Formation Of Electron-dense Deposits Research Articles

Demonstration of Taenia crassiceps Cysteine Proteinase Activity in Tegumentary Lysosome-like Vesicles

Larval stages of Taenia species survive for prolonged periods in the tissues of their intermediate hosts. Other groups have demonstrated that host immunoglobulins are taken up by the cysticerci by adsorptive endocytosis, degraded, and the amino acids incorporated into parasite proteins. We have shown that a 43-kDa cysteine proteinase is the major parasite enzyme that degrades immunoglobulin in vitro. To localize this enzyme in situ, Taenia crassiceps cysticerci were incubated with the peptide substrate Z-Phe-Arg-methoxynaphthylamide. Free methoxynaphthylamide was coupled to p-rosanilin and osmium and visualized by transmission electron microscopy. Initial studies of cysticerci incubated without substrate confirmed the normal microanatomy and absence of significant host inflammation. In comparison to controls with no substrate, sections of cysticerci incubated with substrate revealed electron-dense deposits in round vesicles. The vesicles were found primarily within the tegumentary cytons and internuncial processes, a location similar to that described for vesicles associated with adsorptive endocytosis. There were proportionately more endocytotic vesicles and electron-dense vesicles in smaller cysticerci than larger ones. Formation of electron-dense deposits was inhibited by heat and partially inhibited by the cysteine proteinase inhibitor E-64. These data are consistent with localization of the cysteine proteinase activity to lysosome-like vesicles.

Electron Dense Artefactual Deposits in Tissue Sections: The Role of Ethanol, Uranyl Acetate and Phosphate Buffer

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.

Immunoelectron microscopic study of glomerular lesions using a postembedding method with a protein A-gold complex.

Renal biopsy tissue from 33 children with various glomerular diseases has been investigated by electron microscopy using a postembedding immunostaining technique with a protein A-gold complex in order to establish more precise correlations between immunopathologic and morphologic findings in glomeruli. This technique could detect immunoglobulins (IgG, IgA, and IgM), complement factor (C3c), and fibrinogen-related antigen. The immunoreactivity of these antigens was essentially confined to the mesangial, paramesangial, subendothelial, and subepithelial 'electron-dense deposits' in the glomeruli. Except for IgM and C3c in the case of glomerular sclerosis, the distribution of the mentioned factors was even in the electron-dense deposits, as could be shown by 'double-immunolabeling'. From the above-mentioned findings one can conclude that several of the localized factors are associated with the formation of electron-dense deposits, the ultrastructural hallmarks of glomerular disease.

Precipitating antigen-antibody systems are required for the formation of subepithelial electron-dense immune deposits in rat glomeruli.

This study was conducted to determine whether multivalent, precipitating antigens are required for formation of subepithelial electron-dense immune deposits in glomeruli. 2-nitro-4-azidophenyl (NAP) was conjugated with variable density to human serum albumin (HSA) to yield nonprecipitating (NAP3.1 X HSA and NAP11.4 X HSA) and precipitating (NAP19.7 X HSA) antigens with antibodies to the hapten. These antigen preparations were cationized with ethylene diamine to enhance deposition in renal glomeruli due to interaction with the fixed negative charges in the glomerular capillary wall. Following injection into the left renal artery of rats these antigens alone persisted in the glomeruli for a relatively short time by immunofluorescence microscopy. When antibodies to NAP were injected intravenously after the antigen injection, the nonprecipitating antigens and antibodies were detectable in the glomeruli by immunofluorescence microscopy up to 8 h, comparable to antigen alone. Electron-dense deposits were not formed in these glomeruli. In contrast, when the precipitating antigen was injected and followed by antibodies to the hapten, antigen and antibody were detected by immunofluorescence microscopy through 96 h. In these specimens electron-dense deposits were present from 40 min through 96 h and after 24 h the deposits were present only in the subepithelial area. The same results were obtained when the nonprecipitating hapten-carrier conjugates were followed with antibodies to the carrier molecule. These data indicate that the persistence of immune deposits by immunofluorescence microscopy and the formation of electron-dense deposits in the subepithelial area require a precipitating antigen-antibody system.

Histochemical demonstration of an ATP-dependent Ca2+-pump in bullfrog myocardial cells.

