New tetrazolium method for the histochemical demonstration of gamma-glutamyl transpeptidase.

A new tetrazolium method for the histochemical demonstration of gamma-glutamyl transpeptidase is proposed. The method is based on a newly synthesized substrate-gamma-L-glutamic acid-1-hydroxy-4-naphthylamide, which upon the enzyme hydrolysis liberates 1,4-aminonaphthol--a powerful reducing agent that reduces tetrazolium salts quickly and quantitatively to deeply colored, water-insoluble formazans, precipitating on the sites of the enzyme activity and marking them accurately. The redox reaction is quick enough and does not need any auxiliary electron-acceptor. The method is very fast and convenient for the histochemical visualization of the enzyme.     

The electron histochemical demonstration of cystine-containing proteins in the guinea pig hair follicle

Summary A previously described electron histochemical method for detecting cystine in fully keratinised human hair has been modified for the examination of this amino acid residue amongst the cellular components of anagen guinea pig hair follicles. Using the technique the progressive incorporation of cystine into the hair cuticle and cortex has been observed. Although cystine was absent from the hair medulla and outer root sheath cells at all stages of development, a narrow layer of cystine-containing material was found adjacent to the cell membranes of hardened inner root sheath cells.     

The use of tetrazolium salts in the histochemical demonstration of succinic dehydrogenase activity in plant tissues

Summary Succinic dehydrogenase activity was determined in fresh or cryostat sections of tissues from Allium cepa, Vicia faba, Pisum sativum and Helianthus tuberosus using different tetrazolium salts as electron accepters. In 10 fresh or frozen sections a reaction was obtained with TNBT, NBT, MTT, and INT but not with NT, BT or TTC. In contrast a reaction was obtained with each of the tetrazolium salts in 50–150 fresh or frozen sections. The observed differences in the abilities of the tetrazolium salts to demonstrate succinic dehydrogenase activity are discussed.The sites of acceptance of electrons from the electron transport pathway in plant cells by the tetrazolium salts has been demonstrated cytochemically, and shown to differ from those observed in animal cells in that unlike animal cells, there is no apparent acceptance of electrons by MTT and INT from cytochrome C₁-C region of the pathway.     

Fluorescence methods for the histochemical demonstration of monoamines

Summary The histochemical fluorescence method of Falck and Hillarp for the demonstration of catecholamines and certain tryptamines, e.g. 5-hydroxytryptamine is based on the principle that these amines can be condensed with formaldehyde to yield strongly fluorescent 6,7-dihydroxy-3,4-dihydroisoquinolines and 6-hydroxy-3,4-dihydro--carbolines respectively. The investigation here reported presents the fluorescence characteristics and relative fluorescence yields for formaldehyde treated biogenic monoamines and certain related compounds enclosed in a dried protein layer. The fluorescence properties of some synthetic 6,7-substituted-3,4-dihydroisoquinolines and 3,4-dihydro--carbolines are given, and the fluorescence characteristics in relation to the molecular structure are discussed.Abbreviations used A adrenaline - DA dopamine - DOPA 3,4-dihydroxyphenylalanine - DOPS 3,4-dihydroxyphenyl-serine - 5-HT 5-hydroxytryptamine - 5-HTP 5-hydroxytryptophan - 5-MT 5-methoxytryptamine - -m-DA -methyl-dopamine - -m-DOPA -methyl-3,4-dihydroxyphenylalanine - -m-NA -methyl-noradrenahne - MTA 3-methoxy-tyramine - NA noradrenaline - NM normetanephrine - T Tryptamine - Try Tryptophan     

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV.

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV (DPP IV), based on a newly synthesized substrate Gly-L-Pro-1-hydroxy-4-naphthylamide is proposed. Upon the enzyme hydrolysis of the substrate a strong reducing agent, i.e. 4-amino-1-naphthol is released, which reduces tetrazolium salts to water-insoluble, deeply colored formazans, that precipitate on the sites of enzyme activity, marking them accurately. No auxiliary electron acceptor is needed for the redox reaction. The incubation is performed at the optimal pH of the enzyme. Precise enzyme localization is achieved in all organs studied. Thus, the new method avoids most of the disadvantages of the methods in use and might open new possibilities in peptidases histochemistry.     

