Ultrastructural localization of a lysosomal enzyme in resin-embedded lymphocytes

The intracellular distribution of lysosomal enzymes in lymphocytes has previously been only poorly defined, mainly by cytochemical procedures of low resolution. In the present study we have used a post-embedding immunogold technique to identify the precise ultrastructural localization of a lysosomal enzyme, beta-glucuronidase, in activated lymphocytes embedded in Lowicryl K4M resin. We show that this enzyme is present in the rough endoplasmic reticulum, in the Golgi complex, and in vesicular organelles which probably include lysosomes.     

Direct enzyme transfer from lymphocytes corrects a lysosomal storage disease

Fibroblasts from patients with mannosidosis, the lysosomal storage disease resulting from an inherited deficiency of lysosomal alpha-D-mannosidase (EC 3.2.1.24), accumulate specific mannose-containing oligosaccharides which are characteristic of the disease (1,2). The present study shows that these substances were extensively degraded following transfer of the missing enzyme from normal lymphocytes to mannosidosis fibroblasts on direct contact in tissue culture. Moreover, prolonged correction of the metabolic abnormality of the recipient cells was sustained if contact with fresh donor lymphocytes was periodically renewed. These findings may be highly relevant to lymphocyte function in enzyme replacement therapy by transplantation procedures currently being attempted.     

Ultrastructural localization of lysosomal enzymes in the egg cortex of Brachydanio

The localization of acid phosphatase (E.C. 3.1.3.2), inorganic trimetaphosphatase (E.C. 3.6.1.2), and aryl sulfatase (E.C. 3.1.6.1) in the cortex of unactivated and activated eggs of Brachydanio was examined by ultrastructural cytochemistry. Using a lead capture method, activity for all three acid hydrolases was demonstrated in organelles of the cortex before and after egg activation. Acid phosphatase (AcPase) reaction product was consistently present in primary lysosomes, secondary lysosomes, multivesicular bodies, and yolk bodies. AcPase activity was absent from mitochondria, profiles of the endoplasmic reticulum, coated pits of exocytosed cortical granules, and coated vesicles. Although most cortical granules of the mature, unactivated egg were unreactive for this enzyme, a few showed AcPase reaction product. It is not clear whether the AcPase-positive granules might be an immature form of cortical granules or a subpopulation of these organelles with lysosomal properties. Most cisternae of the Golgi apparatus did not stain for AcPase; however, reaction product was occasionally localized in a single cisterna as well as several small vesicles at the inner face of the Golgi. The intensity of the reaction product and the pattern of distribution of trimetaphosphatase (Tm-Pase) activity was very similar to that of AcPase. However, TmPase was never observed in cortical granules. Cortices of unactivated and activated eggs showed less overall aryl sulfatase (ArSase) activity when compared with AcPase and TmPase. The presence of ArSase reaction product in lysosomes and multivesicular bodies confirmed the acid hydrolytic nature of these organelles. AcPase and TmPase, and to a lesser extent ArSase, are adequate markers of a cortical lysosomal system in the danio egg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural localization of cholesterol by enzyme histochemistry

Summary The histochemical localization of cholesterol using oxidized diaminobenzidine as the final reaction product was studied at the electron microscopical level and compared with the digitonin method of cholesterol localization based on cholesterol digitonide as the final reaction product. Tissue chopper sections of fixed rat adrenal glands were incubated at 37° C in a medium consisting of 0.8 units/ml cholesterol oxidase, 1.4 units/ml cholesterol ester hydrolase, 50 units/ml horseradish peroxidase, 0.5 mg/ml diaminobenzidine, 0.1% v/v Triton X-100 (or Surfal) and an endogenous peroxidase inhibitor in 0.1m phosphate buffer, pH 7.0. An electron-dense osmiophilic reaction product was observed in many lipid droplets, intracellular vesicles and focally around mitochondria. Appropriate control experiments indicated that deposition of reaction product depended on the presence of cholesterol and the necessary enzymes. Comparison studies using digitonin confirmed the presence of cholesterol in the lipid droplets, but ultrastructural distortion limited the resolution of the more discrete deposits of cholesterol such as around mitochondria. The enzyme method permits finer resolution of these discrete deposits of cholesterol than the digitonin method because it does not cause distortion of cellular ultrastructure attributed to the formation of cholesterol digitonide. The enzyme method or a combination of enzyme and digitonin enables localization of free, esterified or total cholesterol.     

