A radioimmunoassay has been developed for measuring protein I, a basic, neuron-specific protein associated with nerve terminals. The procedure utilizes the detergents NaDodSO4 and Nonidet P40 to prevent nonspecific adsorption of this highly charged protein to various surfaces. By use of this procedure, it has been possible to show that protein I comprises approximately 0.4% of'the total protein in-cerebral cortex of several mammalian species. In addition, the amount of protein I was determined in about 40 regions of cat brain. The results suggest that measurement ofprotein I may provide a quantitative method for estimating the density of nerve terminals in various regions of the mammalian nervous system. Protein I is a neuron-specific phosphoprotein (1, 2) that is concentrated in nerve terminals in apparent association with synaptic vesicles (3-5). Recent studies from this laboratory indicate that-protein I may play an important role in neuronal function (2). The phosphorylation ofthis protein is regulated not only by cyclic AMP-dependent and Ca2"-dependent protein kinases in vitro (2, 6-8) but also by neurotransmitters (9, 10) and depolarizing agents (9-11) in intact slices ofnervous tissue. Although the precise physiological role of protein I remains unclear, it seems likely that it is important in some aspect ofthe functioning of synaptic vesicles (2).Many facets ofthe study ofprotein I require a rapid and sensitive method for its quantitation, and one of the most sensitive and specific methods for quantitation of the amount of proteins is radioimmunoassay (RIA). However, protein I has a number of properties that make the use of traditional RIAs impossible. Because it is a strongly basic protein (pI 10.4) (1), it is highly charged at neutral pH and adsorbs nonspecifically to a variety of surfaces (e.g., other proteins, tubes, and pipette tips). Furthermore, protein I is difficult to extract from certain tissues; for example, 1% NaDodSO4 is required to solubilize protein I from rabbit superior cervical ganglion (unpublished data). In addition, protein I represents a very small percentage of the total protein in some tissues; for example, in the adrenal medulla it constitutes 0.001% of the total protein and, thus, must be assayed in the presence oflarge amounts of extraneous material to which it adsorbs. It seemed possible that the use ofdetergents in a RIA might alleviate these problems. Studies (10,12,13) have shown that antigen-antibody complexes are not disrupted by the presence of nonionic detergents. In this paper we describe a detergent-based, competitive, nonsolid phase RIA in which NaDodSO4 and Nonidet P-40 (NP-40) are used to solubilize protein I and to minimize its nonspecific interactions. We have used this assay to quantitate the amount ofprotein I present in the cerebral cortex of various mammalian species and to determine the regional distribution of protein I-in cat brain. MATERIALS AND METHODSMaterials. [y-32P]ATP (5-10 X 107 cpm/nmol) was prepared by the method of Glynn and Chappell (...
The regional and cellular distribution of gua- Considerable evidence now suggests that guanosine 3',5'-cyclic monophosphate (cGMP) may play a role in neuronal function (1-3) and that many effects of this nucleotide are mediated by activation of cGMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) (4). However, the distribution of this enzyme in neuronal tissue has not been closely examined. Cerebellum is the only neuronal tissue that has been reported to contain a high concentration of cGMP-dependent protein kinase (5-7) and of a substrate for this enzyme (8). In addition, the activity of cGMP-dependent protein kinase has been reported to be reduced in the cerebellum of several strains of mutant mice deficient in Purkinje cells (9). In the present study, we have determined the concentration of this enzyme in various regions of brain. We have also studied the cellular localization of cGMP-dependent protein kinase and a substrate (8) animals of the appropriate strain. PCD animals were killed at 3 months of age, nervous and weaver at 2-2.5 months, and staggerer at 21-28 days. Frozen cerebella of X-irradiated rats were a generous gift of Hermes Yeh.Preparation of Tissue Extracts for Photoaffinity Labeling. Adult cats, weighing 4-4.5 kg, were anesthesized with pentobarbital at 30 mg/kg intravenously and ventilated through a tracheotomy tube after administration of Flaxedil at 3 mg/kg. Various locations in the brain were biopsied and each specimen