Edward Essner

THE LOCALIZATION OF PHOSPHATASE ACTIVITIES AT THE LEVEL OF ULTRASTRUCTURE

produ(-ts froni alkaline afl(l aci(l IItosl)hattLSe activities are detectallle in the electron microscope. Since then electron microscopy has demonstrated a number of reactiomi product localizations beyond the resolution of light microscopy. In several iuistaua-es it has removed the subjective element from localizations deduced from light microscopy. Although too little is known, particularly at levels of significance for electron microscopy, regar(liflg adsorption of reaction product or enzyme, and the mode of pre(-ipitation of metal 1)hOsllhates, significant intracellular localizations can now be described with coulsideral)le (-onfidenc(-, as imidicated in Text Fig. 1. In the early work, incubations for enzyme activities were performed on small blocks of tissime pre-fixe(1 iii either osmium tetroxide (briefly) or in formaldehyde-calcium; following incubation for enzyme activity the tissue was

NUCLEOSIDE DIPHOSPHATASE AND THIAMINE PYROPHOSPHATASE ACTIVITIES IN THE ENDOPLASMIC RETICULUM AND GOLGI APPARATUS

The effects of pH, fixatives and divalent ions on nucleoside diphosphatase (NDPase) and thiamine pyrophosphatase (TPPase) activities in the endoplasmic reticulum (ER) and Golgi apparatus (GA) were examined in adult and neonatal hepatocytes and other cell types in the rat. In liver cells TPPase and NDPase both have a similar localization in the rough ER, nuclear envelope and smooth ER but differ in their pH optima; TPPase is most active at pH 8, NDPase at pH 7. TPPase in the GA, unlike its counterpart in the ER, is most active at neutral pH. High levels of NDPase activity are present in the GA of neurons, epididymis and other cells, but not in hepatocytes. TPPase in the ER, but not the GA, is stimulated by the addition of adenosine triphosphate to the medium. These observations show that different conditions are required to demonstrate ER and GA diphosphatase activities. Whether separate enzymes or multiple configurations of a single protein are responsible for these activities cannot be determined by staining procedures.

Distribution of anomalous lysosomes in the beige mouse: a homologue of Chediak-Higashi syndrome.

The beige mouse has been considered as a homologue of Chediak-Higashi syndrome in man. A light microscopic survey of 23 beige mouse tissues, using acid phosphatase as a lysosomal marker, revealed the presence of enlarged (anomalous) lysosomes, often in the form of aggregates, in 15 of the tissues examined. The degree of anomaly varied from one tissue to another and within cells of a given tissue. The most striking alterations occurred in liver parenchymal cells, kidney proximal tubule cells, Purkinje cells and granulocytes. Electron microscopy of liver and kidney revealed enlarged lysosomes containing numerous lipid-like inclusions. The widespread occurrence of anomalous lysosomes strengthens the homology between the beige mouse and Chediak-Higashi syndrome and further supports the concept that lysosomes are intimately involved in this disorder.

Permeability of rat choriocapillaris to hemeproteins. Restriction of tracers by a fenestrated endothelium.

The choriocapillaris is the fenestrated capillary bed in the choroid of the eye and is the major blood supply to the retinal pigment epithelium (RPE) and photoreceptor cells. Bruchs membrane (BM) is a multilaminated basement membrane that separates the choriocapillaris from the RPE. In a previous study (Pino RM, Essner E; Cell Tissue Res 208:21, 1980) we found that the choriocapillary endothelium restricted the egress of ferritin from the choriocapillaris. In the present study, hemeproteins were used to further establish the permeability characteristics of this capillary bed. Horseradish peroxidase (Einstein-Strokes radius (ESR), 30 A) rapidly crossed the capillary endothelium (less than 5 min) after intravenous administration and after 5 minutes filled BM and the basal infoldings of the RPE. In contrast, hemoglobin (Hg) (ESR, 32 A) and lactoperoxidase (LP) (ESR, approximately 40 A) are markedly restricted at the level of endothelial diaphragmed fenestrae, channels, and intercellular junctions. Little vesicular transport of these proteins was observed. The reaction product of the two hemeprotein activities was not demonstrable in BM for up to 30 min after injection; relatively low levels were detected after 75 min. HG and LP appear to be further restricted by BM, since their reaction products were not demonstrable between the RPE basal infoldings at this time. Catalase (ESR, 52 A) activity was not detected in BM for up to 4 hr after injection. These results indicate that the rat choriocapillary endothelium, unlike the fenestrated endothelia lining other vascular beds, substantially restricts the passage of large tracer molecules.

DISTRIBUTION OF ACID PHOSPHATASE, β-GLUCURONIDASE AND N-ACETYL-β-GLUCOSAMINIDASE ACTIVITIES IN LYMPHOCYTES OF LYMPHATIC TISSUES OF MAN AND RODENTS

Acid phosphatase, β-glucuronidase and N-acetyl-β-glucosaminidase activities were demonstrated in lymphocytes of man and various rodents using formalin-fixed cryostat sections. Lymphocytes were heterogeneous in respect to the activity of these enzymes, depending on the location of cells in the lymphatic tissues. Acid phosphatase activity was demonstrable in lymphocytes located in the diffuse lymphatic tissue of the cortex of the lymph node and around the central arteriole of the splenic white pulp of all species studied. In man and in rat, the majority of lymphocytes in these areas also showed β-glucuronidase activity whereas, in the mouse, none had β-glucuronidase but many had N-acetyl-β-glucosaminidase activity. Most of the lymphocytes in the mantle of the germinal centers and in the lymphatic nodules of the splenic white pulp were negative for all three enzymes studied. The thymic lymphocytes were positive for acid phosphatase, but usually negative for β-glucuronidase and N-acetyl-β-glucosaminidase. The possible significance of these differences is discussed.

