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.     

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.     

X-ray microanalytical (EDX) investigation of potassium distributions in mesophyll cells of non-acclimated and cold-acclimated rye leaves

Electron probe X-ray microanalysis was used to analyse the effects of sub-zero temperatures on K⁺ distribution in compartments within non-acclimated and cold acclimated rye (Secale cereale L. cv Voima) leaf cells and to evaluate membrane leakage of ions caused by freezing-injury. The specimens were rapidly frozen from growing temperatures and from two different sub-zero temperatures (LT₅₀ and LT₁₀₀) to which the leaves had already been slowly cooled. Measurements were made in the cytoplasm, vacuole and cell walls in freeze-substituted mesophyll cells. At ambient temperatures, the mean K⁺ concentration in the cytoplasm (100 mol m^?3) differed significantly from that of the vacuole (49 mol m^?3) in the non-acclimated (NA) cells, while in cold acclimated (A) cells, the concentrations were similar (109 vs 93 mol m^?3, respectively). At LT₅₀ temperatures, the K⁺ concentration in NA-cells decreased significantly in the cytoplasm (59 mol m^?3) but increased in the cell walls. In the A-cells, on the other hand, the mean K⁺ concentration increased significantly (about three-fold) in all major compartments. At LT₁₀₀ temperatures, K⁺ concentrations in the cytoplasm and cell walls decreased when compared with corresponding LT₅₀ values in the A-cells but increased in the NA-cells. The increased potassium concentration in the cytoplasm of A-cells at LT₅₀ temperature is compatible with the observed cell shrinkage and an absence of plasma membrane damage. The decreased potassium concentration in the cytoplasm of NA-cells at LT₅₀ temperature is compatible with the slight cell shrinkage and suggests that the plasma membrane in these cells shows increased permeability due to freeze injury.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

X-ray microanalytical detection of iron in pigmentation of oral mucosa.

A patient was referred to the Charles Clifford Dental Hospital with a grey metallic pigmentation of the hard palate. Conventional histopathological examination was inconclusive, suggesting the presence of either an ephelis (freckle) of pigmentation resulting from a stainless steel upper denture. Material from the pathological specimen was dewaxed and reembedded in Spurr's resin. Examination in the TEM revealed electron dense deposits in the cytoplasm of macrophages. Energy dispersive X-ray microanalysis demonstrated that these inclusions contained iron. The results suggested that the iron was in a form similar to haemosiderin and had arisen from the steel denture.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

A novel flat-embedding method to prepare ultrathin cryosections from cultured cells in their in situ orientation.

Immunogold labeling of ultrathin cryosections provides a sensitive and quantitative method to localize proteins at the ultrastructural level. An obligatory step in the routine preparation of cryosections from cultured cells is the detachment of cells from their substrate and subsequent pelleting. This procedure precludes visualization of cells in their in situ orientation and hampers the study of polarized cells. Here we describe a method to sample cultured cells from a petri dish or coverslip by embedding them in a 12% gelatin slab. Subsequently, sections can be prepared in parallel or perpendicular to the plane of growth. Our method extends the cryosectioning technique to applications in studying polarized cells and correlative light-electron microscopy.     

Histochemical localization of monoamine oxidase in whole-body, freeze-dried sections of mice

Summary Monoamine oxidase was investigated histochemically in tissues of the mouse by incubating freeze-dried, whole-body sections with tryptamine, serotonin, tyramine, -phenylethylamine, or benzylamine as substrate and Nitroblue tetrazolium as the final electron acceptor. The most intense staining with tryptamine was exhibited by intestinal epithelium and adrenal cortex; moderate staining was noted in the epithelium of the nose, bronchi, oesophagus, and upper stomach and in preputial gland, pancreas, nerve, spinal cord and brain. Weak staining was seen in the lung, spleen, liver and kidney. The distribution of the formazan deposition was similar, but much less intense, when serotonin and tyramine were used as the substrates. Only very weak staining was observed when -phenylethylamine was the substrate; no staining was seen with benzylamine. Monoamine oxidase activities with tryptamine were greatly inhibited by pretreatment with clorgyline (10 m), while deprenyl (10 m) slightly inhibited activities in all tissues except liver. This staining technique should be useful in further studies on the identification of the multiple forms of monoamine oxidase in tissues of the mouse. Nicotine and nitrosonornicotine were not substrates in any of the tissues; consequently, this enzyme system does not appear to produce the proximal carcinogen from this nitrosamine.     

The position of lysosomes within the cell determines their luminal pH

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H⁺–adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility.     

Sorcin在鳗鱼免疫器官的组织化学研究

目的:探究sorcin蛋白在鳗鱼主要免疫器官中的分布情况,为应用sorcin蛋白研制药用新靶点和应用鱼用疫苗奠定理论基础.方法:运用组织学和组织化学技术,在鳗鱼的主要免疫器官中进行sorcin蛋白的免疫组化定位研究.结果:sorcin蛋白在鳗鱼的心脏、肠、肝脏中均有分布.结论:sorcin蛋白在鳗鱼免疫器官中的分布情况与其功能密切相关.     

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.     

The element distribution in ultrathin cryosections of cultivated fibroblast cells

Summary A preparation method for measurements of the intracellular distribution of elements in tissue culture cells is described which is based on cryofixation, cryoultramicrotomy, cryotransfer and X-ray microanalysis in a scanning transmission electron microscope. Dry weight concentrations of phosphorus, sulfur, chlorine and potassium in the nucleus, the cytoplasm and the mitochondria of L929 fibroblast cells of the mouse are reported. The preparation and quantitation procedures are discussed with respect to present limitations and possible improvements of the method.The paper was presented partly as a poster at the joint meeting on clectron microscopy of the Deutsche Gesellschaft für Elektronenmikroskopie and the Belgische Vereniging voor Elektronenmikroskopie in Antwerpen, 11.–16. 09. 1983