Enzymic heterogeneity of adrenocortical lysosomes: an X-ray microanalytical study

Summary The enzymic heterogeneity of the lysosomal system has been demonstrated earlier. This study was concerned whether or not the lysosomes of the two outer zones of the rat adrenal cortex reveal any population characteristics on the basis of the activity of two enzymes. A double incubation method was used for the simultaneous electron cytochemical demonstration of acid phosphatase and arylsulphatase activity in the zonae glomerulosa and fasciculata of the adrenal. Lysosomes in semi-thin (0.5m) or ultra-thin sections were analysed with an ORTEC energy-dispersive X-ray microanalyser mounted on a JEOL TEMSCAN-100 C electron microscope.Linearity of the amount of reaction deposit with incubation time was found for both enzymes. On the basis of the proportion of the two reaction products in individual lysosomes, three populations were distinguished. One with high acid-phosphatase and low, if any, arylsulphatase activity was only present in the zona glomerulosa. The other two populations exhibited stronger or weaker prevalence of arylsulphatase activity and were common in both zones. The values of the Ba/Pb ratio characteristic of each population changed with the reaction sequence but the principal distribution pattern did not. The nature of the interaction between the two reactions, as well as the possible functional significance of the lysosomal populations in the adrenal cortex, are discussed but are not yet clarified.     

Arterial supply of the oral mucosa.

In 15 human heads, bilateral injection through the common carotid artery was carried out in order to find out whether there are any more or less distinctly limited segments of the oral mucosa supplied by certain arterial branches that would represent the nutritive pedicles of the respective segments. It seems that the gingiva libera of the buccal side in the region of the upper molars and premolars represents such a segment. In spite of small anastomoses between the right and left sublingual arteries, one half of the sublingual mucosa may also be considered as a segment. The same refers to the mucosa of one half of the hard palate. Even smaller units of varying extent of the palatine mucosa may be differentiated as distinct vascular areas. For the majority of the oral mucosa, however, no limitations of vascular segments with distinct arterial pedicles could be discovered.     

X-ray microanalytical studies on cryofixed blood cells of the ascidian Phallusia mammillata

Summary Cryofixed blood morula cells of Phallusia mammillata (Cuvier), which are considered to be vanadium-accumulating cells, were examined by X-ray microanalysis using STEM (scanning transmission electron microscopy) and SEM (scanning electron microscopy). It is thought that cryopreparation preserves the native distribution of diffusible elements such as sodium, chlorine, and potassium, and prevents the displacement of vanadium, all of which may occur during conventional preparation. The results show that morula cell globules contain a large amount of sulphur and chlorine, and some sodium, magnesium, bromium and potassium, but very little or no vanadium.     

Lanthanides and microanalysis. Effects of oral administration of two lanthanides: ultrastructural and microanalytical study

The subcellular localization of cerium and lanthanum in the intestinal mucosa was studied after oral administration of cerium chloride or lanthanum chloride or lanthanum chloride followed 30 minutes after of cerium chloride to young adults Wistar rats. Two methods of observation and microanalysis were used. The transmission electron microscopy revealed the presence of dense electron granulations in the lysosmes of the duodenum enterocyte, when these elements were administrated simultaneously. The ion mass microanalysis permits to detect the presence of La and Ce as bright points outlining the intestinal villi. These points correspond to the lysosomes containing the granulations previously described. These granulations are formed by the cerium and the lanthanum associated to the phosphor and forming probably insoluble salts of Ce/La phosphate.     

Attempts at localizing calcium in relation to synaptic transmission: an X-ray microanalytical study

1. In view of the importance of calcium in triggering the transmitter secretion during nerve stimulation or depolarization, the localization of calcium binding sites was studied in stimulated nerve endings that were fixed in calcium-containing s-collidine buffered paraformaldehyde solution. 2. In cholinergic synapses, such as the superior cervical ganglion of cat, the myoneural junction of rat diaphragm and the electric organ of Torpedo marmorata, a stimulation-dependent accumulation of calcium was found in the mitochondria of presynaptic nerve endings. 3. In synaptosomes prepared from rat cerebral cortex, the activity-dependent accumulation of calcium in mitochondria of pinched off nerve endings was also observed. The mitochondrial accumulation of calcium in synaptosomes was dependent on ATP, temperature and could be inhibited by quercetin. 4. In stimulated synapses, only the mitochondria seemed to accumulate calcium in appreciable amounts, whereas other intra-terminal structures, such as the smooth endoplasmic reticulum and synaptic vesicles were devoid of calcium.     