Effects of mitogenic stimulation of lymphocytes on lysosomal enzyme activity

Changes in the activities of several lysosomal enzymes were studied during transformation of mouse spleen cells in vitro. The activity of beta-glucuronidase increased during culture in the presence of T or B-cell mitogens, and lymphoblasts contained higher levels of activity than did small, non-transformed lymphocytes. Moreover, lymphoblasts in well-transformed cultures had higher activities than those in poorly-transformed cultures. The activities of other lysosomal enzymes (N-acetyl-beta-glucosaminidase, alpha-mannosidase, beta-glucosidase) also increased during mitogenic stimulation, but each at different rates, although aryl sulphatase was unaffected. Such differences may be of importance when lymphocytes are used for diagnosis of inherited lysosomal deficiency diseases.     

Ultrastructural investigations on the lysosomal system of lymph node lymphocytes in transplantation immunity.

Activation of the lysosomal system in T lymphocytes is demonstrated in connexion with immune stimulation, by electron microscopic study of the lymph nodes draining skin allografts. The development of primary lysosomes from enlarged Golgi apparatus, their fusion with autophagic and secretory vacuoles forming secondary lysosomes, of the cytolysome and crinophagosome type, were identified by the ultrahistochemical acid phosphatase reaction, suggesting the involvement of these organelles either in blastic transformation or in the effector function of lymphocytes, in connexion with the elaboration of active factors and cytotoxic events.     

Ultrastructural localization of adenosine triphosphatase activity in lymphocytes activated in vitro by phytohaemagglutinin

Summary The ultrastructural localization of Ca²⁺, Mg²⁺-activated ATPase was studied in phytohaemagglutinin activated lymphocytes and in normal unstimulated lymphocytes. Cells, fixed in paraformaldehyde-glutaraldehyde, were incubated in a medium containing 3mm ATP, 5mm CaCl₂ and 2.4mm Pb(NO₃)₂ in 0.1m tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized i the endoplasmic reticulum, nuclear membrane, Golgi apparatus and mitochondria and on the membrane surrounding large electron-dense bodies. Cytoplasmic vesicles and the plasma membrane were negative. Activity in unstimulated lymphocytes showed a similar localization but the amount of endoplasmic reticulum was much less than in activated lymphocytes.The pH of the medium was critical for the localization of the enzyme. At pH 7.5, the cytoplasmic reaction was almost completely inhibited but a dense precipitate was present on the outer surface of the plasma membrane. The reaction was stimulated by either Ca²⁺ or Mg²⁺ and was greatly decreased in the absence of these cations or in the presence ofp-chloromercuribenzoate orN-ethylmaleimide. Oligomycin inhibited selectively the reaction in mitochondria but not the reaction at other sites. While the reaction in mitochondria showed complete substrate specificity, a mild reaction was obtained at the other sites with uridine diphosphate or sodium -glycophosphate as substrate. ATP was, however, the preferential substate.     

Ultrastructural localization of cellulase in Trichoderma reesei using immunocytochemistry and enzyme cytochemistry

Two components of the cellulase complex (E.C. 3.2.1.4) of the fungus Trichoderma reesei were localized at the ultrastructural level. Immunocytochemistry and enzyme cytochemistry demonstrated that cellobiohydrolase and beta-1,4 glucanase were localized within cisternae of endoplasmic reticulum and within membrane complexes of cellulose-grown hyphae. Both enzymes were also present in the culture medium. Glucose-grown control hyphae lacked enzyme-specific staining, and no enzyme activity was detected in the growth medium.     