The possibilities and limitations of membrane methods for the histochemical demonstration of cholinesterases

Summary The thiocholine method for the histochemical detection of cholinesterases according to Karnovsky-Roots was adapted for unfixed cryostat sections by addition of the agar solution to the incubation mixture and by using the semipermeable membrane interposed between the section and the incubation medium. The procedure prevents the leakage of the enzyme activity of the section and is suitable for tissues where the cholinesterase activity is low.     

The histochemical demonstration of peptidases by natural substrates

Summary Methods for the demonstration of peptidase activities in situ using natural peptides as substrates are described and evaluated. They are based on the use of the l-amino acid oxidase of Vipera ammodytes venom (or of similar snake venom) which is able to catalyze efficiently the oxidation of Leu, Met, Phe, Ileu, Tyr, and Try set free by the action of the particular peptidase(s). Accordingly, peptides from which the mentioned aminoacids are set free can be used as substrates. Cryostat sections adherent to slides are employed which can be preextracted with chloroform-acetone mixture (, 4°C, 5 min) to eliminate lipids. All substances needed for this multistep reaction are prepared and applied in a suitable gel layer which impedes the diffusion of soluble peptidases and also of l-amino acids set free by their action. The enzyme activity is assessed from the difference between the coloration of a pair of parallel sections one of which was incubated with the substrate and the other without it.There are two possible methods: a) The method using l-amino acid oxidase-phenazonium methosulphate and NitroBT (AAO-PMS-NBT method) works in the pH range 6.5–8 and requires anaerobic conditions. The medium consists of buffered substrate, Vipera ammodytes venom, PMS, NBT and KCN and is prepared preferentially in 10% gelatine. It is allowed to set on coverslips which are picked up by slides with sections and incubated at room temperature in the dark. In addition to a quick information on the firmness of the structure association of the peptidase splitting the substrate and on the influence of the fixation on its activity the method enables its cellular localization. Microdensitometric evaluation of formazan deposition during incubation is possible (there is a linear increase in density at least up to 20 min of incubation). b) The method using l-amino acid oxidaseperoxidase-3,3-diamino benzidine hydrochloride (AAO-PO-DAB method) works in the pH range 6–9 and requires aerobic conditions. The medium consists of buffered substrate, Vipera ammodytes venom, PO and DAB and is prepared preferentially in 1% agar solution which is applied on sections either directly or as a sandwich after gelification on a slide. The incubation proceeds at 37°C. The method furnishes similar information as the preceding one, however it is less sensitive and enables histological localization only.The suitability of these methods was shown among others on the demonstration of peptidases of the gastrointestinal tract and of the arterial wall. Biochemical findings reported so far were not only confirmed but entirely new data ascertained.In the human and rat intestinal mucosa the bulk of activities directed towards dipeptides (Leu-Leu, Leu-Gly, Gly-Leu, Ser-Met) is soluble. Peptidases splitting Leu-Leu-Leu are somewhat more structure bound in the human than in the rat intestine. Activities of these peptidases which are present even in crypt enterocytes increase during the process of the differentiation of enterocytes so that enterocytes covering sites and tops of villi display the highest staining intensity. The contribution of cells of the propria to the overall activity of the mucosa of the small intestine is not decisive. As against aminopeptidases A and M, dipetidyl peptidase IV and endopeptidase enzymes splitting natural di-and tripeptides can be demonstrated also in the gastric epithelium. However, in enterocytes the corresponding activity (activities) is (are) substantially higher already in the duodenal bulb. The differences in the staining intensity between gastric epithelium and enterocytes are lower in the case when Ser-Met is used as substrate than in the case when Leu-Leu; Gly-Leu, Leu-Gly and Leu-Leu-Leu are used as substrates. In patients suffering coeliac sprue the activity of peptidases residing in enterocytes splitting the substrates given above is lower in the acute stage of the disease than in normal persons. However, these soluble peptidases seem to be affected less than the brush border peptidases (in decreasing order): endopeptidase, -glutamyl-transferase, dipeptidyl peptidase IV, aminopeptidase A and aminopeptidase M.In the human aortae the highest activity was recorded using Ser-Met and Leu-Met as substrates, followed (in the decreasing order) by Leu-Leu, Leu-Leu-Leu, Met-Met-Met, Met-Val and Ser-Leu-Leu. No activity was recorded when N-CBZ-Leu-Met was used as the substrate. In normal human aorta the highest activity resides in vasa vasorum followed by muscle cells of the media. In cells of atherosclerotic plaques the activity is usually lower, except in cells of fatty streaks and small lipoid plaques where the activity can surpass that found in other cells of the aorta.     

