Ultrastructural demonstration of cartilage acid glycosaminoglycans

Synopsis A method for the demonstration of cartilage acid glycosaminoglycans by light and electron microscopy is described. Rabbit ear cartilage was fixed in cacodylate buffered 2.5% methanol-free formaldehyde with 0.001 M Ruthenium Red andp-chloromercuribenzoate (PCMB). Dehydration was carried out in ethylene glycol followed by embedding in the water-soluble glycol methacrylate (GMA). In some experiments unfixed cartilage was rapidly dehydrated. Sections, 1 thick, and ultrathin sections from the same blocks were stained with 0.001 M Ruthenium Red. Semi-thin sections from cartilage fixed without heavy metal additives were, in addition, stained with the acidophilic fluorochrome Berberine sulphate. It was found that Ruthenium Red intensely stained the same pericellular zone that stained metachromatically with Toluidine Blue or fluoresced after staining with Berberine sulphate. Prior treatment with 0.05% cetylpyridinium chloride entirely blocked the three reactions. Previous digestion with 0.2 mg hyaluronidase/ml for 30 min at 37°C led to the abolition of the fluorescence reaction with Berberine sulphate. It is concluded that Ruthenium Red selectively stains cartilage acid glycosaminoglycans. With the electron microscope the pericellular zones were found to be built up of a three-dimensional branched meshwork of fibrils covered with a mantle of electron-dense material, presumably acid glycosaminoglycans bound to Ruthenium Red.     

Ultrastructural localization of acid protein polysaccharides and calcium in the vacuoles of the chicken thrombocyte.

The coexistence of acid protein polysaccharides and calcium in the vacuoles of chicken thrombocytes were studied by means of ultrahistochemical methods and electron probe X-ray microanalysis. The thrombocytes possessed large vacuoles of a surface connected membrane system. After both ruthenium red staining and tannic acid fixation the innersurface coat of vacuoles was always strongly and continously visualized. Electron microscopic X-ray microprobe analysis of antimonate precipitates in thrombocytes fixed in K-antimonate-OsO4 revealed calcium localization on the innersurface of vacuoles. From these facts it seems likely that the vacuoles of the surface connected membrane system may take part in the pool or the transport of calcium.     

Biphasic mode of epithelial regeneration in murine pulmonary alveoli

After administration to mice of butylated hydroxytoluene, the pulmonary alveolar epithelium adopts a biphasic pattern of regenerative proliferation. This hitherto-unnoticed pattern of epithelial repair in the lung was revealed by the investigation of stereologic parameters. The earliest evidence of epithelial injury involved the type I pneumocytes, whose necrosis and disappearance from the septal surface was shown by a lowered surface density (SV). Proliferation of the type II pneumocytes ensued: the volume density (VV) rose above normal soon after the onset of necrosis, only to decrease as the cells slowly differentiated into intermediate and then type I pneumocytes. A second peak of type II pneumocytes appeared as the denuding of septa persisted. This twofold proliferation was also shown by the numerical density count (NV). Differentiation into an intermediate pneumocyte was itself documented by the raised VV and SV values. These observations of a biphasic mode of proliferation of type II pneumocytes raise the question of an unsuspected, persistent action of the toxic agent within pulmonary alveoli and serve to document the homeostasis of epithelial regeneration.     

New AMP-forming acid:CoA ligases from Streptomyces lividans,some of which are posttranslationally regulated by reversible lysine acetylation

In nature, organic acids are a commonly used source of carbon and energy. Many bacteria use AMP-forming acid:CoA ligases to convert organic acids into their corresponding acyl-CoA derivatives, which can then enter metabolism. The soil environment contains a broad diversity of organic acids, so it is not surprising that bacteria such as Streptomyces lividans can activate many of the available organic acids. Our group has shown that the activity of many acid:CoA ligases is posttranslationally controlled by acylation of an active-site lysine. In some cases, the modification is reversed by deacylases of different types. We identified eight new acid:CoA ligases in S. lividans TK24. Here, we report the range of organic acids that each of these enzymes can activate, and determined that two of the newly identified CoA ligases were under NAD⁺-dependent sirtuin deacylase reversible lysine (de)acetylation control, four were not acetylated by two acetyltransferases used in this work, and two were acetylated but not deacetylated by sirtuin. This work provides insights into the broad organic-acid metabolic capabilities of S. lividans, and sheds light into the control of the activities of CoA ligases involved in the activation of organic acids in this bacterium.     