Dehydrogenase enzyme histochemistry on freezedried or fixed resin-embedded tissue

Summary A method has been developed for the histochemical demonstration of a variety of dehydrogenases in freeze-dried or fixed resin-embedded tissue. Seven dehydrogenases were studied. Lactate dehydrogenase, NADH dehydrogenase and NADPH tetrazolium reductase were all demonstrable in sections of paraformaldehyde-fixed resin-embedded tissue. Freeze-dried specimens were embedded, without fixation, in glycol methacrylate resin or LR Gold resin at either 4°C or –20°C. All the dehydrogenases except succinate dehydrogenase retained their activity in freeze-dried, resin-embedded tissue. Enzyme activity was maximally preserved by embedding the freeze-dried tissue specimens in glycol methacrylate resin at –20°C. The dehydrogenases were accurately localized without any diffusion when the tissue sections were incubated in aqueous media. Addition of a colloid stabilizer to the incubating medium was not required. Freeze-drying combined with low-temperature resin embedding permits accurate enzyme localization without diffusion, maintenance of enzyme activity and excellent tissue morphology.     

Hydrolysis of phospholipids by a lysosomal enzyme

The phospholipid-hydrolyzing activity of rat liver lysosomes has been studied. These lysosomes contain a phospholipase that cleaves both fatty acid ester linkages of lecithin and of phosphatidyl ethanolamine and releases free fatty acids from both positional isomers of lysolecithin. The enzyme does not require calcium for maximum activity, and is inhibited by diethyl ether and sodium deoxycholate. Mercuric ions and cetyltrimethyl ammonium bromide also inhibit the hydrolysis. Compared with lipase activity, this enzyme is relatively stable to heat. The specific activity of the hydrolysis of lecithin by the lysosomal enzyme is considerably higher than those reported for mitochondrial and microsomal phospholipases. The enzyme resembles other hydrolases of the lysosome in that it has an acid pH optimum (pH 4.5). This enzymic activity is present in both the lysosomal soluble enzyme fraction and in the lysosomal membrane fraction. The enzyme may participate in the intracellular digestion of mitochondria that is carried out by the intact lysosome in vivo. Localized inflammation and changes in vascular permeability following tissue damage could be catalyzed by this phospholipase.     

Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts

The activity of a lysosomal enzyme, alpha-D-mannosidase (EC 3.2.1.24), increased markedly in normal lymphocytes when they were cultured together with fibroblasts from a patient with an inherited deficiency of this enzyme. Cell-to-cell contact was obligatory for this increase in activity, which also required new protein synthesis. The enzyme induced in the co-cultured lymphocytes was a high molecular weight form of alpha-D-mannosidase that was not detected in lymphocytes cultured alone, which had only the low molecular weight mature enzyme. It was this precursor form alone that was directly transferred to the mannosidosis fibroblasts, where it was present initially in organelles of low density. When the culture period was extended the lymphocyte precursor enzyme was transported to the heavy lysosomes in the recipient cells, and correctly processed to the functionally effective mature enzyme.     

Angiotensin I converting enzyme in human intestine and kidney. Ultrastructural immunohistochemical localization

The localization of immunoreactive angiotensin I-converting enzyme (ACE) has been investigated at the optical and ultrastructural level with anti-human ACE antibodies in the human kidney and small intestine. In both tissues ACE was found in blood vessels and in extravascular situation in the absorptive epithelial cells of intestinal mucosa and renal proximal tubules. Ultrastructural immunohistochemistry showed that in intestinal and renal proximal tubular cells ACE was prominent in microvilli and brush borders. In the kidney ACE was also present on the basolateral part of the plasmalemmal membrane, where it may contribute to the regulation of angiotensin II-dependent absorption processes. Intracellular positivities were also observed inside the renal vascular endothelial and proximal tubular cell in endoplasmic reticulum and nuclear envelope reflecting the synthesis and the cellular processing of ACE. The intestinal microvascular endothelium was strongly labeled suggesting that the mesenteric circulation is an important site for the production of angiotensin II. Vascular endothelial ACE was also detected in the peritubular but not glomerular capillaries of the kidney.     

