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T Shirao

Publications and source records attributed to T Shirao.

51 records · Page 3Linked to original sources

Nucleotide sequences of two embryonic drebrins, developmentally regulated brain proteins, and developmental change in their mRNAs.

Drebrins are developmentally regulated proteins found in chicken brain and are classified into two forms of the embryonic type (E1 and E2) and one form of the adult type (A). Although the time courses of their appearance are different from each other, the structures of the 3 forms are closely related. Two kinds of drebrin cDNA, designated gDcw6 and gDcw17, were isolated from the cDNA library of the chicken embryo and their nucleotide sequences were determined. Their sequences were entirely identical except for a deletion of an internal 129-nucleotide sequence, and the gDcw17 insert contained an open reading frame capable of encoding 607 amino acids. These cDNAs seemed to correspond to two embryonic forms of drebrin mRNAs. The predicted drebrin molecules are highly hydrophilic and have proline-rich sequences and long stretches of glutamate in the carboxyl-terminal region. RNA dot-blot analysis using the drebrin cDNA as a probe demonstrated that the amounts of drebrin mRNAs were also developmentally regulated as those of drebrins. Southern blot analysis showed that the chicken genome has a single copy of the drebrin gene per haploid complement. These findings suggest that the multiple forms of drebrins result from alternative splicing of the single drebrin gene during neural development.

Amino Acid Sequence↗

Localization of a developmentally regulated neuron-specific protein S54 in dendrites as revealed by immunoelectron microscopy.

We sought to determine the ultrastructural localization of the developmentally regulated neuron-specific protein S54 in the chicken cerebellar cortex and optic tectum. The brains were fixed by perfusion with paraformaldehyde and glutaraldehyde. Frozen sections were immunocytochemically labeled with a monoclonal antibody to S54 protein. The immunoreactivity for S54 protein was localized in dendrites. No immunoreactivity for S54 protein was detected in axons and their presynaptic terminals.

Animals↗

Four synaptic vesicle-specific proteins: identification by monoclonal antibodies and distribution in the nervous tissue and the adrenal medulla.

Synaptic vesicles from the guinea-pig cerebrum were isolated and administered to mice for the production of monoclonal antibodies (MAb). Four vesicle-associated proteins in the guinea-pig nervous tissue were specifically and differentially recognized by MAbs thus obtained. These proteins had molecular weights of 30,000, 36,000, 38,000 and 65,000 Da and were named SVPs (synaptic vesicle proteins) 30, 36, 38 and 65, respectively. Immunohistochemistry demonstrated that all SVPs were localized in the synaptic regions throughout the central nervous system and in the adrenal medulla. Nerve terminals in skeletal muscle, smooth muscle and sympathetic ganglion contained SVPs 36 and 38. Immunoelectron microscopy of the cerebellar cortex confirmed the localization of SVPs in the synaptic vesicles and the adjacent membranes of the presynaptic nerve terminals. Fractionation of the cerebral tissue and treatment with various agents showed that SVPs were localized in the synaptic vesicles and the synaptic plasma membrane and that SVPs were integrated within the membrane and liberated only after solubilization of the membrane.

Adrenal Medulla↗

Molecular events associated with neural development.

Analyses of the chick embryo using two-dimensional gel electrophoresis or immunohistochemistry with monoclonal antibodies (MAbs) revealed several molecules which were highly concentrated in the nervous tissue. The properties and developmental changes of these substances were investigated. Three acidic proteins (molecular weights 95, 100 and 110 kDa, respectively) appeared characteristically during the embryonic development. One of them predominated alternatively at each developmental stage. They showed a high degree of homology in the peptide map and complete cross-reactivity with any specific antibodies. Therefore these proteins were considered to make a protein family and were collectively named drebrins. MAbs were produced against the membrane fraction of the neural tubes and somites of three-day chick embryos. Five MAbs stained the migrating neural crest cells and their progeny in histochemistry. In addition, the otic vesicle, including the differentiated labyrinth, and subtypes of human lymphocytes were recognized by these MAbs.

Animals↗

Identification of a synaptic vesicle-specific 38,000-dalton protein by monoclonal antibodies.

