Günther Geyer

Toluidine blue staining of cartilage proteoglycan subunits

Selective binding of toluidine blue to basophilic proteoglycans is the first stage of a staining method which proceeds to the formation of a heavy metal salt of the dyestuff. By means of this procedure matrix complexes of hyalin cartilage were stained such as to display granular subunits about 25 to 35 A in diameter. The findings are interpreted as the demonstration of proteoglycan constituents of chondromucin aggregates.

Untersuchungen über die anisotrope Toluidinblaufärbung der Glykokalyx

SummaryThe study was concerned with the molecular structure of the erythrocyte membrane and its anisotropic staining with toluidine blue.Enzymatic or chemical degradation impairs the topo-optical staining. Decreased anisotropy results also from digitonin treatment of glutaraldehyde fixed cells. Incubation in a mixture of chloroform and methanol abolishes the anisotropic staining with toluidine blue, but it has been restored by the aldehyde bisulfite-procedure. The efficacy of a treatment with chloroform-methanol is greatly reduced in the case of agglutinated erythrocytes. The reversibly collapsed glycocalyx is devoid of topo-optical staining.The findings refer to the spatial orientation of glycoproteins at the cell surface. Degradation or loss and rearrangement, respectively, change the glycocalyx structure and likewise deteriorate the conditions for the anisotropic staining. Alterations of the lipid layer of the membrane may result in similar effects. There was, however, no indication of an intercalation into the lipid layer of dyestuff molecules. In conclusion, the topo-optical toluidine blue staining is considered a powerful method aiding studies of the glycocalyx of erythrocytes (and presumably of other types of cells too).ZusammenfassungAn der Erythrocytenmembran wird die Beziehung zwischen anisotroper Toluidinblaufärbung und Membranstruktur untersucht.Enzymatischer oder chemischer Abbau der Glykokalyx stört die topo-optische Färbung. Eine Abschwächung wird auch durch Digitoninbehandlung Glutaraldehyd-fixierter Zellen erzielt. Durch Chloroform-Methanol-Inkubation wird die anisotrope Toluidinblaufärbung gelöscht, aber sie kann nach Aldehyd-Bisulfitbildung wieder nachgewiesen werden. An agglutinierten Erythrocyten ist die Wirkung einer Chloroform-Methanol-Behandlung stark eingeschränkt. Die reversibel kollabierte Glykokalyx zeigt keine topo-optische Färbung.Die Befunde werden als Hinweis auf die Bedeutung der räumlichen Orientierung der Glykoproteine an der Zelloberfläche gedeutet. Störungen im Aufbau der Glykokalyx durch Hydrolyse bzw. Verlust oder durch Umverteilung von Komponenten verändern die Bedingungen für die anisotrope Toluidinblaufärbung. Eingriffe an der Lipidschicht können mit solchen Veränderungen einhergehen; für die Interkalation von Farbstoffmolekülen in die Lipidschicht ergab sich jedoch kein Anhalt. Die topo-optische Färbung mit Toluidinblau wird auf Grund dieser Ergebnisse als geeignetes Verfahren zur Untersuchung der Glykokalyx von Erythrocyten (und vermutlich auch anderer Zellen) angesehen.

Endolymphatic leakage in case of acute loss of cochlear microphonics

Rapid loss of cochlear microphonics in guinea-pigs previously exposed to high-energy impulse noise was shown to be related to the breakdown of the endolymphatic boundary. The cochlear duct was rendered leaky by deterioration of the reticular membrane, and damage of sensory and supporting cells.

Horseradish peroxidase as a label of injured cells.

SynopsisThe present study is concerned with artifacts likely to occur in a horseradish peroxidase exclusion test. Incubation of murine peritoneal macrophages and lymphocytes with the peroxidase showed a close relationship between the number of living cells and the percentage of cells excluding the tracer. The penetration of the cytoplasm by horseradish peroxidase is attributed to an increase in the permeability of the cell membrane during the incubation (ranging from 10 to 120 min). It was not increased by the presence of tracer throughout the incubation period. However, concomitant fixation of the cell in the presence of horseradish peroxidase caused an increase in the influx of the tracer. The horseradish peroxidase exclusion test applied to the guinea-pig organ of Corti has proved to be valid provided that: (a) mechanical lesions prior to the tracer incubation are avoided; (b) incubation is terminated by removal of the extracellular tracer; (c) fixation is carried out as soon as possible; (d) a low concentration of horseradish peroxidase is used; and (e) specimens are incubated in diaminobenzidine-H2O2 medium for the shortest possible period.Although fixation-induced cytoplasmic infiltration by horseradish peroxidase was not detected in cochlear specimens, the findings call attention to possible sources of error and define the level of significance of the test. Horseradish peroxidase does not appear to be a cytotoxic agent under the conditions used.

