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The early postdenervation depolarization of rat diaphragm muscle fibers (8–10 mV within 3 h in vitro) is substantially smaller (3 mV) when muscles are bathed with 1×10−3 M l-glutamate (Glu) or 1×10−3 M N-methyl-d-aspartate (NMDA). The effects of Glu and NMDA are inhibited in a dose-dependent manner by competitive inhibitor 2-amino-5-phosphonovaleric acid (APV) withK i 6.3×10−4 M, by 2×10−7 M MK-801, which acts as an open channel inhibitor, by 2–3×10−4 Zn2+, which reacts with surface-located sites of the NMDA subtype of the glutamate receptor, and also by glycine-free solutions and 7-Cl-kynurenic acid, which inhibits the glycine binding sites on NMDA receptors. It follows that the effect of glutamate on early postdenervation depolarization is mediated by the NMDA subtype of glutamate receptor with similar pharmacological properties to those found in neurons. The only exception found was the glutamate-like action of 1×10−7 M MK-801, which partially prevented the early postdenervation depolarization when present in the muscle bath during the first 3 h after nerve section.

Human brain levels of glutathione (GSH), glutathione disulfide (GSSG), and vitamin E were measured in neurologically normal control patients and two, groups of patients with neurodegeneration: those with Alzheimer’s disease (AD), and AD with some features of Parkinson’s disease (AD-PD). Control brain samples contained GSH levels more than 50 times higher than GSSG. The levels of GSH were highest in the caudate nucleus and lowest in the medulla. In patients with AD or AD-PD, hippocampal levels of GSH were significantly higher than controls. Patients with AD also demonstrated high GSH levels in the midbrain compared to normal. In contrast, patients with AD-PD did not have significantly elevated GSH levels in this site. GSSG levels were not significantly different in any brain region between controls and diseased patients. In control brains, the medulla had higher levels of vitamin E than any other brain region. The caudate nucleus had the lowest levels, which were about half the levels in the medulla. Control levels of vitamin E in the midbrain were about 18.8, μg/g. In AD patients the midbrain levels of vitamin E doubled to 42.3 μg/g. This doubling also occurred in AD-PD patients where midbrain vitamin E levels increased to 44.0 μg/g. These results may indicate that compensatory increases in GSH and vitamin E levels occur following damage to specific brain regions in patients with AD or AD-PD.

Neurofibromatosis type 1 (NF1) is a common inherited disorder that primarily affects tissues derived from the neural crest. Recent identification and characterization of the human NF1 gene has revealed that it encodes a protein (now called neurofibromin) that is similar in sequence to theras-GTPase activator protein (orras-GAP), suggesting that neurofibromin may be a component of cellular signal transduction pathways regulating cellular proliferation and/or differentiation. To initiate investigations on the role of the NF1 gene product in embryonic development, we have isolated a partial cDNA for chicken neurofibromin. Sequence analysis reveals that the predicted amino acid sequence is highly conserved between chick and human. The chicken cDNA hybridizes to a 12.5-kb transcript on RNA blots, a mol wt similar to that reported for the human and murine mRNAs. Ribonuclease protection assays indicate that NF1 mRNA is expressed in a variety of tissues in the chick embryo; this is confirmed byin situ hybridization analysis. NF1 mRNA expression is detectable as early as embryonic stage 18 in the neural plate. This pattern of expression may suggest a role for neurofibromin during normal development, including that of the nervous system.

The modulation of the binding of muscarinic cholinergic receptor ligands by phosphatidylserine purified from bovine cerebral cortex (BC-PS) was examined in vitro and in vivo. The enrichment of bovine cerebral cortical synaptosomal membranes with BC-PS, using a fusion technique, produced a concentration-dependent decrease in the affinity (increase in K d ) of [3H]quinuclidinyl benzylate (3H-QNB) specific binding to muscarinic acetylcholine receptors (mAChR), without changes in their maximal number (Bmax). Similar results were observed when [3H]oxotremorine (3H-OXO) was used to label a high affinity subpopulation of mAChR. On the other hand, preincubation of BC-PS liposomes with synaptosomal membranes in a nonoptimum fusion condition (at pH 7.4) did not alter the binding properties of both radioligands. Fusion experiments using a pure phosphatidylserine preparation from spinal cord revealed a similar decrement in the affinity of3H-QNB specific binding. Five day’s intraperitoneal (i.p.) administration of 15 mg/kg of BC-PS liposomes in rats increased the maximal number of cerebral cortical binding sites for3H-OXO. Scatchard analysis revealed no changes in the apparent dissociation constant. This modification is selective in relation to the neural structure studied. Thus, BC-PS treatment did not modify3H-OXO binding in the hippocampal formation and cerebellum. In contrast, parallel experiments using the muscarinic antagonist3H-QNB showed no alteration in the binding properties of mAChR. Five day’s i.p. administration of 15 mg/kg/d of phosphatidylcholine from bovine cerebral cortex (BC-PC) liposomes produced quite similar results to those obtained with BC-PS. These results indicate that mAChR are under the modulatory action of phosphatidylserine (PS) and phosphatidylcholine (PC), and suggest that this endogenous phospholipids may play a regulatory role on the mAChR. The possible implications of these findings on the effects of PC or PS treatment in neurological disorders involving a decrease in central cholinergic functions are discussed.

