Per Gøran Krüger

Mast cells and multiple sclerosis: a light and electron microscopic study of mast cells in multiple sclerosis emphasizing staining procedures.

In the brains of 7 patients with multiple sclerosis, mast cells were observed within the demyelinated plaques, in the border zone of the plaques as well as in seemingly normal white matter. The cells were mostly located in close connection with small vessels. The routine staining with toluidine blue for the demonstration of mast cells is not adequate as compared with staining of similar sections in pinacyanol erythrosine. Mast cells may be a hitherto underestimated contributor to the demyelinating process of multiple sclerosis.

Isolation of rat mast cell granules with intact membranes

Abstract A method for controlled sonication of isolated rat peritoneal mast cells and separation of the membrane-bound secretory granules on a self-generating density gradient of Percoll (polyvinyl-pyrrolidone-coated silica gel particles) is described. Ruthenium red, which binds exclusively to the matrix of the membrane-free granules, proved to be an excellent agent for the quantitative assessment of the state of the perigranular membranes in the various preparations. The percentage of intact granules, as observed in the electron microscope, increased from 49% in the primary granule pellet to 75% after separation on Percoll gradient, whereas the corresponding figures as revealed by the ruthenium red method were 69 and 93% respectively. The observed discrepancies were due to difficulties involved in sampling the granule pellets for electron microscopy.

Recovery of rat mast cells after secretion: A morphometric study☆

Granule reconstitution in rat peritoneal mast cells following massive secretion was studied by morphometric techniques. Immediately following secretion, the earliest identifiable mast cells showed a substantial decrease in cell volume associated with granule loss. Cell volume then increased almost to the original level over a period of a month. The size of the Golgi apparatus increased markedly in the week following secretion and then returned to its original size. The total volume of granules increased slowly after the secretory depletion and by 34 days had not returned to the original value although the number of granules had recovered fully. The reconstitution of mast cells after secretion is a prolonged process with several phases resulting in mast cells of varying appearance and content. This heterogeneity generated by reconstitution post secretion must be considered in studies of populations of mast cells in vivo.

Mast cells in brains during experimental allergic encephalomyelitis in Lewis rats

We studied the number of mast cells and their extent of degranulation in brains of Lewis rats with acute experimental allergic encephalomyelitis (EAE), activity induced with guinea pig spinal cord and Freunds complete adjuvant. Non-immunized controls and EAE rats were killed on days 10, 11, 12, and 16 post-immunization (p.i.). The percentage of degranulated mast cells was significantly increased in EAE brains. Signs of degranulation were observed as early as day 10 p.i. Clinical EAE signs appeared from day 10 p.i. A significant change in mast cell number was not observed. The percentage of degranulated cells was largest at day 16 p.i., at a time when the inflammation had reached the thalamus. This indicates that mast cell degranulation may occur as a result of the inflammation. Collectively, the data suggest that mast cells may play a role in the pathogenesis of EAE.

The Histamine Release Process and Concomitant Structural Changes in Rat Peritoneal Mast Cells

Two morphologically different populations of rat peritoneal mast cells were observed in response to incubation with compound 48/80. Type 2 mast cells: cells with distinct contours exhibiting intracellular vacuoles containing altered granules and without signs of granule liberation; Type 3 mast cells: cells with indistinct countours and with varying number of granules liberated. The absence of calcium in the medium, high temperature (37 degrees C) in the presence as well as in the absence of calcium favoured type 2 versus the type 3 cells. The isolated mast cell population was less morphologically heterogeneous than the mixed cell population in response to the addition of compound 48/80. It is concluded that mast cells might release a great part of their histamine content without a concomitant liberation of the granule matrixes. The varying morphological pictures observed after incubating of mast cells with compound 48/80 is due to variable factors inherent to the experimental procedures.

Isolation, Properties and Nucleolytic Degradation of Chromatin from Escherichia coli

A new procedure has been developed for the isolation of the chromosome complex, termed chromatin, from Escherichia coli. The bacteria were subjected to low ionic strength and T4 lysozyme, followed by detergent treatment analogous to that employed for the isolation of eukaryotic chromosomes. The chromatin was an insoluble viscous material which contained approximately equal amounts of DNA and RNA. The protein content of the chromatin was almost three times greater than the nucleic acid content. Electron microscopy revealed that the chromatin was highly condensed, having multiple loops and beaded structures with various diameters. The chromatin could be completely solubilized by both micrococcal nuclease and DNAase I, whereas RNAase had no effect. The initial degradation by micrococcal nuclease resulted in the production of a DNA-protein particle, sedimentation coefficient 10S, and an RNA-protein complex of 24S. Further degradation led to a decrease in sedimentation coefficient of the DNA-protein complex, but not of the RNA-protein particle. The peak size of the DNA of the initial DNA-protein particle was approximately 2400 bp. The action of micrococcal nuclease also resulted in the production of several discrete RNA species of various sizes. Several low molecular weight proteins (12000-27000) were found in the DNA-protein complex. The DNA-binding protein HU was present in the undigested chromatin; varying amounts of HU were, however, detected in the DNA-protein and RNA-protein particles.

