Masahiko Watanabe

Gene cloning, sequence, expression and in situ localization of 80 kDa diacylglycerol kinase specific to oligodendrocyte of rat brain

A 3.1 kbp cDNA clone encoding diacylglycerol (DG) kinase of 80 kDa (80K-DG kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence was 82% homologous to previously identified porcine 80K-DG kinase and contained zinc finger-like sequences, E-F hand motifs and ATP-binding sites similar to the porcine counterpart. By in situ hybridization histochemistry of rat brain at postnatal week 3, the expression signals for 80K-DG kinase mRNA appeared predominantly on somata of discrete cells in the white matter, and the expression pattern was similar to that of the myelin-specific proteins. In immunohistochemistry using the antibody against bacterially expressed DG kinase-fusion protein, numerous fibrous or dot-like structures exhibiting the immunoreactivity were concentrated in the white matter and they were arranged to radiate in the cerebral cortex and the cerebellar granular layer in a pattern almost identical to that of oligodendrocytes. No neuronal cells exhibited the immunoreactivity. The present finding thus strongly suggests that 80K-DG kinase is expressed specifically in the oligodendrocytes, but not neurons, and may be involved in the myelin formation and metabolism. In addition, the intense hybridization signals and the immunoreactivity for this protein were detected in the entire medulla of the thymus and the periarterial lymphatic area of the splenic white pulp both of which represent T-cell-dependent areas.

Molecular cloning of rat cDNAs for β and γ subtypes of 14-3-3 protein and developmental changes in expression of their mRNAs in the nervous system

We isolated cDNAs to beta and gamma subtypes of 14-3-3 protein, a putative regulatory protein for protein kinase C, from the brain and clarified a high homology in sequences of nucleotides and deduced amino acids between the two rat subtypes and the bovine counterparts and even reciprocally between the two rat subtypes. In Northern blot analysis, the gene expression of the two subtypes was detected weakly at E13, increased progressively after birth and reached a maximum at P7-P14. Thereafter it decreased slightly. In situ hybridization analysis allowed detection of the beta but not the gamma subtype in the matrix cells of the ventricular germinal zone of the neural wall. In post-mitotic neurons in the mantle zone and maturing brain loci, genes of the two subtypes were expressed in patterns similar to each other, and three neuron types were identified: type I neurons with high levels of expression throughout development; type II neurons showing high expression during the early developmental stages with a subsequent decrease in the expression at maturing and adult stages; and type III neurons showing consistently low levels of expression throughout development. The wider and more highly-patterned expression of the 14-3-3 protein family than expected suggests that this protein may be involved in the elaborate regulation of some fundamental cellular activities and differentiation of neurons.

Molecular cloning of cDNA to rat 14-3-3 η chain polypeptide and the neuronal expression of the mRNA in the central nervous system

Abstract Activation of tyrosine and tryptophan hydroxylases, key enzymes for the catecholamine and serotonin biosynthesis, requires Ca2+/calmodulin-dependent protein kinase II and 14-3-3 protein which comprises a family of, at least, seven polypeptides in the bovine. Here we show that the amino acid sequence of the rat 14-3-3 η chain deduced from the nucleotide sequence is completely identical to that of bovine counterpart. Using in situ hybridization the expression of mRNA for this protein is detected not only in the monoamine-synthetic neurons but also in many other discrete nuclei which synthesize neither catecholamine nor serotonin. The highly conservative structure between mammalian species and wider expression of this protein than expected in the central nervous system suggest that the 14-3-3 protein exerts some, though yet to be defined, functions fundamental to neuronal activities other than activation of the monoamine biosynthesis.

