LOCALIZATION OF ACETYLCHOLINESTERASE VIA PRODUCTION OF OSMIOPHILIC POLYMERS: NEW BENZENEDIAZONIUM SALTS WITH THIOLACETATE FUNCTIONSMEDNICK, MORTON L.; PETRALI, JOHN P.; THOMAS, NORMAN C.; STERNBERGER, LUDWIG A.; PLAPINGER, ROBERT E.; DAVIS, DONALD A.; WASSERKRUG, HANNAH L.; SELIGMAN, ARNOLD M.
doi: 10.1177/19.3.155pmid: 4100994
By incorporating an enzyme-susceptible thiolester group and a diazonium group into the same molecule, it has been possible to obtain an osmiophilic polymer upon enzymatic hydrolysis of the thiolester, via diazothioether formation linking the units. The thiolacetates with diazonium groups are specific substrates for cholinesterase because of the strong positive charge on the diazonium group. Following osmication, sites of acetylcholinesterase in motor end plate are notable as black deposits in light microscopy and opaque deposits in electron microscopy. This is the first cytochemical demonstration of a hydrolytic enzyme using a single agent as both substrate and capture reagent to form a polymer. The insolubility of the polymer in both water and lipid before osmication provides precision of localization needed for the high resolution of electron microscopy. However, the effectiveness of the new principle for electron microscopy depends as well upon the rate of the capture reaction (polymerization), which in turn depends upon the pKa of the thiol and pH and temperature at which the cytochemical reaction is conducted.
DISCREPANCIES BETWEEN CYTOPHOTOMETRIC FEULGEN VALUES AND DEOXYRIBONUCLEIC ACID CONTENTNOESKE, K.
doi: 10.1177/19.3.169pmid: 5548584
Diploid nuclei of different cell types of the granulocytopoietic and erythropoietic series showed different Feulgen values. The highest values were found in the most immature nuclei, and the lowest values in the most mature cells. Extraction of acid-soluble nuclear proteins brought the different values to the same level. Examples of similar findings in other cell types, as described in the literature, are discussed. The Feulgen reaction and its colored product depend on the functional state of the chromatin in such a way that nuclei with high template activity of deoxyribonucleic acid show high Feulgen values whereas mature, dense nuclei with low template activity have lower Feulgen values, although both kinds of nuclei contain identical amounts of deoxyribonucleic acid.
DEMONSTRATION OF ACID PHOSPHATASE ACTIVITY USING 1-ACETYL-3-INDOLYL PHOSPHATE AS SUBSTRATEHAYASHI, MASANDO
doi: 10.1177/19.3.175pmid: 4100996
A simultaneous coupling azo-indoxyl method for the cytochemical demonstration of acid phosphatase activity using p-toluidine salt of 1-acetyl-3-indolyl phosphate is described. A satisfactory staining for the enzyme activity was obtained following incubation of formol-calcium-fixed frozen sections for 30 min at 25°C in a medium containing 1 mM each of the substrate and hexazonium pararosanilin and adjusted to pH 4.5-5.0 with acetate buffer. The distribution of acid phosphatase activity demonstrated by this method was identical with that obtained either by Gomori's technique using β-glycerophosphate as substrate or by the Barka and Anderson's naphthol AS-BI phosphate-hexazonium pararosanilin method in several tissues of male rats so far examined. However, the adrenal enzyme activity was most prominent in the medulla with 1-acetyl-3-indolyl phosphate and β-glycerophosphate but it was more marked in the cortex with naphthol AS-BI phosphate. An advantage of using 1-acetyl-3-indolyl phosphate as substrate is that the same compound can be used for comparing azo-indoxyl and lead-salt methods. Effects of phospholipase C and Triton X-100 on staining for acid phosphatase were tested by pretreating fixed rat liver and kidney sections with these agents and incubating them in the medium containing 1-acetyl-3-indolyl phosphate and either hexazonium pararosanilin or lead ions as a coupler. The pretreatment did not change discrete lysosomal staining, as seen in untreated controls, using pararosanilin as a coupler, but greatly modified the staining using lead ions. The results indicate that the preciseness of staining for acid phosphatase with lead-salt method is highly dependent on some lipid material which attracts lead in tissues and that appropriately devised azo dye or azo-indoxyl methods demonstrate enzyme sites more accurately than lead-salt method.
ALTERATION OF HISTONES FROM MOUSE EPIDERMAL CELLS AFTER INCUBATION WITH ELASTASE AND HYALURONIDASESEGAL, ALVIN; SCHROEDER, MARGARET; VAN DUUREN, BENJAMIN L.
doi: 10.1177/19.3.182pmid: 5548585
Chromatin was isolated from whole mouse skin, mouse epidermal cells and mouse liver by standard procedures used for isolation of chromatin from other mammalian tissues. Chromatin from whole mouse skin or from mouse epidermal cells had not been isolated or characterized earlier. For the preparation of chromatin from mouse epidermal cells, the latter was separated from dermis by incubation for 30 min at 37°C in a solution containing the enzymes elastase and hyaluronidase. The relative proportions of the chromatin components, the Tm and the ultraviolet absorption spectrum were all similar to that of chromatin from whole mouse skin which was not treated with enzymes and to other mammalian chromatin preparations. Electrophoresis of the histones from epidermal chromatin in polyacrylamide gels revealed the absence of histones F1, F3 and F2a2 and the appearance of a new band. Histones isolated from chromatin prepared from the whole mouse skin had a gel electrophoresis pattern virtually identical with histones isolated from mouse liver chromatin and to reported histone patterns from other mammalian tissues. The alterations in mouse epidermal histones are similar to reported changes in histones from calf thymus nucleohistone previously subjected to incubation at various temperatures. The enzymatic incubation technique can therefore not be used as a method of isolating unaltered mouse epidermal chromatin. The findings illustrate that very subtle chemical alterations can be induced by usual methods of tissue preparation and that these changes can only be detected by highly sensitive analytical techniques.