Electron microscopic demonstration of carbonic anhydrase activity in mouse liver cells

Summary Carbonic anhydrase (CAH) activity was demonstrated ultracytochemically in the mouse liver cells fixed with 1% glutaraldehyde buffered to pH 7.2 with 0.1 M cacodylate buffer containing 0.1 M sucrose and other aldehyde fixatives. After the fixed 25–40 section were incubated in Hansson's incubation medium containing 0.2 M sucrose, the cobalt phosphate formed by the action of CAH was converted to lead phosphate by immersing the incubated sections into 0.1% lead nitrate aqueous solution.The lead phosphate precipitate was observed very well on the plasma membrane of hepatocytes in Disse space and of endothelial cells or erythrocytes, and very slightly on the external coat of microvilli in bili canaliculi.In the tissues fixed with 4% formaldehyde, the deposits were found very barely on the microvilli in the space of Disse and the plasma membrane of the endothelial cells or the erythrocytes.As the -hydroxyadipaldehyde-fixed tissues showed the highest the CAH activity but had not a good preservation of morphology, this fixative is not suitable for the electron microscopic histochemistry of CAH.The tissue incubated in medium containing Diamox exhibited non-specific deposits in all over the cell, which were lost when the tissue was treated in Diamox solution before incubation.     

Electron spin resonance study of peroxidase activity and kinetics.

An electron spin resonance (ESR) assay has been developed for peroxidase activity. The assay measures the formation of the paramagnetic nitroxide Tempol from the oxidation of its hydroxylamine derivative (TOLH) by short-lived radicals produced by peroxidase cycle intermediates, Compounds I and II. Using phenol as a peroxidase electron donor, the ESR approach is suitable for measurements of peroxidase activity ( > or = 0.003 U/ml) and micromolar quantities of H2O2 in sample sizes as small as 2 microliters. In addition, the ESR method can be used to continuously monitor activity in cell suspensions and other media that are susceptible to optical artifacts. The high membrane permeability of TOLH also makes it possible to estimate peroxidase activity in membrane-enclosed compartments, provided that TOLH oxidation rates can be stimulated with exogenous peroxidase reductants, e.g., phenol. Analysis of TOLH oxidation rates under conditions of low electron donor concentrations and high concentrations of H2O2 also shows clear indications of substrate-dependent inhibition and increased catalytic activity. Computer simulations indicate that the results obtained are consistent with the peroxidase reaction scheme proposed by Kohler et al. (1988, Arch. Biochem. Biophys. 264, 438-449) modified to correct for a nitroxide dependent stimulation of peroxidase catalytic activity.     

Electron microscopic study of the cytoskeleton of human podocytes

The ultrastructural study of man's cytoskeleton of podocytes is carried out. Populations of podocytes with two different types of structure of the cytoskeleton in dependence on age (2, 4, 6, 37 and 65 years) is revealed in kidneys. The first type of cytoskeleton of the podocyte is peculiar for children's age and is characterized by branched, high density microfilament network, expressed by system of microtubules and single myofilaments. The intermediate filaments here are either utterly absent or present so feebly they find themselves "disguised" by other strongly developed components of cytoskeleton and revealing them with the help of technique of electron microscope is impossible. In kidneys of adults, and especially of old aged persons podocytes with other type of organization of the cytoskeleton are mainly identified. The distinctive signs of the last are bundle arrangement of microfilaments, plural bundles of intermediate filaments and individual microtubules. This study permits to make a conclusion that during individual development and growing old in kidneys of high animals and man, probably, physiological changes causing morphological reconstruction of cytoskeleton which is accompanied by intensive development of intermediate filaments' system with simultaneous "involution" of microtubules and microfilaments' systems take place.     

Electron microscopic study of the in vivo erythrophagocytosis by alveolar macrophages of the cat

Summary Erythrophagoeytosis in vivo by cat alveolar macrophages was studied under the electron microscope by collecting the macrophages at 2 hours and 48 hours following the intratracheal injection of autologous blood. Considering the progressive ultrastructural modifications of the red blood cell plasma membrane, different successive stages were observed, corresponding to the hemolysis of the erythrocytes: 1. A recently engulfed erythrocyte appears unaltered within the phagocytic vacuole. 2. A dense layer, surrounding the plasma membrane of the red cell, is observed within the phagocytic vacuole. 3. The content of the vacuole is uniformly dense and the plasma membrane of the red cell exhibits discontinuous thickenings. 4. The whole vacuole appears very dense (hyperdense stage) and the plasma membrane is shown altered. The whole process of erythrophagocytosis is accompanied by an active fusion of the phagocytic vacuole with typical lysosomes and lysosomes containing crystal-like material. It is suggested that hemolysis may be explained in terms of enzymic digestion of the proteinic part of the plasma membrane of the erythrocyte.The authors wish to thank Miss Gabrielle Audet for her technical assistance, and Mr. Gaston Chevalier for revision of the English text.     

