Enzyme histochemistry of human melanomas and pigmented naevi with special reference to alpha-D-mannosidase activity

Summary A histochemical study of-d-mannosidase revealed that normal human melanocytes (resting state, activated, lentigo simplex) exhibit either no or just detectable activity, as do melanocytes in the initial phase of lentigo maligna. Junctional, or occasionally zone A naevocytes displayed a very low enzyme activity. On the other hand, melanocytes in the initial stage of neoplastic transformation (dysplastic naevi, advanced stage of lentigo maligna) and also melanoma cells in disorders of low malignant potential (initial naevogenic melanoma, superficial spreading melanoma) displayed a high activity uniformly throughout the cell population. In the malignant forms (nodular melanoma, recurrences, metastases), the enzyme activity was remarkably heterogeneous, suggesting a breakdown of uniformity during malignant transformation. The significance of -mannosidase activity induction in the course of melanocyte neoplastic transformation is not clear at present. The results of biochemical assays suggest that the lysosomal isoenzyme is mainly responsible. Other lysosomal enzymes, and dehydrogenases studied concomitantly, did not display any comparable phenomena of induction or similar behaviour. However, the results of a comparison of-mannosidase with the melanocyte reference enzyme tyrosinase suggested activity patterns in the enzyme pair which may provide a better insight into the biochemical differentiation of human melanocytes in neoplastic disorders. The possible relationship of-mannosidase to melanogenesis is also discussed.     

Enzyme histochemistry of human brain tumors and their tissue cultures with special reference to the oxidoreductases in the glioblastoma multiforme

Summary Fresh tissue and tissue cultures of 80 glioblastoma multiforme and 12 monstrocellular sarcoma were histochemically investigated. The activity of the following enzymes was demonstrated in the biopsies and tissue cultures of every tumor: NADH-tetrazolium reductase, NADPH-tetrazolium reductase, lactic dehydrogenase, succinic dehydrogenase, glutamic acid dehydrogenase and cytochrome oxidase. No major differences in the relative activity pattern was shown when fresh tissue and tissue cultures were compared, nor did the enzymatic pattern change during the four week observation time.In both groups major quantitative differences in the enzymatic activity of the tumor cells in the same tissue area or tissue cultures were frequently a striking finding. Differences in the intracellular localization of the enzymatic activity were also observed. In slowly growing gliomas these histochemical variations are absent.     

Cytologic observations on the pancreatic islets with reference to some endocrine-like cells of the gastrointestinal mucosa

Summary Some findings obtained from cytological studies on the pancreatic islet of man and different animal species by applying several appropriate methods are reported. Three islet cell types were found to be simultaneously demonstrated by toluidine blue after methylation and saponification of tissue sections; metachromasia appeared to be in some way a constant finding of argyrophil D-cells which, at least in duck and man, displayed also a weak PAS-positivity. No one of the three insular cell types was found to show all the morphologic patterns of enterochromaffine cells, nor enterochromaffine cells showed all the patterns of insular cells. The existence of possible morphologic relationships between insular A-cells and some endocrine-like cells in fundic mucosa and, on the other hand, between argyrophil D-cells and some cells in antro-pyloric mucosa of the stomach is emphasized.This work was supported by the Grant N. 04/57/4/7137 of the Consiglio Nazionale delle Ricerche, Rome.     