was immediately immersed in 10 ml of ice-cold "homogenization buffer" containing 10 mM Hepes (pH 7.0), 5 mM 2-mercaptoethanol, 1 mM EDTA, 30,M phenylmethylsulfonyl fluoride, 2% (vol/vol) ethanol, and 0.25-M sucrose. Mice were killed by cervical dislocation and individual cerebella were placed on ice in homogenization buffer. Brain microvessels were prepared from whole rabbit brain by the technique of Nathanson and Glaser (18) and suspended in homogenization buffer. All subsequent steps were carried out at 4°C. The tissue was homogenized by hand in 4-6 vol of homogenization buffer, and the homogenate was centrifuged for 45 min at 150,000 X g. The resulting cytosol was used immediately for photoaffinity labeling or pretreated by a 10-min incubation at 30°C with 50 ,ug of beef heart phosphodiesterase (Boehringer Mannheim) Abbreviations: cGMP, guanosine 3',5'-cyclic monophosphate; cAMP, adenosine 3',5'-cyclic monophosphate; 8-N3-cIMP, 8-azidoinosine 3',5'-cyclic monophosphate; PCD, Purkinje cell degeneration mouse strain; R-I and R-II, regulatory subunits of the type I and type II cyclic AMP-dependent protein kinases, respectively; Dal, dalton; 23-kDal G-substrate, 23,000-dalton substrate for cyclic GMP-dependent protein kinase. 5537The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" intaccordance with 18 U. S. C. §1734 solely to indicate this fact.
To evaluate the effect on neurological outcome of spinal core compression persisting after a closed injury, the authors reviewed 44 of 62 consecutively managed cases of cervical spinal cord and spine injuries at C3-7, inclusive. Decompression within 48 hours of injury confirmed by myelography or open reduction. Neurological status, graded numerically on a spinal trauma scale at admission and at follow-up review (an average of 1 year +/- 2 months after admission), and precent recovery of neurological deficit were compared to canal narrowing (22 severe, greater than or equal to 30%, versus 22 moderate, 11% to 29%; or mild, less than or equal to 10%) and to delay before treatment (30 within 8 hours of injury versus 14 treated 9 to 48 hours after injury). Severe narrowing was equated with compression. Status at admission and at follow-up review was positively correlated. Patients with admission scores of less than 2 recovered a mean of 15% of their deficit, while those with scores more than 2 recovered a mean of 77%. Admission status correlated significantly with spinal canal narrowing but not with vertebral body displacement. Time of treatment had no significant effect upon admission status and percent recovery. No significant difference in the percent of recovery was noted, whether decompression was early (up to 8 hours) or late (9 to 48 hours) after injury. Surgery did not significantly alter the percent of recovery. The findings indicate that the initial injury to the cervical spinal cord and spine remains the primary determinant of neurological outcome. Severe canal narrowing with cord compression thereafter appears to have comparatively little effect. The conclusion that decompression is without effect is not possible without comparison with a group of patients whose spinal canals remained narrowed at follow-up review.
Five consecutively admitted patients with aneurysmal subarachnoid hemorrhage were treated with an indwelling lumbar spinal catheter. Daily samples of cerebrospinal fluid were analyzed for erythrocyte, protein, glucose, dopamine, epinephrine, serotonin, 5-hydroxyindoleacetic acid, tryptophan, histamine, thromboxane, 6-ketoprostaglandin F1 alpha, prostaglandin E, and prostaglandin F2 alpha concentrations. The patients' neurologic grade on admission, hospital course, presence of vasospasm, level of consciousness, computed tomographic and angiographic findings, and outcome were compared with the concentrations of the above substances in the cerebrospinal fluid. All patients had elevated concentrations of serotonin, with the highest levels found early in the hospital course of the patients who developed vasospasm. Tryptophan content increased markedly in association with clinical and angiographic vasospasm. Concentrations of prostaglandin F2 alpha correlated highly with development of and fluctuations in clinical vasospasm, with angiographic findings, with neurologic grade on admission, and with outcome. Our results suggest that prostaglandin F2 alpha may be involved in delayed clinical vasospasm in patients with subarachnoid hemorrhage.
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