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Biomedical subjects

W T Norton

Publications and source records attributed to W T Norton.

At least 19 recordsLinked to original sources
PubMed1978

Proteolytic and peroxidatic reactions of commercial horseradish peroxidase with myelin basic protein.

Degradation of myelin basic protein during incubations with high concentrations of horseradish peroxidase has been demonstrated [Johnson & Cammer (1977) J. Histochem. Cytochem.25, 329-336]. Possible mechanisms for the interaction of the basic protein with peroxidase were investigated in the present study. Because the peroxidase samples previously observed to degrade basic protein were mixtures of isoenzymes, commercial preparations of the separated isoenzymes were tested, and all three degraded basic protein, but to various extents. Three other basic proteins, P(2) protein from peripheral nerve myelin, lysozyme and cytochrome c, were not degraded by horseradish peroxidase under the same conditions. Inhibitor studies suggested a minor peroxidatic component in the reaction. Therefore the peroxidatic reaction with basic protein was studied by using low concentrations of peroxidase along with H(2)O(2). Horseradish peroxidase plus H(2)O(2) caused the destruction of basic protein, a reaction inhibited by cyanide, azide, ferrocyanide, tyrosine, di-iodotyrosine and catalase. Lactoperoxidase plus H(2)O(2) and myoglobin plus H(2)O(2) were also effective in destroying the myelin basic protein. Low concentrations of horseradish peroxidase plus H(2)O(2) were not active against other basic proteins, but did destroy casein and fibrinogen. Although high concentrations of peroxidase alone degraded basic protein to low-molecular-weight products, suggesting the operation of a proteolytic enzyme contaminant in the absence of H(2)O(2), incubations with catalytic concentrations of peroxidase in the presence of H(2)O(2) converted basic protein into products with high molecular weights. Our data suggest a mechanism for the latter, peroxidatic, reaction where polymers would form by linking the tyrosine side chains in basic-protein molecules. These data show that the myelin basic protein is unusually susceptible to peroxidatic reactions.

Chemical Phenomena↗
PubMed1978

Encephalitogenic properties of purified preparations of bovine oligodendrocytes tested in guinea pigs.

The present study has investigated central nervous system disease in guinea pigs inoculated with emulsions containing purified preparations of bovine oligodendroglia and their fractions isolated with or without trypsinization, whole bovine white matter or myelin basic protein (MBP). The MBP content of the oligodendroglial fractions was determined by radioimmunoassay. It was found that oligodendroglia prepared from trypsinized fresh brain contained minute amounts of MBP and did not induce disease. The corresponding cell fraction from non-trypsinized frozen brain was rich in MBP and induced disease. Bovine white matter and MBP induced typical experimental allergic encephalomyelitis (EAE). The structural preservation of the non-encephalitogenic trypsinized MBP-poor cells was very good and that of the encephalitogenic MBP-rich non-trypsinized cells very poor. It has been concluded that the encephalitogenicity observed was due to MBP, rather than to a specific oligodendroglial antigen.

Animals↗
PubMed1978

Characterization of antioligodendrocyte serum.

An antioligodendrocyte serum (AOS) has been raised in rabbits against preparations of isolated bovine oligodendrocytes. The antibody was assayed by two techniques. By complement fixation with isolated oligodendrocytes, the titer of the antibody was 1:64 to 1:128. By indirect immunofluorescence testing of oligodendrocyte suspensions and frozen brain sections, the titer of the AOS was 1:256 to 1:512. When unabsorbed AOS was used, immunofluorescent staining proved it specific for bovine oligodendrocytes in suspension and in sections, and for human oligodendrocytes in sections. In cell suspensions, the staining was membrane related and in sections, cytoplasmic. The oligodendrocyte staining could be totally removed by absorption of AOS against oligodendrocyte suspensions, whereas absorption against bovine myelin, bovine myelin basic protein, and bovine neurons did not affect the staining reaction. AOS also stsined Schwann cells, a property possible related to antigens shared with oligodendrocytes. It is concluded that AOS is specific for oligodendrocytes and can now be applied to fundamental and disease-rel

Animals↗
PubMed1978

Neutral proteinases secreted by macrophages degrade basic protein: a possible mechanism of inflammatory demyelination.