Electron microscopic study of the interaction of Yersinia pseudotuberculosis with macrophages and HeLa cells

The comparative electron-microscopic study of early stages of the interaction of Y. pseudotuberculosis virulent strain (No. 282) with "professional" (macrophages) and "nonprofessional" (HeLa cells) phagocytes has been carried out. The character of the intimate mechanism of this interaction has been found to be essentially different. The common feature for both systems is the adsorption of bacteria and their penetration into cells due to phagocytosis. But the subsequent fate of Y. pseudotuberculosis is different. In HeLa cells they are isolated from the cytoplasm by multilayer membrane structures, thus remaining morphologically intact. In macrophages the destruction of the microbe in phagolysosomes occurs.     

Electron microscopic study of troponin

Troponin and its components or fragments were observed in an electron microscope by the use of the rotary shadowing technique. In freshly prepared troponin with low viscosity, globular particles were mainly observed. The size of the long axis of the particles was 13.2 +/- 1.3 nm and the size perpendicular to the long axis was 9.5 +/- 1.2 nm. The mean axial ratio was 1.4 +/- 0.3. Most of the particles observed in a stored troponin preparation, having a higher viscosity than that of fresh troponin, had a globular head with a thin tail, with the total length of 25.4 +/- 1.4 nm (head-tail type particles). The axial size of the globular portion was 8.3 +/- 1.2 nm and the tail length was 17.1 +/- 1.6 nm. Observation of various particles during the transitional stages indicated that, in the globular particles, the tail region of head-tail type particle was associated along the globular head region. Troponin T was a filamentous particle with 16.9 +/- 1.5 nm length. The 26K fragment of troponin T, which was devoid of the N-terminal 45 residues from troponin T, was a filamentous particle with the length of 14.4 +/- 1.3 nm. Troponin T1, one of two chymotryptic subfragments of troponin T, was a filamentous particle of 11.6 +/- 1.4 nm length. Troponin C.T in the presence of Ca2+ was a particle with a globular head (7 nm in size) and a tail of about 17 nm length. The Fab fragment of anti-troponin T1 formed regular transverse striations along the thin filament of rabbit skeletal muscle with a 38 nm period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic study on amyloid fibril formation in human lymph nodes

The purpose of this investigation was to clarify the mechanisms of amyloid fibril formation in human lymph nodes. In our present study, amyloid deposition was observed diffusely in all compartments of the lymph nodes. The deposition form showed extremely characteristic findings in its morphological features. Namely, amyloid deposits mainly consisted of clusters of round or oval nodules. Each amyloid nodule was frequently enclosed with long-stretched cytoplasmic processes of abutting reticulum cells and/or macrophages. Amyloid fibrils often formed parallel amyloid bundles radiating to outlying sections of the nodule from the center. The amyloid bundles closely adhered to the cytoplasmic membrane of not only the abutting reticulum cells, macrophages and sinus endothelium but also to the lymphocytes and plasma cells. In the central portion of the amyloid nodules, a concentric core was also observed. The most interesting finding was the intracellular formation of amyloid fibrils in all cells, such as macrophages, reticulum cells, foreign body giant cells and lymphocytes in the process of degeneration. Some fibrils localized in the limited area of the cytoplasm and others appeared in all parts of the cells, including the nucleus. Their cell membranes were missing in several areas and the cell organella had gradually dissolved. Finally the cell residuums were completely replaced by amyloid fibrils and transformed into a nodular structure with radiating bundles of amyloid fibrils.     

Electron microscopic observations of young rat liver

Summary Electron microscopic observations on normal liver tissue of four-day-old rats reveal the presence of numerous lamellar structures (lamellar bodies). These can be contained within parenchymal cells or in bile canaliculi, Disse's space, and in the lumen of blood sinusoids. Such bodies can also be found in Kupffer and endothelial cells.The lamellar bodies within hepatic cells are generally seen in very intimate relation to glycogen particles and lipide droplets, but in some to agranular endoplasmic reticulum and Golgi membranes as well.On the basis of this intimate relation to intracellular glycogen granules and lipide droplets, it is presumed that lamellar bodies represent a special intermediate stage in carbohydrate and lipide metabolism.Discontinuities in the endothelial layer of intrahepatic sinusoids are described.This work was supported in part by a N.A.T.O. research fellowship of the Consiglio Nazionale delle Ricerche, Roma.Assistant Professor in the Department of Veterinary Anatomy, Histology and Embryology (Dir.: Prof. A. de Girolamo), University of Naples, Naples, Italy.     

Electron microscopic study of Ag-protein localization in the nucleoli of human lymphocytes

The present study was undertaken to provide more information on the ultrastructural localization of a silver reaction in normal resting and stimulated lymphocytes as well as leukaemic resting lymphocytes. The results obtained indicated that in the ring-shaped nucleoli of normal mature lymphocytes silver stained proteins (SSPs) were present mostly within single fibrillar centers. In the nucleoli of lymphocyte cultures, being in the presence of phytohemagglutinin (PHA) for 6--72 hours, SSPs formed finger or loop-like protrusions from fibrillar centers towards the adjacent areas of the nucleoli. In the ring-shaped nucleoli of mature leukaemic lymphocytes SSPs are present not only within fibrillar centers, but also in protrusions diverging from fibrillar centers into the surrounding peripheral nucleolar ring. In this respect the nucleoli of leukaemic mature lymphocytes were similar to normal lymphocytes shortly after mitogen stimulation.