In the present investigation different finestructural and histochemical procedures were employed to demonstrate normal morphology and Ca2+-transport mechanism in bullfrog atrial myocardium. For normal morphology specimens were fixed in 1% OsO4, 2.5% glutaraldehyde or liquid propane at -185 degrees C before they were prepared for conventional embedding and freeze-etching, respectively. Special interest was focussed on the caveolae system, which is composed of single, spherical membrane invaginations (diameter, 85 nm), randomly distributed at the entire cell periphery. The caveolae enlarge the cell surface by 24.5% and occupy 8.5% of the cellular volume. The caveolae membrane contains a few intramembraneous particles with a diameter of 8.4 nm comparable to the size of ATPases as found in other cells. For histochemistry the specimens were first stabilized by treatment with 50% glycerol, 0.025% glutaraldehyde or 0.15% formaldehyde and then incubated in a medium containing 4 mM CaCl2, 4 mM EGTA, 5 mM MgCl2, 5 mM K2C2O4, 5 mM ATP and 20 mM histidine at pH 7.0. This incubation always succeeded in the formation of electron-dense deposits with elliptical shape, measuring 100-200 nm in length and 10-30 nm in diameter. According to X-ray spectra they deliver a characteristic calcium-peak and can be found within two different cellular compartments: in small invaginations of the sarcolemma, i.e. the caveolae-system, and in the intrafibrillar sarcoplasmic reticulum. The elliptical deposits can be clearly distinguished from round electron-dense granules measuring 16 nm in diameter, which are located within randomly distributed small vesicles and composed of potassium and phosphate. Contrary to the elliptical deposits the round granules are also present in controls and seem to be identical with the so-called atrial granules. In comparison to observations obtained with the same method in other muscular systems and derived from various control experiments the results of this study favour the existence of an ATP-dependent Ca2+-pumping mechanism in frog atrial muscle bound to both, the sarcoplasmic reticulum and the caveolae system.

Ultrastructural localization and X-ray analysis of calcium-induced electron-dense deposits in the skate retina

The technique of Oschman & Wall (1972) was used to visualize the sites at which electron-dense deposits accumulate in neural layers of the skate retina. The method, which involves the addition of calcium to the primary fixative, resulted in the formation of electron-dense deposits along the plasma membranes of horizontal cells, within the outer segments, mitochondria and synaptic vesicles of the visual cells, and at pre- and postsynaptic membranes of the synaptic elements of the outer plexiform layer. Energy dispersive X-ray spectrometry of the electron-dense deposits yielded prominent peaks for calcium and phosphorus. In the dark-adapted retina, the electron-dense deposits were seen more frequently in association with the receptor terminal than with the processes of second-order neurons; after light adaptation, the reverse situation was observed. A light-induced translocation of electron-dense deposits was not found at any other retinal location. The addition of cobalt to the fixative in lieu of calcium produced results equivalent to those obtained with calcium; i.e. electron-dense deposits were formed at simular loci within the retina. The emergence in the X-ray spectrum of a small calcium signal in the presence of a large cobalt peak suggests that the two ions compete for similar membrane sites. On the other hand, the formation of electron-dense deposits was suppressed completely by exposing the retinas to Ca 2+-free solutions, with or without the addition of Mg 2+. These findings are consistent with physiological observations that suggest that Mg 2+ and Co 2+ act at different sites in modifying the actions of Ca 2+ at synapses.

Light-induced changes in the ultrastructure of a plasmodial myxomycete

Light, required to induce sexual sporulation (fruiting) of the plasmodial myxomycete Physaruin polycephalum, causes the formation of electron-dense deposits in the intermembrane spaces of mitochondria and in cytoplasmic vacuoles. Such mitochondrial deposits are also present but to a much smaller extent in the dark. This deposition is nutritionally dependent on calcium. The deposits are rich in calcium as determined by EGTA treatment of sections and by X-ray spectroscopy. Light also induces the formation of these deposits in young plasmodia not yet competent to sporulate upon exposure to light. In addition, growing microplasmodia which are severely inhibited by light also form these characteristic deposits when illuminated. A basic part of the photometabolism appears to be an enhanced movement of calcium and possibly other ions into and within the plasmodium. The physiological response evoked by light, sporulation or growth inhibition, depends on the nutritional history and immediate environment of the plasmodium.