FINE STRUCTURAL LOCALIZATION OF ACETYLCHOLINESTERASE USING ACETYL-β-METHYLTHIOCHOLINE AND ACETYLSELENOCHOLINE AS SUBSTRATESKOKKO, A.; MAUTNER, H. G.; BARRNETT, R. J.
doi: 10.1177/17.10.625pmid: 5384471
Acetyl-β-methylthiocho1ine and acetylselenocholine were used as substrates for fine structural demonstration of acetylcholinesterase activity and were compared with acetylthiocholine. Essentially, the same localization of the enzyme activity was found with all of these substrates in the rat spinal cord. Acetyl-β-methylthiocoline proved to be the most specific for acetylcholinesterase in the cytochemical system used and the final product was deposited most rapidly with acetylselenocholine. When the method of Karnovsky and Roots was modified by substituting tartrate for citrate as a chelating agent, a fine crystalline end product was produced and a sharp localization with little evidence of diffusion was obtained. A fixative containing a mixture of formaldehyde and glutaraldehyde proved to be the best in preserving both enzyme activity and ultrastructure. The substrates were tested biochemically and the results substantiate the cytochemical data.
LEAD AND PHOSPHATE AS SOURCES OF ARTIFACT IN NUCLEOSIDE PHOSPHATASE HISTOCHEMISTRYGANOTE, CHARLES E.; ROSENTII, ALAN S.; MOSES, HAROLD I.; TICE, LOIS W.
doi: 10.1177/17.10.641pmid: 4317882
The contribution of lead and lead phosphate binding to tissue as a possible source of artifact in the Wachstein-Meisel histochemical procedure was considered. In addition, the relevance of lead-catalyzed substrate hydrolysis in tissue water and in the reaction media, as well as the efficacy of the enzyme inhibitors, para-mercurobenzoate and potassium fluoride were examined. Lead-catalyzed hydrolysis of substrate in tissue water does not play a significant role in reaction product deposition. Lead-catalyzed hydrolysis in the reaction media does produce significant amounts of lead phosphate. Furthermore, the enzyme inhibitors in addition to inhibiting enzyme activity alter lead binding to tissue, precipitate lead from solution and decrease tissue staining. Loss of tissue staining may result from a decrease in phosphate-trapping efficiency of lead when the free lead ion concentration is lowered in the reaction medium. Lead phosphate and lead localize as deposits of electron-dense material at tissue sites parallel to those observed with the complete Wachstein reaction mixture. It is concluded that tissue and chemical events other than enzyme activity may contribute to reaction product localization.
THERMAL ANALYSIS OF POLYANION METACHROMASY: EFFECT OF TEMPERATURE ON METACHROMATIC SOLUTIONS OF NUCLEIC ACIDS AND ACID MUCOPOLYSACCHARIDESKELLY, JOHN W.; CHANG, LOUIS; ROTHSTEIN, FRED
doi: 10.1177/17.10.651pmid: 4194353
Spectra of toluidine blue with acid mucopolysaccharides and nucleic acids were recorded over the temperature range 10-70°C. Metachromatic ratios were inverse, linear functions of temperature. Intercepts and slopes of the thermal plots distinguished acid mucopolysaccharides from nucleic acids. Acid mucopolysaccharide reactions were thermally reversible. Nucleic acid reactions were not strictly reversible; such behavior was not attributable to thermal denaturation. An explanation for the unusual γ-peak of ribonucleic acid-toluidine blue metachromasy is offered in terms of terminal phosphate groups of ribonucleic acid. Solution data in which the polyanion-dye ratio was approximately 1, representing typical "metachromasy," were subjected to thermodynamic analysis. These data did not fit a common equation deriving equilibrium constants spectrophotometrically by variation of reactant concentrations. This failure supported the view of metachromasy as a phenomenon primarily involving dye-dye interaction. Another equation based only on temperature variation yielded a value of –4 kcal/mole for the chondroitin sulfate-toluidine blue complex, in good agreement with similar published values obtained both in solutions and stained materials.
THERMAL ANALYSIS OF POLYANION METACHROMASY: TEMPERATURE EFFECTS ON STAINED CELLS, TISSUES AND MODELSKELLY, JOHN W.; CHANG, LOUIS
doi: 10.1177/17.10.658pmid: 4194354
Metachromasy of toluidine blue-stained materials was examined at 5-95°C visually and 30-70°C microspectrophotometrically. Cells and tissues provided sites of acid mucopolysaccharides and nucleic acids, which were also prepared as films and droplets. As in similar studies of aqueous solutions, metachromatic ratios were inverse, linear, reversible functions of temperature, with the possible exception of deoxyribonucleic acid. An aqueous mounting medium (gelatin) supported maximum excursions of metachromasy during heating and cooling, although reversible loss of metachromasy occurs to lesser degrees in conventional media. Removal or denaturation of cartilage matrix protein merely increased over-all metachromasy; slopes of thermal plots were unchanged. All evidence suggests that metachromasy is not a fundamentally different phenomenon in solutions and solid systems. Temperature studies emphasize the role of structured water in metachromasy, interaction of water and other solvents and particularly solvent dielectric constant in relation to dye-dye interaction. The limited literature on temperature and biologic staining is reviewed.
HISTOCHEMICAL DEMONSTRATION OF SULFATED MUCOSUBSTANCES AND CATIONIC PROTEINS IN HUMAN GRANULOCYTES AND PLATELETSDUNN, W. B.; SPICER, S. S.
doi: 10.1177/17.10.668pmid: 4194355
Histochemical staining methods visualize sulfated mucosubstance in numerous small granules of early neutrophil leukocytes in appropriately processed human bone marrow smears. These reactive granules presumably constitute the counterpart in human neutrophils of the mucosaccharide-rich primary granules in rabbit heterophils. Neutrophils at a late developmental stage in human marrow or buffy coat smears reveal few or no granules with such reactivity. Strong staining for sulfated mucosubstance is readily demonstrable in numerous large cytoplasmic granules in human eosinophils. This reactivity diminishes but does not disappear during maturation of the eosinophil and its single population of cytoplasmic granules. Granules of human basophils at all developmental stages stain for sulfated mucosubstance. Platelets in human buffy coat smears reveal evidence for sulfated mucosaccharide. The mucosaccharide-containing granules of the three myeloid series also disclose acidophilia at high pH indicative of the presence of strongly cationic proteins.