Synaptic vesicles were purified from the guinea pig cerebrum by sucrose density gradient centrifugation, and monoclonal antibodies (MAbs) were produced against this vesicle fraction. Seven MAbs (171B5, 171E8, 174D12, 174H11, 177A2, 177H11 and 178D4) recognized a novel acidic protein of about 38,000 daltons which was specific to synaptic vesicles. In immunofluorescence microscopy, the staining pattern of these MAbs corresponded to the distribution of the synapses in the guinea pig central nervous system. These MAbs appeared to stain all synaptic regions, irrespective of their synaptic function or type of neurotransmitters. MAb 171B5 and 174H11 stained the rat, rabbit and bovine synapses similarly to the guinea pig. Two other MAbs (171E8 and 177H11) stained other mammals weakly but the remaining 3 MAbs reacted only with the guinea pig. In immunoelectron microscopy of both the cerebellar tissue and isolated vesicle fraction, these MAbs selectively labeled the synaptic vesicles but not other structures. Immunoblot analysis was performed on electrophoretically separated proteins in vesicle fraction and brain homogenate. All of 7 MAbs reacted with a band at a molecular weight of about 38,000 from the guinea pig. Isoelectric focussing disclosed that this protein was acidic (pI 4.5-5).

Animals↗

Immunochemical homology of 3 developmentally regulated brain proteins and their developmental change in neuronal distribution.

Proteins S5, S6, and S54 (mol. wts. 95,000, 100,000, and 110,000 Da) appear characteristically at certain developmental stages in the chick brain (Shirao, T. and Obata, K., J. Neurochem., 44 (1985) 1210-1216). In the present study polyclonal and monoclonal antibodies were developed against electrophoretically purified S5 and S6 proteins. Each polyclonal and monoclonal antibody specifically recognized all 3 proteins, S5, S6, and S54, by immunoblot analysis. The tissue specificities of these proteins were examined by immunoblot analysis with these antibodies. Proteins S5 and S6 were found in the neural tissue and in some non-neural tissues of chick embryo. In the adult chicken, however, they were detected neither in neural nor in non-neural tissues with the exception of the spinal ganglion. Protein S54, on the other hand, was found both in late embryonic and adult neural tissues. It was detected neither in embryonic nor in adult non-neural tissues. Immunohistochemical analysis of adult nervous system showed that S54 protein was present only in neurons. Therefore it is concluded that S54 protein is a neuron-specific protein. Developmental changes of localization of these proteins were then examined by immunohistochemistry. In the developing brain, immunostaining was first observed in newly differentiated neurons, later becoming localized in the neuronal processes. In the adult brain, the immunoreactivity was mainly localized in certain types of synaptic regions, but it was also observed in a small population of neuronal somata.

Animals↗

Two acidic proteins associated with brain development in chick embryo.

The developmental changes in protein composition of the chick optic tectum were analyzed by two-dimensional gel electrophoresis. Staining with Coomassie Brilliant Blue R revealed 54 major proteins, eight of which remarkably changed their abundance during development: Four of these proteins (S8, S14, S30, and S54) increased and two of them (S7 and S37) decreased in the course of the brain development. The other two proteins (S5 and S6) appeared at specific embryonic stages and were not detected in the adult. The abundance of S5 protein was highest at day 7, and that of S6 protein at days 9-18. The two proteins were present in other regions of the embryonic brain but were not detected in the embryonic liver. The proteins were purified from the soluble fraction of embryonic chick brains by pH 5.5 precipitation, DEAE-Sepharose column chromatography, ammonium sulfate precipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of S5 and S6 proteins were 95,000 and 100,000, respectively, and their isoelectric points were about 4.5. They were compared by peptide mapping using V8 protease and found to share 11 common peptides out of 17 distinct ones. This indicates a strong degree of structural homology between these two proteins.

Animals↗

Prinzmetal's variant angina associated with subarachnoid hemorrhage: A case report.

Prinzmetal's variant of angina occurred in a 48-year-old man who sustained two attacks of subarachnoid hemorrhage within 10 days. The first anginal pain started at the same time that the second cerebrovascular accident developed, but subsequent anginal episodes were not accompanied by other symptoms or signs that indicated new development of subarachnoid hemorrhage. Twelve days later, when nuchal rigidity was fairly improved, the episodes of chest pain ended. A vasospasm of the large coronary arteries--probably due to the derangement of the autonomic nervous system caused by subarachnoid hemorrhage--was presumed to contribute to the occurrence of the variant angina. Based on this case and on review of the literature, we propose that coronary arterial spasm is one of several causes of the cardiac changes seen in subarachnoid hemorrhage.

Angina Pectoris↗