Batch purification of the myelin basic protein (MBP) in the lipidbound form was obtained from bovine brain white matter by using the slightly polydisperse nonionic detergent,n-octyl-pentaoxyethylene (octyl-POE) and hydroxyapatite. This large-scale procedure can also be carried out in laboratories without chromatographic equipment, and is applicable to small amounts of myelin. More interestingly, removal and inhibition of the proteolytic activity associated with myelin allowed us to obtain more stable and intact forms of the protein when compared with MBP isolated in the lipid-bound form by our previous method. Since it retains binding to all myelin lipids, this purified MBP may be considered as being in a native-like form. In this article, we suggest why this more intact form of MBP could be used to advantage as an alternative to lipid-free, water-soluble MBP in the study, detection, and treatment of myelin damage in pathology.

Nitric oxide (NO) has been proposed as a neuronal messenger molecule in hypoxic/ischemic cell injury (Nowicki et al., 1991; Trifiletti, 1992). We conducted studies in a model of combined glucose-oxygen deprivation using cultured rat cerebellar granule cells. Experiments were designed to test the hypothesis that sustained elevation of cytosolic calcium ([Ca2+]i) and NO generation act in concert to trigger neuronal injury after anoxic insult. A hypoxic state was achieved by perfusing the cells with medium pre-equilibrated with argon gas. [Ca2+]i was monitored using digital-imaging fluorescence microscopy in cells loaded with fura-2 AM. Under short-term hypoxic conditions, cells displayed a progressive and sustained, moderate increase of [Ca2+]i, which returned to near basal levels on restoration of O2-containing medium. Prolonged hypoxic conditions (>60 min) caused irreversible elevation of [Ca2+]i followed by disruption of cell membrane integrity, as indicated by severe swelling, loss of regular cell shape and processes, leakage of dye fura-2, and propidium iodide uptake (“point of no return”). Pretreatment withN G-nitro-l-arginine methyl ester (l-NAME, 100 μM), a specific NO synthase inhibitor, markedly delayed the onset of intensity of the rise of [Ca2+]i. The hypoxia-induced elevation of [Ca2+]i was also greatly attenuated ifl-NAME (100 μM) was added to the argon-perfused medium before the cells demonstrated signs of irreversible injury. Prolonged or repeated hypoxic conditions, however, caused a rapid and intense increase of [Ca2+]i, which could not be blocked by inhibition of NO synthase (NOS). In addition, reoxygenation after the “point of no return”, as characterized above, greatly potentiated [Ca2+]i overload and facilitated the process of cell injury. The potentiation and facilitation of cell damage, as demonstrated by rapid massive increase of [Ca2+]i and subsequent cell death, was not blocked by NOS inhibitor,l-NAME.

In previous studies, we have used histological methods to characterize cellular changes, and validated the use of the myeloperoxidase (MPO) activity assay to quantitate increased neutrophil infiltration in ischemic stroke. We also identified increased leukotriene B4 (LTB4) binding sites as a potential marker for neutrophil infiltration into, focal ischemic tissue. However, these studies were conducted at only one time-point, 24 h after ischemia. In the present study, we examined the full time-course of MPO activity and LTB4 receptor binding following middle cerebral artery occlusion (MCAO) made permanently (PMCAO) or transiently (160 min followed by reperfusion; TMCAO) in spontaneously hypertensive rats, and compared the results to previously characterized histologic changes in these models. Ischemic and contralateral (control) cortical tissue samples were assayed for MPO (U/g wet wt) and [3H]LTB4 receptor binding (fmol/mg protein). Following PMCAO, MPO activity significantly increased as early as 12 h and continued to increase over the next 5 d (p<0.05). Following TMCAO, MPO activity was significantly elevated already after only 6 h of reperfusion and also continued to increase over the next 5 d of reperfusion (p<0.05). LTB4 receptor binding and MPO activity were highly correlated during periods when both measures increased together (i.e., 0.5–5dp<0.01). However, by 15 d post-MCAO, LTB4 receptor binding remained elevated after MPO activity levels had returned to normal. This persistent LTB4 binding was associated with the significant gliosis that was characterized previously to persist in these models. The time-course of increased MPO activity and initially increased LTB4 binding post-MCAO correspond very well to our previous histological data that characterized the time-course for leukocyte infiltration under these conditions. Therefore, the increased MPO activity over time was associated with initial neutrophil and later mononuclear cell infiltration into ischemic tissue in these models. In addition, the present studies utilized histochemical analysis to demonstrate peroxidase activity in macrophages within the cerebral infarct following MCAO, thus validating that MPO activity originates from the later infiltrating mononuclear cells in addition to the early infiltrating neutrophils that had been previously characterized in the same manner. TMCAO produces a significantly larger and earlier increase in ischemic cortex MPO activity and a similar later increase in MPO activity compared to PMCAO treatment. Clearly, reperfusion of cerebral tissue following ischemia greatly exacerbates the degree of cerebral tissue inflammation. These biochemical assays, especially the MPO activity assay, have now been validated for quantitating the early and late phases of the cerebral inflammatory reaction to tissue injury.