A substrate-film method for the histochemical demonstration of cellulase

Summary A substrate-film method has been devized for the histochemical demonstration of cellulase. The substrate film is made of sodium carboxymethyl-cellulose, which is made insoluble in water by fixation in acid ethanol. The tissue is briefly fixed in cold formalin, washed, and sectioned with a freezing microtome. The sections are mounted on slides, and covered with a piece of carboxymethyl-cellulose film, and the slide is incubated in a warm, moist atmosphere. After incubation, the film is stained with toluidine blue, and sites of cellulase activity appear as pale or clear patches in the film.In the digestive systems of certain molluscs, cellulase has been found in the lumens of the crop and stomach, and in the lumen and absorptive cells of the digestive gland tubules. The salivary glands, and the epithelia of the crop and stomach, show no reaction.Sections of control tissue, inactivated by boiling in water, do not show any reaction.I thank the Nobel Division of Imperial Chemical Industries Ltd. for information on their product Cellofas B 10, and for permission to publish that information.     

A histochemical method for the demonstration of unsaturated lipids

Summary A method has been developed for the histochemical demonstration of unsaturated lipids in light microscopy. It is a peracetic acid-thiocarbohydrazide-silver protein sequence followed by a physical development procedure. In the present study on paraffin and cryostat sections of liver, brain and ovary, unsaturated lipids were visualized as distinct reaction products coloured various shades of brown and black. The reaction products are easier to see and the method is more efficient than the peracetic acid-Schiff method.     

The demonstration of mouse lung lactate-yellow tetrazolium reductase

Summary Yellow tetrazolium has been examined for use as a qualitative histochemical reagent in light microscopy. Experimental conditions for the demonstration of the lactate-yellow tetrazolium reductase system in mouse lung are described, and the possibility of subsequently demonstrating either NADH-MTT reductase or 3-glycerophosphate-MTT reductase in the same section has also been investigated. The yellow colour of the deposited formazan is uniquely favourable for counterstaining with blue nuclear stains.This paper forms part of a thesis for the Ph. D. degree submitted to the Council for National Academic Awards by J.E.E.     

Studies on ATP hydrolysis in medium for histochemical demonstration of ATPase activity

Summary Nonenzymatic ATP hydrolysis in medium of Wachstein and Meisel for histochemical demonstration of ATPase activity was investigated. In this medium considerable amounts of phosphorus are released without the participation of the enzyme. ATP hydrolysis in Wachstein-Meisel's medium increase with the concentration of Pb⁺⁺ and decrease at its small concentrations. The degree of ATP hydrolysis appeared to increase with increase both temperature and pH. At high concentration of ATP (5.76 mM) the degree of ATP hydrolysis in Wachstein-Meisel's medium is lower than at 1.44 mM ATP. 10.0 mM Ca⁺⁺ or 3.6 mM Fe⁺⁺ speed up ATP hydrolysis after 30- and 60-minute incubation. In the presence of 3.6 mM Co⁺⁺ or 2.6 mM Cu⁺⁺ ATP hydrolysis in Wachstein-Meisel's medium increased throughout the whole period examined. On the contrary, 3.6 mM Fe⁺⁺⁺ decreases ATP hydrolysis in this medium.10.0 mM F^– raises the degree of ATP hydrolysis which is, however, lowered in the presence of 2.5 mM pCMB or 3.6 mM KCN. 2.0 mM cysteine highly inhibits the process of nonenzymatic ATP hydrolysis in Wachstein-Meisel's medium.These data show that the histochemical reaction for ATPase activity in Wachstein-Meisel's medium does not originate exclusively from the hydrolysis of ATP in the presence of Pb⁺⁺, but take rise, above all, as a result of an enzymatic reaction.     

The possibilities and limitations of membrane methods for the histochemical demonstration of cholinesterases.

The thiocholine method for the histochemical detection of cholinesterases according to Karnovsky-Roots was adapted for unfixed cryostat sections by addition of the agar solution to the incubation mixture and by using the semipermeable membrane interposed between the section and the incubation medium. The procedure prevents the leakage of the enzyme activity of the section and is suitable for tissues where the cholinesterase activity is low.