Gene expression of Ca2+/calmodulin-dependent protein kinase of the cerebellar granule cell type or type IV in the mature and developing rat brain

The localization and ontogenic changes of expression of the mRNA for Ca2+/calmodulin-dependent protein kinase of the cerebellar granule cell type or type IV (CaM kinase Gr or IV) in the rat brain were examined by in situ hybridization histochemistry. At the young adult stage, intense expression signals for this kinase mRNA were detected in the cerebellar granule cells, the hippocampal pyramidal cells, the dentate granule cells, and the piriform cortex. Moderate levels of the mRNA were expressed in the thalamic nuclei and the cerebral cortex. No distinct expression signals were detected in the Purkinje cells and most brainstem nuclei except for the pontine nuclei, locus ceruleus and inferior olive which showed weak expression. During development, two chronological patterns of changes in the gene expression for this kinase were discerned. The first was a high and persistent expression from the developing stages till the adult stage, which was observed in the cerebellar granule cells, the hippocampal pyramidal cells and the dentate granule cells. The other was a transiently high expression during limited developmental periods, which was observed in the Purkinje cells, neurons in the inferior olive, various brain stem nuclei, and the subventricular neuronal cells. These findings suggest that this Ca2+/calmodulin-dependent protein kinase is involved differentially in multiple Ca2+ signaling pathways in different developing and mature neurons.

Developmental regulation of neuronal expression for the η subtype of the 14-3-3 protein, a putative regulatory protein for protein kinase C

An in situ hybridization technique was applied to rat nervous tissues, to analyse the developmental changes in expression for the eta subtype mRNA of 14-3-3 protein, a putative regulatory protein for protein kinase C. Although signal levels of the eta subtype mRNA were low in mitotic cells in the ventricular zone, most neurons displayed a marked increase at their definitive location in the mantle zone. In general, neurons in the spinal ventral horn and peripheral ganglia showed this increase at E13-E15, those in the telencephalon, diencephalon, midbrain, pons and medulla oblongata at E18-P1, and the cerebellar Purkinje cells at P7-P14. It is at these developmental stages when neuronal differentiation including axonal and dendritic growth and ramification occurs actively. Subsequently high levels of the eta subtype mRNA were maintained until the adult stage in projection type neurons possessing larger cell bodies and highly developed dendritic fields, such as the olfactory mitral cells, hippocampal pyramidal cells, cerebellar Purkinje cells, and motor neurons in the brainstem and spinal cord. However, the signal levels decreased until the adult stage in smaller projection type neurons. On the other hand, the signal levels in local circuit type neurons were consistently low throughout development. These findings suggest that gene expression for the eta subtype mRNA of the 14-3-3 protein is regulated in close relation to both neuron types and their cytodifferentiation.

Localization of gene expression of calbindin in the brain of adult rats

Localization of gene expression of calbindin, a cytosolic calcium-binding protein, was examined throughout the adult rat brain by in situ hybridization with cDNA probes. The gene was expressed most intensely in the Purkinje cells in the cerebellum, intensely in the granule cells of the dentate gyrus, and moderately in the inferior olivary nucleus, in the nuclei of the trapezoid body, in the medial part of the lateral habenular nuclei, entorhinal cortex and in the mammillary nuclei. In addition, weak expression of the gene was widespread in the forebrain and brainstem gray matter, and also in small cells in the spinal posterior horn as well as the ependymal cells. The widespread and heterogeneous expression of the gene in the brain suggests that calbindin is differentially involved in calcium-regulated phenomena in different neurons.

Developmental Changes in Expression of a Calcium-binding Protein (Spot 35-calbindin) in the Nervus Terminalis and the Vomeronasal and Olfactory Receptor Cells

The detailed localization of spot 35-calbindin and its ontogenic change were studied in Nervus terminalis, the vomeronasal organ and the olfactory epithelium of the rat by immunohistochemistry. At the embryonic days 12 and 13 (E 12-13), calbindin-immunoreactive cells were found in the outermost layer of the presumptive olfactory bulb and within the olfactory placode. At E 14 to the postnatal day 1 (P 1), intense calbindin-immunoreactivity was localized in ganglionated fiber bundles of Nervus terminalis coursing through the mesenchymal spaces on both sides of the nasal septum. Nervus terminalis decreased the immunoreactivity abruptly after P 1 and it showed no distinct immunoreactivity for calbindin at P 7 and thereafter. On the other hand, numerous receptor cells in the olfactory epithelium and the thicker vomeronasal epithelium exhibited weak to moderate immunoreactivity for calbindin at E 18-P 1. Their immunoreactivity decreased in intensity progressively after P 7 and no distinct immunoreactivity for calbindin was detected in most of the receptor cells, whereas moderate immunoreactivity was detected in most of the vomeronasal and olfactory nerves at P 28 and P 63.