In the inflammatory demyelinating diseases, such as multiple sclerosis, Landry-Guillain-Barré syndrome and experimental allergic encephalomyelitis, demyelination occurs in the vicinity of infiltrating mononuclear cells. Although the histopathology is characteristic of each disease, the general observation that myelin destruction in inflammatory lesions begins prior to phagocytosis suggests a common mechanism for myelinolysis in these diseases. Recent studies show that stimulated macrophages secrete several neutral proteinases, including plasminogen (Plg) activator. We have tested the possibility that these proteinases could, directly or indirectly, initiate myelin destruction. Isolated brain myelin was incubated with supernatant media from cultures of stimulated mouse peritoneal macrophages in the presence and absence of Plg. Cell supernatants alone caused some degradation of basic protein (BP) in myelin. The amount degraded was considerably enhanced in the presence of Plg. The other myelin proteins remained essentially intact. While the Plg-independent proteolytic activity in the supernatants was abolished by EDTA, known to inhibit the neutral proteinases, the Plg-dependent hydrolysis was inhibited by p-nitrophenylguanidinobenzoate, an inhibitor of Plg activator and plasmin. These results suggested that the Plg activator secreted by the macrophages generated plasmin, which selectively degraded BP. This interpretation was confirmed by the observation that urokinase, a Plg activator, plus Plg was effective in degrading BP in myelin. We propose that the action of neutral proteinases released by stimulated macrophages, and its amplification by the Plg-plasmin system, may play a significant role in several inflammatory demyelinating diseases; and that the relative specificity of these reactions for myelin lies in the extreme susceptibility of BP to proteolysis.

Animals↗
PubMed1978

Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages: a possible mechanism of inflammatory demyelination.

In inflammatory demyelinating diseases such as multiple sclerosis and experimental allergic encephalomyelitis, myelin destruction occurs in the vicinity of infiltrating mononuclear cells. The observations that myelin can be altered prior to phagocytosis and in areas not contiguous with inflammatory cells suggests a common mechanism for the initial stages of demyelination. Because stimulated macrophages secrete several neutral proteases, including plasminogen activator, we have investigated the possibility that myelinolysis could be mediated directly or indirectly by these enzymes. Isolated myelin was incubated with conditioned media from cultures of thioglycollate-stimulated mouse peritoneal macrophages in the presence and absence of plasminogen. Myelin appeared to be vulnerable to attack by at least two proteolytic activities secreted by the macrophages, a plasminogen-dependent and a plasminogen-independent activity; of the major proteins in myelin, the basic protein was most susceptible. The direct myelinolytic activity of macrophage-conditioned media was abolished by EDTA, and the plasminogen-dependent hydrolysis was abolished by p-nitrophenylguanidinobenzoate, an inhibitor of plasminogen activator and plasmin. These results suggest that the plasminogen activator released by the stimulated macrophages generated plasmin which hydrolyzed basic protein in intact myelin. This interpretation was confirmed by the observation that urokinase, a plasminogen activator, in the presence of plasminogen brought about marked degradation of basic protein in myelin. We propose that the release of neutral proteases by stimulated macrophages involved in cell-mediated reactions, and its amplification by the plasminogen-plasmin system, may play a significant role in the demyelination observed in several inflammatory demyelinating diseases.

Animals↗
PubMed1978

Isolation and characterization of glial filaments from human brain.

Intermediate (8--9 nm) filaments of human central nervous system astrocytes were isolated from the gliosed white matter of cases of adrenoleukodystrophy (ALD). This hereditary lipidosis is characterized pathologically by demyelination, loss of axons, and replacement of the white matter of the caudal cerebrum by a glial scar. Glial filaments were composed largely of a single protein component with a mol wt of about 49,000 daltons. Smaller components (44,000--39,000 daltons) were detected in some samples, and appear to represent degradation products of the filament protein. Human neurofilaments were isolated from the normal frontal white matter of ALD cases by the standard myelin-free axon technique. Isolated glial and neurofilament proteins comigrated during acrylamide gel electrophoresis in SDS. Polypeptides resulting from cyanogen bromide cleavage of the two filament proteins were the same. Both proteins reacted with rabbit antisera raised against isolated bovine neurofilament protein and human glial fibrillary acidic protein.

Astrocytes↗
PubMed1978

Huntington disease: normal lipid composition of purified neuronal perikarya and whole cortex.

This is the first report of the lipid composition of human neurons. Neuronoal perikarya were isolated from frozen samples of the cerebral cortex of persons with Huntington disease and two normal controls. These were analyzed for total lipid, individual lipids, and gangliosides. No differences were detected between diseased and normal cells. In addition, gray matter samples from the same patients, and one additional patient and control sample, were analyzed and found not to differ. Thus the ultrastructural abnormalities seen in cortical biopsies are not reflected in the concentration of the major lipid classes.

