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Neurotransmitter analysis of clonal cell lines from Drosophila melanogaster larval CNS
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NEURONAL EXPRESSION OF mRNA FOR RAT 14-3-3 ETA CHAIN POLYPEPTIDE IN THE CENTRAL AND P E R I P H E R A L NERVOUS SYSTEM. M A S A H I K O WATANABE, AND HISATAKE KONDO, Department of Anatomyt Tohoku U n i v e r s i t y School of Medicine, 2-I Seiryou-cho, Aoba-ku, Sendai 980, Japan. To examine w h e t h e r the physiological function of 14-3-3 protein is confined to the a c t i v a t i o n of tyrosine- and t r y p t o p h a n - h y d r o x y l a s e s in the presence of calciu m / c a l m o d u l i n - p r o t e i n kinase II as reported previously, cDNA encoding rat 14-3-3 eta subunit was isolated from the rat brain, and using the cloned cDNA as a specific h y b r i d i z a t i o n probe the e x p r e s s i o n of the mRNA in the nervous system was a n a l y s e d in relation to monoamine neurons. The cloned cDNA coded 246 amino acids (28kDa) whose sequence was c o m p l e t e l y identical to the bovine counterpart. By Northern blot analysis, an e x t r a o r d i n a r i l y high level of the mRNA expression was detected in the brain as compared with non-nervous tissues. By in situ hybridization histochemistry, the expression of mRNA for this protein was detected not only in the m o n o a m i n e - s y n t h e t i c neurons but also in many other discrete nuclei and ganglia without any relation to the synthesis of c a t e c h o l a m i n e or serotonin. The highly conservative structure of this p r o t e i n b e t w e e n the two m a m m a l i a m species and its wider expression than expected in the central and peripheral nervous system suggest that the 14-3-3 protein exerts some, though yet to be defined, functions fundamental to neuronal a c t i v i t i e s other than the a c t i v a t i o n of m o n o a m i n e biosynthesis.
NEUROTRANSMITTER ANALYSIS O F CLONAL CELL LINES FROM D R O S O P H I L A M E L A N O G A S T E R LARVAL CNS. K U M I K O UI *I, MASAFUMI SAKUMA *I. YUKO W A T A N A B E ~I . SHOKO N I S H I H A R A ~2. SHIN TOGASH1.2, TADASHI M I Y A K E ~2, AND YUHEI M I Y A T A l, iDeDartment of Pharmacolo~,' Nippon Medlcal School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, D e p a r t m e n t of Molecular Biology, Mitsubishi Kasei Institute of Life Sciences, ii M i n a m i o o y a t ~achida-shi, Tokyo 194, Japan. We have e s t a b l i s h e d ii clonal cell lines from D r o s o p h i l a m e l a n o g a s t e r larval central nervous system(CNS). These clonal cell lines show r e a c t i v i t y to an antibody against HRP, a specific neuronal m a r k e r in insects. In this study, w e attempted to c h a r a c t e r i z e the phenotype of n e u r o t r a n s m i t t e r s in these clones. Among ii clonal cell lines, 6 and 7 lines c o n t a i n e d acetylcholine(ACh) and L-DOPA, respectively. Four of them had both substances. However, dopamine, noradrenaline, adrenaline, s e r o t o n i n and o c t o p a m i n e c o u l d not be d e t e c t e d in any cell lines. It was found that 2 0 - h y d r o x y e c d y s o n e ( 2 0 - H O E ) , the insect m o l t i n g hormone, changed the t r a n s m i t t e r phenotype in some cell lines. That is, L-DOPA, which was contained normally, could not be d e t e c t e d in some cell lines after treatment with 20-HOE. Furthermore, 20-HOE i n d u c e d a decrease of a s p a r t i c acid and an increase of arginine. The latter amino acid was r e c e n t l y found to act as a precursor of a new type of neuronal messenger, nitric oxide. These changes are e x p e c t e d to occur in vivo d u r i n g metamorphosis.
PURIFICATION AND CHARACTERIZATION OF 150 kDa H-ANCHORED PROTEIN WITH HNK-1 EPITOPE IN RAT BRAIN. YOSHIHIR0 YOSHIHARA, KEN~AKU MORI, SHOG0 OKA, YASUYOSHI WATANABE. Department of Neuros¢ience. Osaka Bioscience Institute. 6-2-4 Furu~dai, Suita-shi. Osaka 565. Japan, Complicated but highly-ordered neuronal networks are organized during development according to the expression of molecules in time- and locus-specific manners. Cell surface adhesion and recognition molecules play critical roles in a variety of developmental events. HNK-1 carbohydrate antigen is a common epitope expressed on such molecules involved in cell-cell interactions in the nervous system. In the present study, we found and purified a novel phosphatidylinositol (PI)anchored glycoprotein belonging to HNK-1 family in rat brain. The molecule (PI-GP150) was detected by combination of PI-specific phospholipase C treatment of brain membranes and Western blot analysis with monoclonal antibody HNK-1. The expression of HNK-1 epitope on PI-GP150 was developmentally and spatially regulated. In newborn rats, HNK-l-epitope was expressed on PI-GP150 throughout the brain. The level of HNK-l-positive PI-GP150, however, decreases after postnatal 7 days only in hindbrain, and becomes completely absent in adult myelencephalon and metencephalon, while HNK-1 epltope on PI-GP150 was constitutively expressed in telencephalon. This developmental change resulted in formation of rostro-caudal gradient of HNK-1 expression on PI-GP150 in adult brain.
