Journal of Histochemistry & Cytochemistry

SMITH, ROBERT E.; FARQUHAR, MARILYN G.

doi: 10.1177/18.4.237pmid: 4314886

The modulations in nucleoside diphosphatase (NDPase) activity which occur during protein secretion have been investigated in mammotrophic hormone-producing cells (MT) of the female rat adenohypophysis. Pituitaries were fixed by perfusion or immersion in glutaraldehyde, and nonfrozen sections were incubated for NDPase. Tissue was examined from lactating and estrogen-treated animals in which mammotrophic hormone secretion (MTH) is high and from postlactating rats in which secretion of MTH is suppressed. In all experimental groups, NDPase reaction product was found: (a) in Golgi cisternae, (b) around forming and mature secretion granules, (c) in pericapillary and intercellular spaces and (d) along the outer surface of endothelial and MT cell membranes. The staining intensity of both the intracellular and extracellular sites paralleled the level of MTH secretion; it was greatest after estrogen treatment, moderate during lactation and minimal in the postlactating animal. In animals given estrogen, the Golgi apparatus was increased in size and up to six successive cisternae along the concave Golgi face were filled with reaction product in virtually every cell. In postlactating animals, the Golgi apparatus was small, and there was only patchy staining of a few cisternae in some cells. From its location and fluctuations with secretion, it seems likely that NDPase activity may be associated with the condensation and/or discharge of secretory granules.

ERNST, STEPHEN A.; PHILPOTT, CHARLES W.

doi: 10.1177/18.4.251pmid: 4245438

The effect of glutaraldehyde and formaldehyde fixation on the level of biochemically demonstrable Na-K-adenosine triphosphatase (Na-K-ATPase) and Mg-ATPase of avian salt glands and teleost gill filaments was studied. Sections, 100-200 µ, prepared with the Smith-Farquhar tissue chopper, were fixed for varying periods, homogenized and assayed for ATPase activity. Fixation of salt gland tissue with 0.5% glutaraldehyde for 40-60 min completely inhibited the Na-K-ATPase activity and reduced the level of Mg-ATPase by 85%. In contrast, fixation with 2 or 3% formaldehyde, prepared from paraformaldehyde, for 60-90 min resulted in a loss of only 30% of the Na-K-ATPase activity and 65% of the Mg-ATPase activity. Similar results were obtained with gill filaments. In addition, Na-K-ATPase of formaldehyde-fixed tissue retained an obligatory requirement for Na+ and K+ and was fully sensitive to ouabain. Electron microscopic examination of formaldehyde-fixed tissue, sectioned with either the tissue chopper or in the cryostat and incubated in the Wachstein-Meisel medium, showed excellent morphologic preservation. Reaction product deposition (presumably due to Mg-ATPase) was associated with the extracellular side of the plasma membrane in the secretory cells of the salt gland and over the mitochondrial matrix of chloride cells present in the gill epithelium.

BRENNAN, PATRICIA C.; PERAINO, C.; FRY, R. J. M.; SWICK, R. W.

doi: 10.1177/18.4.264pmid: 4985873

Ornithine aminotransferase (l-ornithine:2-oxoacid aminotransferase) (EC 2.6.1.13) was localized in the livers of rats fed diets differing in protein content with an indirect fluorescent antibody technique. The ornithine aminotransferase specific fluorescence in livers from rats fed a 60% protein diet was distributed throughout the cytoplasm of hepatic cells, and numerous fluorescent cells were observed throughout the lobules. Stained hepatic cells of rats fed a standard laboratory diet (24% protein) appeared to be less numerous and the fluorescence less intense than in rats fed the 60% diet. Livers from rats fed the 0% protein diet showed no specific fluorescence. The identification of ornithine aminotransferase in situ with the fluorescent antibody technique is compatible with measurements of the activity of enzyme extracted from the livers of rats fed similar diets.