Ultrastructure and enzyme histochemistry of the pancreatic islets in the horse

Summary The characteristic localization of the silver-negative A₂ cells in the central part of the pancreatic islets in the horse offers a good opportunity to study the ultrastructure and histochemistry of this type of islet cell. Electron microscopical analyses revealed that the A₂ cells contained dense spherical granules varying considerably in size. Light and dark A₂ cells were identified. The presence of numerous secretory granules of very low density was the most conspicous feature of the B cells. These cells also showed considerable differences in density. A second type of peripheral islet cell was characterized by a very high content of mitochondria and ribosomes. These small islet cells contained tiny granules and are probably identical with the A₁ cells.Negative reactions for alkaline and acid phosphatases were obtained throughout the islet tissue, while a strong glucose-6-phosphatase activity was displayed by the peripheral cells. The diphosphopyridine and triphosphopyridine nucleotide diaphorase activities were high in the peripheral cells, considerably weaker reactions being noted in the A₂ cells. On the whole there was a low succinic dehydrogenase activity in the islet tissue with a somewhat weaker enzyme staining in the A₂ than in the peripheral cells. The reactions for glucose-6-phosphate dehydrogenase and lactic dehydrogenase were also less pronounced in the A₂ cells than in the intensely reacting peripheral cells.The following abbreviations are used DPN Diphosphopyridine nucleotide - DPND Diphosphopyridine nucleotide diaphorase - DPNH Diphosphopyridine nucleotide, reduced form - G-6-PD Glucose-6-phosphate dehydrogenase - LD Lactic dehydrogenase - MTT 3,5-diphenyl-2-(4,5-dimethylthiazol-2-yl)-tetrazolium bromide - Nitro-BT 2,2-di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-biphenylene)-ditetrazolium chloride - SD Succinic dehydrogenase - TPN Triphosphopyridine nucleotide - TPND Triphosphopyridine nucleotide diaphorase - TPNH Triphosphopyridine nucleotide, reduced form Supported by the Swedish Medical Research Council and the research grant A-5759 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.     

Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.

Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative manner in relation to pathophysiological processes to understand its relevance and the potential impact of its targeting by drugs. Proteases, in particular, are crucial in every aspect of life and death of an organism and are therefore important targets. Enzyme activity in living cells can be studied with various tools. These can be endogenous fluorescent metabolites or synthetic chromogenic or fluorogenic substrates. The use of endogenous metabolites is rather limited and nonspecific because they are involved in many biological processes, but novel chromogenic and fluorogenic substrates have been developed to monitor activity of enzymes, and particularly proteases, in living cells and tissues. This review discusses these substrates and the methods in which they are applied, as well as their advantages and disadvantages for metabolic mapping in living cells.     

The islets of Langerhans in ducks and chickens with special reference to the argyrophil reaction

Summary The pancreas of birds is a suitable object for studying the A and B cells separately, since the two cell systems are topografically almost entirely segregated in the form of light (= B cells) and dark (= A cells) islets of Langerhans.On the whole in the chicken and duck the actual distribution of the light islets into different size classes followed the same regular pattern previously found in the rat and man. In the body of the pancreas, containing the great majority of islets, the volume distribution curves thus appeared symmetrical.With the silver impregnation method used a distinct argyrophil reaction in both types of islets was obtained on paraffin sections of the pancreas. According to the presence or absence of blackening, the cells of the dark islets could be divided into two distinct fractions. Especially in the duck the silver-positive cells were grouped in a characteristic way along the walls of the capillaries. Ducks and chickens are not the only animals in which it is possible to identify an argyrophil fraction in what the usual granule stains had shown to be A cells. Parallel studies of various mammals are in complete agreement with these observations. It is, however, still uncertain whether we are here dealing with differences in function, age etc. in one and the same type of cell or with two completely different kinds. No correlation between the argyrophil reaction in the dark islet cells and their content of SH and SS groups or tryptophane could be established.This work was aided by grants from the Swedish Medical Research Council.     

Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase

Summary This paper deals with the progress made over the last few years in our understanding of enzyme cytochemical staining methods as studied using a fundamental approach with the aid of a model system of thin gel films. Although model films with a matrix of polyacrylamide have been mostly used, the properties and possible applications of other matrices are also reviewed. The chemical aspects of the entrapment of enzyme molecules into a matrix are summarized. Special attention has been paid in model film studies to the principles of the trapping reaction of a diffusable precursor resulting from the enzymatic conversion of a substrate. They are considered here as they concern the cytochemical demonstration of acid phosphatase activity with a lead salt. The effect of fixatives on different enzyme activities, the diffusion rate of substrates and chromogenic compounds to the enzyme site, and enzyme kinetics under cytochemical conditions are also discussed, since they are factors which influence the final results of the staining procedures. The advantage of model film studies in enabling the direct correlation of cytochemical and biochemical results is outlined with special reference to the cytochemical determination of glucose-6-phosphate dehydrogenase with Tetra Nitro BT. A method for determining enzyme activities in the soluble fraction of isolated cells after incorporation in model films is described for the first time. This method has proved to be highly appropriate for microscopical observations of glucose-6-phosphate dehydrogenase activity in single cells, because it results in a good morphology and no formazan precipitaties outside the cells. On the other hand, this type of model film forms a bridge between fundamental model film studies using purified enzyme and quantitative enzyme cytochemistry performedin situ.     

Determination of calcium in microgram amounts of dried biological material by flameless atomic absorption spectrophotometry with special reference to the pancreatic islets.

Flameless atomic absorption was used for direct measurements of calcium in microgram amounts of dried biological material. Sufficient analytical precision was reached with a commercial instrument complemented with a device allowing proper control of the heating of the graphite tube furnace. A comparison of different organs in the mouse revealed particularly high concentrations of calcium in the pancreatic islets. β cell-rich pancreatic islets from obob mice contained 60 mmol of calcium/kg dry weight. This value was reduced by 20 mmol during 120 min of washing in physiological saline. There was less calcium in the endocrine and exocrine parts of the directly excised pancreas than in samples incubated in Krebs-Ringer bicarbonate buffer. Islets microdissected from genetically diabetic mice were similar to those from nondiabetic controls with regard to the contents of both readily mobilizable and slowly exchangeable calcium.     

An immunocytochemical study of the distribution of pancreatic endocrine cells in chicks,with special reference to the relationship between pancreatic polypeptide and somatostatin-immunoreactive cells

Summary Araldite sections of formalin-fixed pancreas from chicks at hatching were treated by an indirect immuno-enzyme technique to reveal cells containing APP, somatostatin, glucagon and insulin.APP cells were found scattered in the exocrine parenchyma. A few were associated with insulin-containing B islets and occasional cells occurred in and around glucagon-containing A islets. Somatostatin-immunoreactive cells were distributed peripherally in A and B islets and were dispersed in the exocrine tissue. APP cells were roughly as numerous in the exocrine parenchyma as somatostatin-immunoreactive cells.Since certain published observations point to the possible occurrence of APP and somatostatin in the same cells, consecutive sections were stained for these hormones. In no case did the two peptides occur in the same cell. Sections subjected to double-staining confirmed this result. Therefore it is likely that the described differences between APP and somatostatin-immunoreactive cells are valid.     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

An immunocytochemical study of the distribution of pancreatic endocrine cells in chicks, with special reference to the relationship between pancreatic polypeptide and somatostatin-immunoreactive cells.

Araldite sections of formalin-fixed pancreas from chicks at hatching were treated by an indirect immuno-enzyme technique to reveal cells containing APP, somatostatin, glucagon and insulin. APP cells were found scattered in the exocrine parenchyma. A few were associated with insulin-containing B islets and occasional cells occurred in and around glucagon-containing A islets. Somatostatin-immunoreactive cells were distributed peripherally in A and B islets and were dispersed in the exocrine tissue. APP cells were roughly as numerous in the exocrine parenchyma as somatostatin-immunoreactive cells. Since certain published observations point to the possible occurrence of APP and somatostatin in the same cells, consecutive sections were stained for these hormones. In no case did the two peptides occur in the same cell. Sections subjected to double-staining confirmed this result. Therefore it is likely that the described differences between APP and somatostatin-immunoreactive cells are valid.