Localization of carbohydrate components in rat colon with fluoresceinated lectins.

Cryostat sections of rat descending colon were studied by fluorescence microscopy after exposure to conjugates of fluorescein isothicoyanate with lectins from Glycine max (soybean), Triticum vulgaris (wheat germ), Ricinus communis (castor bean), Ulex europaeus, (gorse), Dolichos biflorus (horse gram) and Canavalia ensiformis (concanavalin A) (Jack bean). No two lectins showed identical patterns of fluorescence. FITC-conjugates of soybean and D. biflorus lectins reacted strongly with the mucus present in the crypt lumens and with the surface (as well as cytoplasm) of the epithelial cells suggesting that these sites are rich in terminal, non-reducing, N-acetylgalactosamine residues. Wheat germ, R. communis, U. europaeus and concanavalin A-FITC conjugates did not stain mucus but showed fluorescence in the cytoplasm of absorptive cells as well as in the lamina propria and submucosa. The FITC-R. communis conjugate also reacted with structures in the apical portion of epithelial cells that may correspond to the Golgi apparatus.

Location and chemical composition of anionic sites in Bruch's membrane of the rat.

The location and chemical composition of anionic sites in Bruchs membrane (BM) were examined using cationic probe molecules demonstrable in electron microscopic preparations and tissue digestion with specific degradative enzymes. Ruthenium red and native lysozyme revealed densities distributed at regular intervals in two major components of BM: the basal laminae of the retinal pigment epithelium (RPE) and choriocapillary endothelium (EN). Staining was not observed with succinylated lysozyme (anionic). Colloidal iron also failed to stain BM components. Following crude heparinase treatment at 43 degrees C (specific for heparan sulfate) anionic sites in the RPE basal lamina were not demonstrable with either ruthenium red or native lysozyme. Sites in the EN basal lamina were not affected. Chondroitinase treatment removed almost all of the ruthenium red-positive material in the EN basal lamina; lysozyme binding here was markedly reduced. No changes were observed in the RPE basal lamina after chondroitinase digestion. There was no morphological evidence for site removal by either neuraminidase or leech hyaluronidase, although a detachment of the RPE from BM often occurred after incubation of eye tissue in the latter. Pronase E removed all stainable material. These findings indicate that anionic sites in BM consist to a large extent of chondroitin sulfates and heparan sulfate.

Localization of peroxidase activity in microbodies of fetal mouse liver.

The peroxidase activity of microbodies in fetal mouse liver was studied by light and electron microscopy. Two types of microbodies were present; a small population of bodies that lacked a nucleoid, predominant on the 16th day of gestation, and a larger population of nucleoid-bearing microbodies, predominant on the 19th day, in association with the rough endoplasmic reticulum from which they probably originate. Both types of bodies were visualized when incubated for peroxidase activity but were negative (19th day) for acid phosphatase activity. The findings suggest that the anucleoid- and nucleoid-bearing organelles together constitute the microbody population of the fetal liver.

OBSERVATIONS ON HEPATIC AND RENAL PEROXISOMES (MICROBODIES) IN THE DEVELOPING CHICK

Peroxisomes (microbodies) in developing liver and metanephric kidney of the chick were studied with a staining method that visualizes these organelles by light and electron microscopy. In both tissues nucleoid and anucleoid peroxisomes were evident at all stages studied (15th day of incubation to the adult). Hepatic peroxisomes showed no special topologic relationship to glycogen areas and were mainly confined to other areas of the cytoplasm. Nucleoid type peroxisomes were more apparent before and after hatching and rarely observed in the adult. Peroxisomes were present in kidney proximal tubule cells, but absent from cells of distal convoluted tubules. They appeared to increase in number during development and were most conspicuous in the adult. The structure of the nucleoid in renal peroxisomes resembled that of the hepatocyte but contained a more distinct series of parallel dense laminae. Diaminobenzidine (DAB) oxidative activity, although relatively weak, visualized nucleoid and anucleoid peroxisomes in liver and kidney at all stages of development. Anucleoid peroxisomes could be distinguished from various dense bodies in hepatocytes and from droplets in proximal convoluted tubule cells, both of which lacked DAB oxidative activity. The DAB reaction in peroxisomes was not suppressed in the absence of hydrogen peroxide or in the presence of the catalase inhibitor, 3-amino-1,2,4 triazole. DAB reaction, sensitive to potassium cyanide, was localized to cristae, but not to the outer membrane, of hepatic and renal mitochondria. Mitochondria of distal tubule cells showed a stronger reaction. The possible significance of nucleoid-bearing peroxisomes in the developing chick is discussed in terms of nitrogen metabolism, uric acid excretion and the presence of the uricolytic enzymes. The nature of the DAB reaction in peroxisomes and in mitochondria is considered.

FURTHER OBSERVATIONS ON THE PEROXIDATIC ACTIVITIES OF MICROBODIES (PEROXISOMES)

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