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Biomedical subjects

W Cammer

Publications and source records attributed to W Cammer.

90 records · Page 5Linked 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

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↗
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

Commercial horseradish peroxidase degrades myelin encephalitogenic protein during coupling for immunohistochemical studies.

Conjugates of myelin encephalitogenic basic protein (EP) and commercial horseradish peroxidase (HRP) have been used for immunohistochemical demonstrations of anti-EP antibody in animals with experimental allergic encephalomyelitis. We performed gel electrophoresis studies on EP-HRP conjugates prepared with glutaraldehyde and on mixtures of EP and HRP incubated without glutaraldehyde. The results show that under conditions of one-and two-step coupling HRP causes rapid loss of the native EP band, apparently due to EP degradation. The EP-HRP mixtures are not encephalitogenic in rabbits, or encephalitogenic activity is lost during processing. The immunohistochemical reactivity of conjugates, however, signals some preservation of antibody-combining sites. The mechanism of the HRP effect on EP is unknown. The possibilities of a contaminating proteinase or direct peroxidatic attack are suggested. Until this action of HRP can be overcome, the effect of coupling procedures on the biological activities of EP will be difficult to assess, and EP-HRP conjugates cannot be expected to reveal sites that may bind encephalitogenic portions of the EP molecule.

Animals↗
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

Neurotoxicity of hexachlorophene: new pathological and biochemical observations.

Twenty female rats with nursing litters were fed a diet containing 500 p.p.m. of hexachlorophene (HCP). The blood concentrations of hexachlorophene in mother rats were 4.7-6.0 mug/ml and in infant rats 2.0-2.5 mug/ml. Fifty percent of mother rats and 75% of infant rats died. Most of the surviving animals were asymptomatic apart from imparied visual function. Ten adult and 20 infant rats were killed by perfusion. Light- and electron-microscopic studies showed vacuolation of myelin in the central and peripheral nervous system and axonal degeneration which was especially severe in the optic nerves. After withdrawal of HCP hydrocephalus ex vacuo was present in the chronically intoxicated infant rats and the optic nerves showed marked atrophy and gliosis. Studies with liver mitochondria prepared from lactating hexachlorophene-fed rats showed a 50-75% inhibition of respiration with succinate as substrate.

Animals↗
PubMedDate not supplied

Hexachlorophene retinopathy in suckling rats. A light- and electron-microscopic study of short-term and long-term effect of hexachlorophene intoxication on the retina of young rats.

Hexachlorophene (HCP) in very large doses has a toxic effect on the retina of adult rats. The present study examined the sensitivity of the developing retina to smaller doses and its ability to recover from neurotoxic damage. Suckling rats were intoxicated with HCP added to the mothers' diet. The retinae of the pups were examined by light and electron microscopy for early and late effects. All dose levels above 100 ppm caused vacuolar degeneration of the outer segments of photoreceptor cells within 14 days of intoxication, followed by degeneration of the inner segments after 28 days. The cell bodies of photoreceptor cells and all other retinal layers remained normal. Suckling intoxicated with HCP (100 - 500 ppm) added to the mothers' diet for 28 days and then withdrawn, showed no recovery of the outer or inner segments o photoreceptors when examined up to 150 days later. In addition, extensive degeneration and loss of photoreceptor cell occurred after withdrawal of HCP and the degenerative process extended to involve the inner nuclear layer. The rate of appearance of pathological changes was proportional to the total amount of ingested HCP. This study showed that photoreceptor cells in newborn rats are highly sensitive to HCP. The retinal lesions were irreversibly an continued to progress by continued trans-synaptic degeneration after withdrawal of the toxic agents.

Animals↗