Ultrastructural localization of a breast tumor-associated antigen

We used a monoclonal antibody to a high molecular weight glycoprotein of the milk fat globule membrane to study at the ultrastructural level the sites of accumulation of immunoreactive material in breast tumors. By light microscopy, the antigen was found only on the luminal membrane of normal resting or lactating breast cells. In tumors, antigen could be found in the cytoplasm. We used the avidin-biotin-peroxidase complex immunoperoxidase technique to localize the site of cytoplasmic accumulation of immunoreactive material in breast tumors. This technique showed that antigen is found on the membrane of cytoplasmic vesicles. Some of these vesicles were collapsed and gave rise to what appeared to be clumps of free cytoplasmic immunoreactive material. We have also documented that in some tumors the entire cytoplasmic membrane bears antigen, whereas in other tumors only areas of cytoplasmic membrane that form microscopic lumina express antigen.     

Ultrastructural localization of gamma-chain and immunoglobulin G in human lymphocytes using enzyme-labeled Fab fragment.

By the use of rabbit antibodies against the heavy chain of human immunoglobulin G (IgG), the gamma-chain and IgG molecules were successfully localized at the ultrastructural level in human peripheral lymphocytes. The rabbit Fab fragment was coupled to horseradish peroxidase by means of glutaraldehyde and the resulting conjugate could penetrate the intact plasma membrane. Discernible reaction product was observed in cisternae of the nuclear envelope, rough endoplasmic reticulum and Golgi apparatus as well as on the surface of the lymphocytes. In normal human individuals under no specific antigenic stimulation, only a few peripheral lymphocytes showed a rare positive intractoplasmic reaction. Reaction product may represent either the whole IgG molecule, the half molecule consisting of one heavy and one light chain or nascent gamma-chain.     

Acquisition of a lysosomal enzyme by myoblasts in tissue culture

Skeletal muscle myoblasts from different sources acquired high levels of the lysosomal enzyme beta-glucuronidase, when they were cultured together with mitogen-activated lymphocytes. Immunofluorescent staining, thermal stability, and electrophoretic mobility showed that the increase in enzyme activity in the myoblasts was due to the presence of the lymphocyte form of the enzyme. Although myoblasts were able to take up exogenous beta-glucuronidase from the culture medium by mannose 6-phosphate receptor-mediated endocytosis, enzyme acquisition during co-culture with lymphocytes was independent of this pathway. Enzyme transfer from the lymphocytes was found to require direct cell-cell contact with the muscle cells, and was accompanied by an increase in beta-glucuronidase activity in the lymphocytes themselves. Since this additional activity was also due to the presence of the lymphocyte form of the enzyme, these results indicate that interaction with the muscle cells induced the de novo synthesis of beta-glucuronidase in the lymphocytes.     

Ultrastructural localization of acetylcholinesterase

Summary A method is described allowing localization of acetylcholinesterase (AChE) by both light and electron microscopy. During the reaction lead thio-diacetyl is decomposed, and therefore precipitated as PbS in the presence of native-SH group produced by the hydrolysis of acetylthiocholine perchlorate. The reaction takes place at neutral pH, since improves the sensitivity of AChE localizations. Application of the method to parasympathetic neurons showed that AChE was mainly localized in the rough endoplasmic reticulum of the perikaryons. No reaction was visible in glial cells. AChE was also localized on the plasma membrane of parasympathetic neurons. In mouse embryo muscles AChE activity was seen to be high and was not yet restricted to the synaptic area. The well developed Schwann cells accompanying the neurites displayed constant AChE activity on their plasma membrane.Supported by a grant of INSERM C.R.L. N⁰ 79-5-318-6