| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1987: ¥100,000 (Direct Cost: ¥100,000)
Fiscal Year 1986: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Glutamine synthetase(GS) is know to be a key enzyme in a small glutamate compartment in the brain that is engaged in the metabolism of putative neurotransmitters, <gamma>-aminobutyric acid, and glutamic acid. We studied the isolation of human brain GS which formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000 The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of <gamma>-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than in the sheep enzyme. Antibodies were raised in rabbit against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated form human, sheep, and rat brains, but the enzymes were
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notimmunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytesof the liver.
As a part of inquiry into the pathogenesis of Hirschsprung's disease, immunocytochemical investigations were carried out on surgical specimens from normal human guts, ganglionic and aganglionic portions Hirschsprung's guts using three glial markers; GFAP, S-100 and GS. In control human gut, GFAP, S-100 and GS immunostaining disclosed intensely immunoreactive supporting cells in the myenteric and submucosal plexuses. The supporting cells of the interconnecting nerve fiber bundles of the plexuses and fine nerve strands in the muscular layer were also immunostained with these markers. Extrinsic nerve fibers in the subserosa of the control gut and coarse, mostly aberrantly proliferated, nerve fiber bundles in the aganglionic portion of Hirschsprung's disease were also immunoreactive for GFAP and S-100 but not for GS. Thus, GS immunocytochemistry seemed to be a useful tool for the distinction of extrinsic and intrinsic neural components in the ENS and may provide an important clue for histopathological diagnosis of Hirschsprung's disease. Less
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