2 mM ATP increased the uptake 6.6 times and 4.5 times, respectively. The cation, ATP-dependent uptake reached its half-maximal level within 10 min after the initiation of incubation at 25 degrees C, and little Leu-enkephalin was taken up at 0 degree C. The apparent Km for the uptake of [3H]Leu-enkephalin was 1.8 x 10(-7) M. Guanosine triphosphate stimulated the uptake as well as ATP, whereas CTP and ITP were only one-fourth effective of ATP. The cation, ATP-dependent uptake was inhibited by 25% and 20% in the presence of 0.1 mM colchicine and 1 microM reserpine, respectively.

Identification of the novel developmentally regulated gene, Bdm2, which is highly expressed in fetal rat brain.

Most of the neurogenesis take place during the embryonic stage; the genes expressed predominantly in this stage may play important roles in the control of development of the central nervous system. Using a differential display method, we identified the novel rat gene, brain development-related molecule 2 (Bdm2), that is expressed more abundantly in the embryonic brain than in the adult brain. Full-length Bdm2 cDNA consists of 1842 base pairs (bp) and contains an open reading frame of 1260 bp. Northern blot analysis demonstrated that Bdm2 was strongly expressed in the late embryonic brain and was still detected at lower levels in an early postnatal period; in adults, Bdm2 mRNA was decreased to an undetectable level in brain, though the expression of this mRNA was revealed in other tissues. Level of Bdm2 mRNA was maintained during neuronal differentiation of mouse embryonal carcinoma cell P19, but decreased during the differentiation to glial and unidentified non-neuronal cells. In situ hybridization study demonstrated the wide distribution of Bdm2 mRNA in the embryonic brain; in the adult brain, the hybridization signals became more restricted to the hippocampus, olfactory bulb, cerebellum, and neocortex, almost coinciding with the regions where nascent and immature neurons are present. Thus, it appears likely that Bdm2 encodes a protein that is involved in both the regulation of growth of undifferentiated neural cells and the terminal differentiation of neuronal cells.

Distribution and co-existence of Met-enkephalin-like and mesotocin-like immunoreactivity in the neural lobe of the pituitary of the frog

Content and distribution of Met-enkephalin (Met-ENK)-like immunoreactivity in the hypothalamo-hypophysial system of bovine, rat and frog were examined using specific radioimmunoassay and immunohistochemistry. Ultrastructural localization and co-existence of Met-ENK-, mesotocin (MT)- and vasotocin (VT)-like immunoreactivity in the neural lobe of the frog pituitary was examined by a method combining pre-embedding peroxidase-antiperoxidase immunostaining for Met-ENK with post-embedding immunocolloidal gold staining for MT or VT. The highest concentrations of immunoassayable Met-ENK were present in the neural lobe of the pituitary of the frog. In addition to nerve fibers showing only MT-like or Met-ENK-like immunoreactivity, nerve fibers containing neurosecretory granules showing both MT- and Met-ENK-like immunoreactivities were very rich. But VT-like and Met-ENK-like immunoreactivity was confirmed separately in different axon terminals.