Cerebral Cortex↗
PubMed1977

Quantitation of myelin carbonic anhydrase-development and subfractionation of rat brain myelin and comparison with myelin from other species.

A number of related studies have been performed to characterize further the carbonic anhydrase activity of myelin. Recent assertions that carbonic anhydrase activity is intrinsic to the myelin sheath were subjected to the additional test of isolation of rat brain myelin in the presence of purified carbonic anhydrase. This procedure did not increase the carbonic anhydrase activity in myelin above the endogenous level, indicating that this enzyme does not stick to myelin membranes. A developmental study of rat brain carbonic anhydrase showed that the enzyme activity increased in whole brain homogenates and in myelin, with the greatest increments in enzyme activity occurring before the animals were 60 days old. When myelin from adult rat brains was fractionated on a density gradient, carbonic anhydrase activity was relatively enriched in the heavy subfraction but was present in all three layers. This finding suggested that the activity in myelin preparations was not due to contamination with a carbonic anhydrase-rich membrane fragment. Carbonic anhydrase in myelin was not confined to the rat. Beef brain homogenates and myelin had low activities of the enzyme, but myelin from rabbit, cat, monkey and mouse had carbonic anhydrase activities comparable to that of the rat, accounting for 6.3--13.6% of the respective homogenate activities.

Age Factors↗
PubMed1977

The isolation of cerebral neurons with partial retention of processes.

A novel tissue disaggregation technique has been devised which permits the isolation of neurons with fairly extensive processes attached. Cortex is dissociated by aspiration through nozzles of decreasing size followed by agitation on a vortex mixer, rather than by the usual technique of forcing tissue through sieves. After each aspiration step, dissociated cells are separated from undisrupted tissue by coarse filtration and the latter is subjected to repeated treatment. This prevents unnecessary trauma to the free cells. After disruption is complete, small pieces of undisrupted tissue are removed from the cell suspension by floating on the foam created by degassing the suspension under vacuum. Cells are purified by conventional velocity-gradient centrifugation. This procedure has been applied successfully to fresh rat brain, with or without a preincubation with trypsin, frozen human brain and frozen bovine brain. The cell yields from rat brain were comparable to or better than, those obtained by other procedures (37 X 10(6) cells/g brain) while the purity was comparable. Cell yields from human brain were similar to those from rat brain but the purity was lower. The lowered particle purity of human and bovine cells can probably be attributed to the conditions of storage of the tissue and to trapping of free nuclei in the meshwork of dendritic processes. Values are given for the amount of protein, RNA and DNA per cell.

Animals↗
PubMed1977

Studies on the encephalitogenic effects of purified preparations of human and bovine oligodendrocytes.

Bulk-isolated human and bovine oligodendroglia, practically free from myelin, have been used in attempts to elicit an autoimmune response which has been compared with acute experimental allergic encephalomyelitis (EAE). For these experiments, a total of 20 Hartley guinea pigs, 33 Lewis rats and 16 rabbits have been studied. Animals were inoculated with a range of doses of purified preparations of both human and bovine oligodendroglial cells in complete Freund's adjuvant (CFA) and compared with others challenged with whole white matter in CFA. The latter animals all developed clinical and histological signs of experimental allergic encephalomyelitis (EAE) 2-3 weeks post-inoculation. In general, oligodendroglial cells were encephalitogenically less potent than white matter. Guinea pigs were the most susceptible to inoculations of oligodendroglia. In several given human oligodendroglia 14 days earlier, a paraparesis indistinguishable from conventional EAE was seen. Animals receiving bovine cells showed no clinical signs. Histologically, the CNS of afflicted guinea pigs displayed severe inflammation but, in contrast to conventional EAE in the same species, demyelination was rare in the small group of animals tested. After sensitization with oligodendroglia, rats displayed no clinical disease. Histologically, some given human cells had positive evidence of disease while bovine cells in others gave a mild response. Rabbits showed no clinical and very little histological disease. Although more extensive studies are needed to confirm the findings, from the animals studied it appears that (1) variation in response to inocula containing oligodendroglia exists among the species tested, (2) that human oligodendroglia are more potent immunologically than bovine cells, (3) that CNS lesions produced by these cells in guinea pigs, lack a strong demyelinative component and (4) a specific antigen might exist in oligodendrocytes which is distinct from myelin basic protein. The possible reasons underlying our findings are discussed.