Ultrastructural localization of calcium in post-mortem bovine muscle: A cytochemical and X-ray microanalytical study

Summary Calcium localization was demonstrated in bovine longissimus muscle using the antimonate precipitation technique in combination with electron probe X-ray microanalysis. Samples were taken each hour during the first 24 h post-mortem, and then after a storage period of 8 and 15 days. For all sampling times analysed, heavy precipitates were seen in dense parts of nuclei and on N-lines of myofibrils. Up to 18–20 h post-mortem, deposits were observed in sarcoplasmic reticulum at the level of triads. In comparison with the earlier post-mortem samples, myoplasmic precipitates were strongly increased at 4 h post-mortem, and just before rigor onset, at 19 h where intermyofibrillar spaces were completely blackened and triads were no more visible. These localizations of precipitates were still observed up to 15 days post-mortem. At these storage times, myofibril disruptions were seen at the level of N-lines. Wavelength-dispersive and energy-dispersive spectrometric analyses indicated that significant amounts of calcium occurred in the dense precipitates observed.     

Biomechanics of prehensile oral mucosa

Large dogs are able to deliver a powerful bite that generates considerable stress in the anterior, prehensile part of the jaws. In the upper jaw most of the biting force is borne by the anterior teeth. The palatal mucosa provides little resistance to deformation. It is easily compressed and rather mobile. In the lower jaw, the mucosa covering the upper surface of the symphysis receives a sizeable portion of the biting force. It is firmly attached to the underlying bone and possesses special connective tissue arrangements that enable it to transduce locally applied pressure to tension distributed over a broad area.     

Functional morphology of phagocytosing alveolar macrophages. Long-term electron microscopic and X-ray microanalytical investigations on the rat model

A single instillation of 1 ml iron dextran (containing 191.3 mg iron(III)hydroxide and 200 mg dextran) was administered under anaesthesia by a polyvinyl catheter into the lower lobe of the right lung in one hundred 4-week-old wistar rats. The animals were killed at intervals ranging between 1 min and 4 weeks. The lower lobe of the right lung was examined by light and electron (transmission and scanning) microscopy. In addition, X-ray microanalyses were performed on tissue sections in the transmission and scanning electron microscopes. The process of phagocytosis of iron dextran by alveolar macrophages can be subdivided into three stages, which we have termed the "phase of attachment" (from 1 to 5 min), followed by the "phase of phagocytosis" (from 5 to 20 min) and finally the "resident macrophage stage" (from 1 to 24 h). X-ray microanalysis shows a high phosphorus content even if iron dextran is concentrated on the surface of macrophages. Phagocytosis of particles between 15 and 40 A in size occurs within minutes, the particles being engulfed in phagosomes, which form as double-layered invaginations of the cell membrane into the interior of the cell. The fusion of phagosomes with lysosomes produces phagolysosomes (type 2 lysosomes) in which iron dextran is broken down into lamellar residual bodies. In these lamellar bodies X-ray microanalysis shows that in addition to abundant iron, there is a high phosphorus content, which may indicate the involvement of surfactant. Only 1 h after instillation, free particles of iron dextran can no longer be demonstrated in the alveoli, although a proportion of the iron dextran remains in resident macrophages (pulmonary tissue macrophages) and some is also found in splenic macrophages.     

An X-ray microanalytical azo dye technique for the localization of acid phosphatase activity

Summary A new method is described for the histochemical localization of acid phosphatase. Naphthol AS BI, enzymatically released from naphthyl AS BI phosphoric acid, is coupled with diazotized 2,5-dibromoaniline to produce a fine insoluble red azo dye. The histochemical and cytochemical localization of this final reaction product in rat liver is described. In the electron microscope, sites of the azo dye can be detected by X-ray microanalysis of ultrathin cryosections of reactive tissue.This research was supported by Scientific Research Council Grant No. B/RG/67527     

An X-ray, microanalytical and ultrastructural study of cadmium absorption and transport in rat liver