Structural Features of Histamine Release in Rat Peritoneal Mast Cells

Cellular events correlated to the histamine release process were studied in rat peritoneal mast cells applying the dye toluidine blue as histamine-discharging agent. The effects of toluidine blue were studied both in the presence and absence of calcium. Histamine release was clearly dose-dependent within the range 2X10––5 m toluidine blue to 4X10––4 m. Whereas spontaneous histamine release values were in the order of a few percent, maximum release in the presence of calcium amounted to 65–80%. Histamine release was accompanied by characteristic morphologic changes in the cells: granule fine structure was markedly altered, perigranular vacuoles were formed and, through repeated fusions of the vacuole-delimiting membranes, large caveolae containing granule matrices arose. In fortuitous sections, these cavities were seen to communicate with the extracellular space through pores, often of substantial size. Granule extrusion from the cellular domain to the surrounding medium, although it did occur to a certain extent, was not a prominent feature of the histamine release process. The dose-response curve for histamine release was lowered by roughly one half in the absence of calcium. This was reflected in mast cell morphology by a decreased tendency towards granule alteration and vacuole formation. The present observations are viewed in the light of various suggested models for the mode of histamine release from mast cells.

Effect of Methyl Methanesulphonate on the Nucleoid Structure of Escherichia coli

Incubation of a strain of Escherichia coli K12 with 25 mM-methyl methanesulphonate (MMS) for 1 h changed the sedimentation coefficient of the nucleoids from 1600S to 850S. When isolated nucleoids were treated with MMS under identical conditions in vitro there was no change in the sedimentation coefficient. Alkaline sucrose-gradient centrifugation of DNA from cells treated with 25 mM-MMS for 1 h indicated that there were approximately 100 breaks plus apurinic sites per chromosome. Titration with ethidium bromide of nucleoids from MMS-treated cells showed that almost all supercoiling had been lost, suggesting that the breaks plus apurinic sites consisted mostly of breaks. Further experiments showed that the apurinic sites were probably created by non-enzymic depurination and that little non-enzymic strand breakage had occurred. The depurinated sites thus created could then serve as substrates for the apurinic-specific endonucleases of the cell, with the result that strand breakage occurred. MMS treatment did not cause any changes in the DNA:RNA ratio of the nucleoids. Removal of MMS followed by a period of incubation resulted in a decrease in the number of breaks plus apurinic sites and an increase in the sedimentation coefficient of the nucleoids. After 2 h incubation in MMS-free medium the sedimentation coefficient of the nucleoids from MMS-treated cells was the same as that of the control; the supercoiling was also partially restored. The effect of MMS on two MMS-sensitive mutants of E. coli, one a polA and the other a recA mutant, was also studied. In both cases MMS caused complete collapse of the nucleoid structure.

Enigma of Disodium Cromoglycate Action on Mast Cells

In rat peritoneal mast cells, disodium cromoglycate showed no inhibitory effect on histamine release values if the cells were preincubated with the drug for 40 min prior to stimulation with antigen, compound 48/80 or ATP. If the drug was added simultaneously with antigen or a low dose of compound 48/80, a repression of histamine release occurred. No such effect was noted if ATP was employed as histamine liberator.

Mitomycin-C-induced changes in the nucleoid of Escherichia coli K12

The influence of low concentrations of mitomycin-C on the structure of the envelope-free nucleoid was studied in several strains of Escherichia coli K12. The wild-type strain AB1157 uvr+ rec+ and 3 mitomycin-C-sensitive derivatives carrying mutations in the uvrA, uvrB and recA genes, were used. Treatment of the control strain with mitomycin-C, 0.5 microgram/ml, followed by incubation in drug-free medium resulted in the formation of a transient fast-sedimenting nucleoid with a sedimentation coefficient of 2200 S. A fraction of 25% of the nucleoids had attained the normal sedimentation coefficient of 1570 S 3 h after removal of mitomycin-C. With the uvr- strains, mitomycin-C induced a slow, almost linear increase in the S value of the envelope-free nucleoid. In these cases the S value continued to increase during post-incubation and was 2050 S 3 h after removal of the drug. Post-incubation of recA- cells resulted in loss of supercoiling, decrease in S value of the nucleoid and degradation of DNA. Results obtained with phase-contrast and electron microscopy were in good agreement with the hydrodynamic data.