Journal of Histochemistry & Cytochemistry

SCHLAEPFER, WILLIAM W.; TORACK, RICHARD M.

doi: 10.1177/14.5.369pmid: 5961993

Ultrastructural localization of cholinesterase activity was demonstrated in rat sciatic nerve using thiocholine esters as substrate. This enzymatic activity was limited to axons, where it was located on limiting axonal membranes, and on axonal vesicles of all unmyelinated and some myelinated nerve fibers. No accentuation of activity was present at nodes of Ranvier. The use of acetyl, propionyl and butyrylthiocholine as substrate resulted in decreasing amounts of end product precipitate, respectively. The cholinesterase activity was sensitive to heat, resistant to N-ethyl maleimide, and inhibited byeserine, DFP, E600, RO2-1250 and BW 62C47. Studies of specific inhibition sensitivities and substrate preferences suggested that the axonal cholinesterase of rat sciatic nerve is a specific acetylcholinesterase which is relatively homogeneous.

DARŻYNKIEWICZ, Z.; ROGERS, A. W.; BARNARD, E. A.

doi: 10.1177/14.5.379pmid: 5961994

Megakaryocytes from bone marrow of immature rats have been treated with 3H-DFP, and the grain density over them measured in autoradiographs. The uptake of labeled DFP is reduced by one-third by eserine and by the AChase inhibitor 284C51, and by reactivation with pyridine-2-aldoxime. It is concluded that one-third of the DFP taken up by megakaryocytes represents binding to true AChase. The other esterases present include no significant amount of pseudocholinesterase.

ADAMS, C. W. M.; ABDULLA, Y. H.; BAYLISS, O. B.; WELLER, R. O.

doi: 10.1177/14.5.385pmid: 5961995

A histochemical method for triglyceride esters is described that depends on hydrolysis of triglycerides to fatty acids by pancreatic lipase, precipitation of the released fatty acid as calcium soap and formation of lead sulphide by the conventional Gomori technique. The specificity of the lipase for triglyceride was investigated by chromatography and activation-inhibition studies. The enzyme preparation used hydrolyzed triglycerides and waxes but did not split cholesterol esters or phosphoglycerides. Positive histochemical reactions were obtained with this pancreatic lipase method in medium sized and fine lipid droplets in frozen sections. Positive reactions were observed in rat brown fat; in various adipose tissues in man, rabbit and rat; in the spermatogonia of rat testis; in human epidermis and sebaceous glands; in solitary unidentified adrenocortical cells in man and rat; in human fatty liver and in fine droplets in senile human myocardium. A slight variable reaction was obtained in human atherosclerotic plaques. The reaction of brown fat was also investigated with the electron microscope. No triglyceride was demonstrated in unfixed tissue sectons when an active preparation of lipoprotein lipase was used in place of pancreatic lipase. This failure was probably due to diffusion of lipoprotein from unfixed sections. Lipoprotein lipase is inactive against serum lipoproteins fixed with formaldehyde or glutaraldehyde.

MAEIR, DAVID M.; ZAIMAN, HERMAN

doi: 10.1177/14.5.396pmid: 5961996

Rat striated muscle (gastroenemius) containing encysted Trichinella larvae was studied histochemically for hydrolases associated with lysosomes. Activity of the enzymes studied (acid phosphatase, esterase, aminopeptidase), not demonstrable in appreciable amounts in normal striated muscle, appears in the altered muscle fibers in granules which by various criteria are demonstrated to be lysosomes. The increase in lysosomal enzyme activity is accompanied by increased prominence of the Golgi apparatus, as demonstrated by thiamine pyrophosphatase activity, and by an increase in the ribonucleoprotein content of the muscle fibers. These changes illustrate the facultative development of lysosomes and their associated ferments during a regenerative process. They suggest the need for a revision of the classic concept of the primarily degenerative nature of the trichinous lesion as well as a possible role of the developing lysosomes in this process.

FELGENHAUER, K.; GLENNER, G. G.

doi: 10.1177/14.5.401pmid: 5961997

In order to determine the relationship and characteristics of the soluble and tissue-bound (so-called "lyo" and "desmo") components of enzymes in histochemical and biochemical systems, a biochemical investigation of the hydrolysis of l-leucyl β-naphthylamide in rat kidney was undertaken. By ultracentrifugation, autolysis, salt and solvent fractionation procedures and DEAE-cellulose column chromatography primary soluble and tissuue-bound enzymes were separated and partially purified. The soluble enzyme was found to be inhibited by p-chloromercuribenzoate and reactivated by cysteine. The tissue-bound enzyme was found to be inhibited by o-phenanthroline and reactivated by Co++, and hydrolyzed peptides and amino acid β-naphthylamides at rates differing markedly from those of the soluble enzyme and commercial leucine aminopeptidase. Disc electrophoresis of the tissue-bound enzyme preparation demonstrated two protein bands, both revealing enzymic activity with almost identical hydrolytic characteristics, i.e., isozymes. This evidence established the identity of the tissue-bound Co++-activated enzyme with that found histochemically in the brush border of the proximal tubuli, and indicated its functional capacity as that of an aminopolypeptidase. It was also concluded that in the system investigated the "lyo" and "desmo" components represented, in reality, distinct enzymes with unique characteristics.

APPLETON, T. C.

doi: 10.1177/14.5.414pmid: 5961998

The resolving power, sensitivity and latent image fading have been studied in autoradiographs prepared by a technique that retains soluble compounds. A resolution of 2-4 µ was obtained in preparations containing 3H-thymidine and 9-13 µ in those containing 22NaCl. Results showed that there was no significant diffusion of soluble compounds. Sensitivity studies revealed that –25°C was the optimum exposure temperature. There was no significant latent image fading in preparations exposed for periods up to 330 days.

LILLIE, R. D.; PALMER, R. W.; GUTIÉRREZ, A.

doi: 10.1177/14.5.421pmid: 4164107

When azo coupling of tissue with freshly diazotized sulfanilic acid at 20-25 mM concentration in a bicarbonate solution is followed by acid washing and staining with dilute azure A or safranium O solutions at pH 1, the azo coupling sites in tissue are demonstrated with green to greenish black or red colors of sufficient intensity to render the method useful for the study of the distribution of reactive proteins. Erythrocytes react more strongly by this method than with most other diazonium salts. The method also affords a procedure for creating readily demonstrable sulfonic acid groups in tissue sections, for the study of their reactions in comparison with those of ester sulfuric acid groups.

SILVERMAN, LLOYD; GLICK, DAVID

doi: 10.1177/14.5.425pmid: 4164108

Bromsulfalein has been shown to provide an intense blue stain for protein with some advantages over other acid dyes that have been used similarly. The procedure is simple and requires no unusual materials. The previously established stoichiometric nature of the reaction between the anionic dye and the cationic groups of protein offer the potential use of the stain for measurement.

MCCURDY, L. E.; BURSTONE, M. S.

doi: 10.1177/14.5.427pmid: 5336049

Several new water-soluble mounting media derived from polyvinyl alcohol and acrylic resins have been reported. These are of use in the preservation of various azo dye products, fats and histological stains. They may also offer an approach to new embedding techniques.

HUGON, J.; BORGERS, M.

doi: 10.1177/14.5.429pmid: 5961999