Animals↗
PubMed1976

Mucolipidosis IV. Clinical, ultrastructural, histochemical, and chemical studies of a case, including a brain biopsy.

A 7-year-old Ashkenazi Jewish boy with normal early development started to regress at 8 months of age and made no further developmental progress. Corneal clouding was noted at age 10 months. Corneal and conjunctival biopsy at 14 months, cerebral biopsy at 24 months, and fibroblast cultures at 32 months showed lysosomal inclusions, suggesting the storage of lipid-like and mucopolysaccharide-like material. In the brain, dense fluorescent inclusions resembled those in ceroid-lipofuscinosis. Total ganglioside content of white matter was raised, but the pattern was normal. The level of nonlipid hexosamine in the brain was normal. The cornea and conjunctiva contained electronlucent vacuoles resembling those in the mucopolysaccharidoses. Cornea, brain, and lymphocytes contained concentric membranous lamellar structures reminiscent of those in the gangliosidoses. The clinical picture and ultrastructural findings support the impression that this case belongs to a new variant of the mucolipidoses, mucolipidosis IV.

Cerebral Cortex↗
PubMed1975

Biochemical and pathological studies of myelin in hexachlorophene intoxication.

Adult female rats were fed a diet containing 500 ppm hexachlorophene (HCP). Morphological study of brains from these animals showed vacuolation of the myelin sheaths due to separation of myelin lamellae at the minor dense line. However, myelin could be isolated from the brains of these animals in normal yield. The myelin isolated from HCP-fed animals had normal lipid and protein compositions as shown by analyses of the individual lipids and by disc gel electrophoresis of the proteins. Assay of the myelin-specific enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase, showed normal specific activity in myelin obtained from HCP-fed rats. Brains of HCP-fed rats showed an increase in wet weight and a decrease in dry weight, with the chloroform-methanol insoluble fraction showing the greatest weight loss. During isolation of myelin from HCP-fed rats material was found floating over 0.32 M sucrose. This "floating fraction" contained a higher ratio of lipid to protein but the same relative proportions of the individual lipids as are found in myelin. The yield of "floating fraction" from each HCP-fed rat was less than 10 percent of the yield of myelin. Disc gel electrophoresis demonstrated the presence of the usual myelin proteins in this fraction, but with a slight increase in the relative amount of the low molecular weight basic protein. The data were compared to reports on the biochemistry of triethyltin poisoning, and it was concluded that vacuolation of myelin in HCP poisoning is probably due to increased permeability of myelin lamellae to water and electrolytes.

Animals↗
PubMed1975

The lipid composition of isolated brain cells and axons.

The current status of the published work on the lipid composition of isolated brain cells is reviewed and some new work on the sphingolipids of these cells is presented. In spite of considerable differences in isolation techniques between different groups, the lipid analyses of different cell preparations are similar enough to permit several generalizations. This fact is an encouraging sign that cell separation methods have considerable usefulness in defining the composition of normal brain cells. It is a general finding that astrocytes have more lipid than neuronal perikarya but that the gross lipid composition of these two cell types is surprisingly similar. Oligodendroglial lipids are quite different from those of the other two cell types and are characterized by a high galactolipid content. Although such a lipid pattern might be expected in oligodendroglia, which are myelin-forming cells, axonal lipids have an even higher galactolipid content. In an effort to find more cell-specific patterns, the glycosphingolipids were examined in more detail. Differences were seen in the distribution and fatty-acid patterns of these minor lipids in neurons and astrocytes, although it may be premature to conclude that these differences will prove to be cell-specific. All of the isolated cells were found to contain galactosylceramide, sulfatide, glucosylceramide, dihexosylceramide, and gangliosides. The distribution of these lipids in the normal cells was found to differ considerably from that reported in cultured neuroblastoma cells or astrocytoma cells. Not only were gangliosides present in all cells but the ganglioside patterns of neurons and astrocytes were nearly identical. The fatty-acid patterns of the neuronal and astroglial sphingolipids generally do not resemble each other, and both are quite different from those found in oligodendroglia and axons. However, the fatty-acid composition of the sphingolipids from bovine oligodendroglia and from axons are similar and resemble those of myelin lipids. The fatty acids of glucosylceramide and dihexosylceramide are similar in all three cell types. They have rather large amounts of 16:0 and acids longer than C18; thus they are considerably different from the ganglioside fatty acids (which have mostly 15:0) isolated from the same fractions.

Age Factors↗