Localization of gene expression of calreticulin in the brain of adult mouse.

The localization of gene expression of calreticulin, a calcium-binding protein in the endoplasmic reticulum, was examined throughout the entire brain of adult mice by in situ hybridization. Calreticulin mRNA is expressed widely and heterogeneously in discrete neurons throughout the brain, but the white matters expressed it weekly or faintly. In the olfactory bulb, the mRNA is expresses moderately in the mitral cells, but weakly in the periglomerular cells and internal granule cells. In the cerebrum, the gene is expressed intensely in the piriform cortex, but weakly in neocortex, the entorhinal cortex and the amygdaloid nuclei. In the hippocampal formation, calreticulin mRNA is expressed intensely in the CA1-CA3 regions but less intensely in the granule cells of the dentate gyrus. The caudate-putamen, thalamic and hypothalamic nuclei, and mammillary nuclei express the mRNA weakly or faintly. In the mesencephalon, pons and medulla, moderate expression of the mRNA is detected in the pontine nuclei and the locus ceruleus. Weak expression of the mRNA is detected in several discrete nuclei and zones such as the substantia nigra, the superior colliculus and the central gray. Expression signals of calreticulin mRNA are faint in the inferior olive. In the cerebellum, calreticulin mRNA is expressed moderately in the Purkinje cells whereas no significant expression is detected in the granule cells. The plexus choroideus of the lateral, third and fourth ventriculi express calreticulin mRNA intensely although no distinct expression of the mRNA is discerned in the ependyma.

The effect of methamphetamine on the mRNA level for 14·3·3 ⥈ chain in the human cultured cells

Abstract14·3·3 protein, a brain-specific protein, is an activator of tyrosine and tryptophan hydroxylases, key enzymes for biosynthesis of dopamine and serotonin. In this article, we describe cloning of cDNA for human brain 14·3·3 ν chain and expression of 14·3·3 ν chain mRNA in some human cultured cells. The cloned cDNA is 1730 bp long and contains 191 bp of a 5′-noncoding region, the complete 738 bp of coding region, and 801 bp of a 3′-noncoding region, containing three polyadenylation signals. This cDNA encoded a polypeptide of 246 amino acids (M, 28,196). Furthermore, usingin situ hybridization histochemistry, the expression of mRNA for this protein was examined in the rat central nervous system.In situ hybridization histochemistry indicated that 14·3·3 ⥈ chain mRNA is detected not only in the monoamine-synthetic neurons, but also in other neurons in the discrete nuclei, which synthesize neither cathecholamine nor serotonin. Northern blot analysis demonstrated that the addition of methamphetamine into the cultured medium increased the mRNA level for 14·3·3 ⥈ chain in U-251 cells, but did not increase that of GFAP.

Partial down-regulation at post-transcriptional level of the gene expression for preproenkephalin in the superior cervical ganglion of the maturing rat

In rats at the postnatal week 3 (P3W), enkephalin-immunoreactivity was detected in subsets of principal ganglion cells in the superior cervical ganglion and nerve fibers within the submandibular gland, one of the targets for the ganglion, whereas it disappeared from them at P8W. Enkephalin-immunoreactive ganglion cells and intraglandular fibers were detected again after the colchicine-pretreatment at P8W. By in situ hybridization the population density of ganglion cells expressing mRNA for preproenkephalin and the expression intensity were similar in both ganglia at P3W and P8W. These findings suggest that the post-transcriptional down-regulation of gene expression for preproenkephalin is involved in the disappearance of enkephalin-immunoreactivity in the adult ganglion.