HIRSCH, HILDE E.

doi: 10.1177/18.4.268pmid: 5440660

By quantitative histochemical methods, activities of (α-naphthyl) acid phosphatase and β-galactosidase, and of malate and lactate dehydrogenase, were measured in individual human anterior horn nerve cell bodies selected for high and low lipofuscin content. No significant differences were found. However, when single neurons were subdivided into pigmented and unpigmented portions, the two lysosomal enzymes were considerably more active, and malate and lactate dehydrogenase less active, in the lipofuscin-rich part of the cell. These results are consistent with the view that lipofuscin granules are derived from lysosomes.

SHUTER, ELI R.; ROBINS, ELI; FREEMAN, MARY LOU; JUNGALWALA, FIROZE B.

doi: 10.1177/18.4.271pmid: 4985874

A modification of an existing fluorimetric method for the assay of β-glucosaminidase and β-galactosaminidase, based upon the hydrolysis of the corresponding 4-methylumbelliferyl glycosaminides, is described. The method is simple and convenient; its sensitivity is 3 x 10–8M for the assay of β-glucosaminidase and 1 x 10–8M for β-galactosaminidase. The results obtained on brain homogenates and dissected sections of cerebellar cortex and subjacent white matter of rat, rabbit and monkey were found to be similar to those obtained with the standard colorimetric procedures using p-nitrophenyl substrates. In each species, in each layer of cerebellar cortex and subjacent white matter, there were only minimal differences in the ratios of β-glucosaminidase to β-galactosaminidase activity, suggesting that a single enzyme possesses both activities. β-Glucosaminidase and β-galactosaminidase in the cerebellum were most active in the cellular granular layer, suggesting an association of β-hexosaminidase activity with neuronal cell bodies as has been found with other lysomal enzymes.

VRENSEN, G. F. J. M.; DE GROOT, D.

doi: 10.1177/18.4.278pmid: 5440661

Quantitative aspects of submicroscopic autoradiography, using the flat substrate technique, were studied. Using Kodak D19b as developer efficiency values of about 37 for the Ilford LA emulsion and about 17 for the Gevaert-Agfa Scientia Nuc 3.07 emulsion were obtained with H3-methacrylate sections of gray interference colors. Theoretical and experimental data revealed that the high standard deviations of efficiency values obtained are caused mainly by variations in section thickness within ribbons of sections, which on account of identical interference colors were considered to be equally thick. Measurements of efficiency on sections with thickness variations between 400 and 2500 Å revealed that self-absorption is an important factor in tritium autoradiography at the electron microscopic level.

GALJAARD, H.; BUYS, J.; VAN DUUREN, M.; GIESEN, J.

doi: 10.1177/18.4.291pmid: 4314887

The effect of low doses of x-irradiation (50-400 R) on the activity of various enzymes in rat intestinal epithelium has been investigated by histochemical staining methods and quantitative microchemical analyses of crypts and villi dissected from frozen-dried sections. Irradiation had no effect on the activities of enzymes which in nonirradiated animals are present exclusively or mainly in the villus epithelium (aminopeptidase, various phosphatases) or are evenly distributed along the epithelium of crypts and villi (various dehydrogenases). However, nonspecific esterase activity decreased markedly both in crypt epithelium and villus epithelium. This occurred 36 hr after irradiation, independent of the radiation dose. The number of crypt cells with reduced esterase activity and the duration of the effect increased with higher radiation doses. These results were confirmed by quantitative analyses which also showed that esterase activity is 5 times higher in the villus than in the crypt. The remarkable correspondance between the period of reduced esterase activity in the crypt and the period of increased proliferative activity after various radiation doses suggest a relationship between changes in crypt cell population dynamics and esterase activity; the functional consequences for the villus epithelium of changes in the crypt cells after irradiation are discussed.

ZACKS, S. I.; SAITO, A.

doi: 10.1177/18.4.302pmid: 5440662

doi: 10.1177/18.4.305pmid: N/A

doi: 10.1177/18.4.312pmid: N/A

In the January issue, Volume 18, Number 1, of this Journal, in the article by Paul K. Nakane on page 9, the sentence beginning in the fifth line of the abstract should read "TSH cells were scarce at the periphery of the gland."