Accumulation of cadmium in the liver was demonstrated by X-ray microanalysis in every type of experiment, i.e. after injecting Cd into the ligated intestine and after the peroral acute single and combined, subchronic and chronic administration of Cd. Half an hour after its injection, Cd was localized diffusely in the liver; one hour after injection its increased accumulation in the cells caused generalized changes in the endoplasmic reticulum, mitochondria and nuclei. In acute and chronic peroral tests, the hepatocytes of the intermedial and peripheral zone of the lobes were the main storage region. After an acute dose of Cd, the cells in the centrolobular zone were hydropic, or single-cell necrosis developed; after the longer effect of combined doses the latter was manifested as centrolobular focal necrosis. Cd was not demonstrated in the lesions. Chronic administration did not lead to manifest severe degenerative changes in the liver. Accretions in the mitochondria and on the membranes of the endoplasmic reticulum were identified by means of X-ray analysis with cadmium peaks. Cadmium showed up clearly as L alpha- and L beta-lines at 3.135-3.320 keV. We presume that cadmium is bound in the ribosomes of the endoplasmic reticulum, as well as the mitochondria, and is released by the invagination of swelling mitochondria of the peripheral hepatocytes into Disse's spaces.     

Distribution of calcium in the suctorian Discophrya collini: An X-ray microanalytical study

Discophrya collini is a free-living suctorian with tentacles which can be induced to contract by means of a range of experimental stimuli, including the application of CaCl₂ and MgCl₂ but not BaCl₂. X-ray microanalysis of glutaraldehydeonly fixed cells shows Ca to be present in the cytoplasmic ground substance and elongate dense bodies (EDB). In 10^?1 M CaCl₂-treated cells, calcium levels remain unchanged except for a three-fold increase in the EDB. Treatment of cells with 10^?1 M MgCl₂ and 10^?1 M BaCl₂ does not result in their detection in the cell. It is suggested that EDB may act as reservoirs controlling levels of calcium.     

Production and characterization of an in vitro engineered human oral mucosa.

The role of epithelial cells in oral pathologies is poorly understood. Until now, most studies have used normal or transformed epithelial cell monolayers, a system that largely bypasses oral mucosal complexity. To overcome these limitations, an engineered human oral mucosa (EHOM) model has been produced and characterized. Following histological and immunohistochemical analyses, EHOM showed well-organized and stratified tissues in which epithelial cells expressed proliferating keratins such as Ki-67, K14, and K19 and also differentiating keratin (K10). In this model, epithelial cells interacted with fibroblasts in the lamina propria by secreting basement membrane proteins (laminins) and by expressing integrins (beta1 and alpha2beta1). Cytokine analyses using cultured supernatants showed that cells in EHOM were able to secrete interleukins (IL) including IL-1beta and IL-8 and tumor necrosis factor alpha (TNF-alpha). Finally, cells in this engineered model were able to secrete different metalloproteinases such as gelatinase-A and gelatinase-B. In conclusion, using tissue engineering technology, we produced well-organized EHOM tissues. It is anticipated that this model will be useful for examining mechanisms involved in oral diseases under controlled conditions by modeling the interactions between mucosa and microorganisms in the oral cavity.     

Role of intestinal mucus in crystal biogenesis: an electron-microscopical,diffraction and X-ray microanalytical study

Summary In the posterior intestine of the sea-water eel, mucus plays an important role in biocrystallization of calcium ions. By means of transmission and scanning electron microscopy associated with X-ray microanalysis and X-ray diffraction it has been possible to determine the role of mucous fibers as nucleation sites. Biocrystallization occurs in 2 steps: (1) Calcification of mucus. As soon as mucus is excreted in the intestinal lumen, it is loaded with calcium, as shown by lanthanum affinity and X-ray microanalysis on freeze-dried tissues. (2) Genesis of crystals. Needleshaped crystallites build up in coalescent spherites in the intestinal lumen near the microvilli. Genesis occurs as follows: (a) crystallite mineralization by nucleation in an organic matrix composed of glycoproteinaceous mucous fibers, followed by the appearance of spherites; (b) coalescence in spherites and association of spherites in rhombohedra; (c) extrusion of organic material during the final step of crystallization.