Erythrocyte membrane topo-optical staining reflects glycoprotein conformational changes.

The study is concerned with the relationship of induced birefringence to conformational alterations of glycocalyx glycoproteins. Human erythrocytes washed with phosphate‐buffered saline (PBS) and fixed with glutaraldehyde exhibit the most intense topo‐optical staining, which is thought to reflect the lipid bound state of glycophorins. Incubations at reduced pH, in hypotonic media or in the presence of procaine resulted in a significant decline of induced anisotropy. The effects were not due to degradation of glycocalyx constituents. Erythrocytes subjected to treatments with procaine or at pH 6Ṁ2 suffered from loss of electrophoretic velocity, a finding indicative of rearrangement of cell surface glycoproteins. By example of the red blood cell the findings of this study confirm the premised suggestion of the sensible detection by topo‐optical toluidine blue staining of the conformational state of glycoproteins of the glycocalyx.

A polyacrylamide gel method for the cytochemical demonstration of glucose-6-phosphate dehydrogenase activity in mouse sperm

Solubility and low activity of glucose-6-phosphate dehydrogenase let both the aqueous and agarose gel cytochemical incubation techniques fail in mouse sperm. By means of a polyacrylamide gel medium G6PDH was constantly demonstrated in the midpieces of sperm cells prefixed with cold acetone. Although this technique prevented the dislocation of the enzyme, diffusion artifacts due to delayed formation of formazan may occur from sites of high dehydrogenase activity, e.g., epididymal epithelium encountered in sperm smears.

A peroxidase exclusion test for cell viability

Summary Similar to ruthenium red horseradish peroxidase may be employed in the light and electron microscopic determination of live and dead cells.

Reversible conformational changes of plasmalemmal glycoproteins.

Repeated incubations of human red blood cells in low ionic isotonic sucrose result in an instantaneous agglutination. In the same medium which had caused the agglutination, erythrocytes completely disagglutinate within 60 to 90 min. Disagglutination is accompanied by the efflux of cellular ions, which causes a 500-fold increase of extracellular K+. Decomposition of agglutinates occurs at once upon addition to the medium of about 3 mM KCL. It will be inhibited for hours, if the medium is renewed twice an hour. Erythrocytes washed successively with phosphate buffered saline and isotonic sucrose are devoid of adhering blood plasma proteins. If these cells were fixed with glutaraldehyde in isotonic sucrose they had lost a) their anisotropic staining with toluidine blue, and b) most of their colloidal iron binding capacity. The staining with ruthenium red and the electrophoretic velocity of these erythrocytes apparently were identical with the controls. The findings are considered evidence of the reversible unfolding of glycocalyx glycoproteins in the low ionic medium.

Immunologische und enzymatische Aktivität nach Fixierung mit Diimidoester Verbindungen

Zusammenfassung In der vorliegenden Arbeit wird uber die Fixierungseigenschaften zweier Diimidoester fur histoehemische Untersuchungen berichtet. Es wird gezeigt, das nach diimidoesterinduzierten Vernetzungsroaktionen an RBC die Receptoraktivitat dieser Zellen weitgehend erhalten bleibt. Diese Eigenschaften machen es moglich, amidinierte RBC als Immunadsorbens fur Hamagglutinations-reaktionen einzusetzen. An peroxidasestimulierten Plasmazellen wird beobachtet, das die Reaktivitat intracellularer Antiperoxidase-Antikorper nach Amidiniorung ebenfalls sehr hoch ist. Am Beispiel der histochemischen Nachweisreaktionen fur SDH, alkalische bzw. saure Phosphatase und unspezifische Esterase in amidinierten Gewebsschnitten wird die Uberlegenheit der Diimidoesterfixierung gegenuber der konventionellen Aldehydfixation verdeutlicht. Die fur licht- und elektronenmikroskopisch-histochemische Nachweisverfahren erforderlichen Amidinierungsbedingungen werden vorgestellt.

Adsorbed proteins mask negatively charged sites of the erythrocyte glycocalyx

Protein masking of charged sites of the erythrocyte glycocalyx was studied by means of the colloidal iron affinity. Washed red cells were fixed with glutaraldehyde such as to stabilize their glycocalyx and to inhibit conformational changes during posttreatment. Following the incubation in an ionic protein solution, proteins adsorbed to the cell surface were insolubilized by repeated treatment with low ionic isotonic surcose. Erythrocytes coated with precipitated proteins exhibited a rough surface. Their iron binding capacity was reduced considerably. In comparison with serum albumin, masking by gamma globulin was more efficient, apparently, because of its insolubility in low ionic media. High ionic incubation of coated erythrocytes resolubilized adsorbed proteins and unmasked negatively charged groups of the glycocalyx.