NEURONAL EXPRESSION OF mRNA FOR RAT 14-3-3 ETA CHAIN POLYPEPTIDE IN THE CENTRAL AND P E R I P H E R A L NERVOUS SYSTEM. M A S A H I K O WATANABE, AND HISATAKE KONDO, Department of Anatomyt Tohoku U n i v e r s i t y School of Medicine, 2-I Seiryou-cho, Aoba-ku, Sendai 980, Japan. To examine w h e t h e r the physiological function of 14-3-3 protein is confined to the a c t i v a t i o n of tyrosine- and t r y p t o p h a n - h y d r o x y l a s e s in the presence of calciu m / c a l m o d u l i n - p r o t e i n kinase II as reported previously, cDNA encoding rat 14-3-3 eta subunit was isolated from the rat brain, and using the cloned cDNA as a specific h y b r i d i z a t i o n probe the e x p r e s s i o n of the mRNA in the nervous system was a n a l y s e d in relation to monoamine neurons. The cloned cDNA coded 246 amino acids (28kDa) whose sequence was c o m p l e t e l y identical to the bovine counterpart. By Northern blot analysis, an e x t r a o r d i n a r i l y high level of the mRNA expression was detected in the brain as compared with non-nervous tissues. By in situ hybridization histochemistry, the expression of mRNA for this protein was detected not only in the m o n o a m i n e - s y n t h e t i c neurons but also in many other discrete nuclei and ganglia without any relation to the synthesis of c a t e c h o l a m i n e or serotonin. The highly conservative structure of this p r o t e i n b e t w e e n the two m a m m a l i a m species and its wider expression than expected in the central and peripheral nervous system suggest that the 14-3-3 protein exerts some, though yet to be defined, functions fundamental to neuronal a c t i v i t i e s other than the a c t i v a t i o n of m o n o a m i n e biosynthesis.
NEUROTRANSMITTER ANALYSIS O F CLONAL CELL LINES FROM D R O S O P H I L A M E L A N O G A S T E R LARVAL CNS. K U M I K O UI *I, MASAFUMI SAKUMA *I. YUKO W A T A N A B E ~I . SHOKO N I S H I H A R A ~2. SHIN TOGASH1.2, TADASHI M I Y A K E ~2, AND YUHEI M I Y A T A l, iDeDartment of Pharmacolo~,' Nippon Medlcal School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113, D e p a r t m e n t of Molecular Biology, Mitsubishi Kasei Institute of Life Sciences, ii M i n a m i o o y a t ~achida-shi, Tokyo 194, Japan. We have e s t a b l i s h e d ii clonal cell lines from D r o s o p h i l a m e l a n o g a s t e r larval central nervous system(CNS). These clonal cell lines show r e a c t i v i t y to an antibody against HRP, a specific neuronal m a r k e r in insects. In this study, w e attempted to c h a r a c t e r i z e the phenotype of n e u r o t r a n s m i t t e r s in these clones. Among ii clonal cell lines, 6 and 7 lines c o n t a i n e d acetylcholine(ACh) and L-DOPA, respectively. Four of them had both substances. However, dopamine, noradrenaline, adrenaline, s e r o t o n i n and o c t o p a m i n e c o u l d not be d e t e c t e d in any cell lines. It was found that 2 0 - h y d r o x y e c d y s o n e ( 2 0 - H O E ) , the insect m o l t i n g hormone, changed the t r a n s m i t t e r phenotype in some cell lines. That is, L-DOPA, which was contained normally, could not be d e t e c t e d in some cell lines after treatment with 20-HOE. Furthermore, 20-HOE i n d u c e d a decrease of a s p a r t i c acid and an increase of arginine. The latter amino acid was r e c e n t l y found to act as a precursor of a new type of neuronal messenger, nitric oxide. These changes are e x p e c t e d to occur in vivo d u r i n g metamorphosis.
PURIFICATION AND CHARACTERIZATION OF 150 kDa H-ANCHORED PROTEIN WITH HNK-1 EPITOPE IN RAT BRAIN. YOSHIHIR0 YOSHIHARA, KEN~AKU MORI, SHOG0 OKA, YASUYOSHI WATANABE. Department of Neuros¢ience. Osaka Bioscience Institute. 6-2-4 Furu~dai, Suita-shi. Osaka 565. Japan, Complicated but highly-ordered neuronal networks are organized during development according to the expression of molecules in time- and locus-specific manners. Cell surface adhesion and recognition molecules play critical roles in a variety of developmental events. HNK-1 carbohydrate antigen is a common epitope expressed on such molecules involved in cell-cell interactions in the nervous system. In the present study, we found and purified a novel phosphatidylinositol (PI)anchored glycoprotein belonging to HNK-1 family in rat brain. The molecule (PI-GP150) was detected by combination of PI-specific phospholipase C treatment of brain membranes and Western blot analysis with monoclonal antibody HNK-1. The expression of HNK-1 epitope on PI-GP150 was developmentally and spatially regulated. In newborn rats, HNK-l-epitope was expressed on PI-GP150 throughout the brain. The level of HNK-l-positive PI-GP150, however, decreases after postnatal 7 days only in hindbrain, and becomes completely absent in adult myelencephalon and metencephalon, while HNK-1 epltope on PI-GP150 was constitutively expressed in telencephalon. This developmental change resulted in formation of rostro-caudal gradient of HNK-1 expression on PI-GP150 in adult brain.