Ultrastructural localization of polysaccharides in the motile diatomNavicula cuspidata

Summary Generation of movement in benthic diatoms is thought to be intimately associated with secretion at the raphe, a slit in the silica cell wall. The presence and distribution of extracellular substances and their source was investigated cytochemically by transmission electron microscopy. Extracellular material, possibly-acid mucopolysaccharide, was observed consistently within the entire length of the raphe of both valves and also as a sheath enveloping the silica frustule. Such quantities of extracellular material are absent in conventionally fixed motile diatoms. Numerous cytoplasmic vesicles, with fibrillar contents, distributed peripherally but concentrated along the raphe and at the cell poles, react strongly with a polysaccharide specific stain; their distribution in the cell and polysaccharide content suggest these may be the source of raphe and sheath material. Results support the most recent theories on the mechanism of locomotion in outline only; the details cannot be clarified. Localization procedures using alcian blue and silver staining of peroxidised sections are discussed briefly.     

Effect of exogenous pulmonary surfactant preparations on the structure of pulmonary alveoli in newborn rats

Treatment of pre-term newborns with exogenous surfactant preparation is a well established part of the therapy for respiratory distress syndrome of the newborns (RDS). Since the introduction of surfactant into clinical practice in 1980, hundreds of studies have been published describing beneficial effects of such treatment. There is only limited number of morphological publications reporting adverse effects of surfactant administration. The aim of the present study is to describe morphological changes in the lung after surfactant administration to healthy newborn rats. Two types of surfactant were used: Exosurf (Glaxo Wellcome, England) and Survanta (Abbott Laboratories, USA). Surfactant preparation were given intratracheally in single dose (bolus) (100 mg of lipids per kg b.w.). Animals from control group received 0.9% saline in equivalent volume. Lung specimens were taken 15, 20, 25 and 30 minutes after drug administration and evaluated by light and electron microscopy. There was no damage in lungs from the control group. Tissue specimens from the Exosurf group revealed severe pathological changes: foci of atelectasis, frank edema in the parenchyma, focal disruption of air-blood barrier, hemorrhages in many alveoli, surfactant particles in many alveolar capillaries, and strongly activated alveolar macrophages. In this group changes appeared as early as 15 min after surfactant administration and intensity of lung injury increased with time. Also, Survanta administration caused damage to the lung tissue. However, the changes were less intense and appeared later (20-25 minutes after Survanta treatment). In conclusion, the presented morphological findings proved that exogenous surfactant administration to healthy rat newborns caused lung damage. Comparing two different surfactant preparation, Exosurf and Survanta, it was shown that the former one produced stronger and faster damage to lung alveoli than the latter one.     

Configuration of the acetal carbon atom of pyruvic acid acetals in some bacterial polysaccharides

The configuration at the acetal carbon atom of pyruvic acid acetals present in some extracellular bacterial polysaccharides has been investigated. Assignment of the absolute configuration was made by comparing signals in the ¹³C- and ¹H-n.m.r. spectra of the polysaccharides with those of model substances. The S-configuration was demonstrated in eight polysaccharides in which pyruvic acid is linked to O-4 and O-6 of D-glucopyranosyl or D-mannopyranosyl residues. The R-configuration was demonstrated in four polysaccharides in which pyruvic acid is linked to O-4 and O-6 of D-galactopyranosyl residues. Consequently, in each of these acetals, which form 1,3-dioxane rings, the methyl group is equatorial and the carboxyl group axial. The S-form was further demonstrated in four polysaccharides in which the pyruvic acid is linked to O-3 and O-4 of D-galactopyranosyl groups.     

Ultrastructural demonstration of amine granules in the adrenal medullary cells of the rat using acid permanganate fixation

Summary Potassium permanganate fixative is usually employed at pH 7.0. At this pH the amines in the granules of the adrenal medullary cells do not react with permanganate. When the pH was adjusted to 5.0, electron dense precipitates were seen in the amine granules of part of the medullary cells, probably noradrenalin containing cells.Supported by the grant from the Finnish Cultural Foundation     

Immune complex receptors on cell surface. I. Ultrastructural demonstration of macrophages.

A method is described for ultrastructural localization of immune complex receptors on the surface of viable peritoneal exudate cells. The technique entails incubation with a soluble complex of horseradish peroxidase (HRP) and specific antibody to HRP at 4 degrees C followed by exposure to diaminobenzidine and processing for electron microscopy. The bound immune complexes were evident as focal deposits of HRP reaction product, adhering closely to the external surface of macrophages with an uninterrupted periodicity varying between 30 and 120 nm. Following incubation with an insoluble immune complex containing a higher proportion of antibody, receptor sites stained frequently, but large aggregates adhered to the cells. Rinsing cells after staining with soluble complexes partially displaced the bound immune complexes. Fixation prior to exposure to immune complexes largely eliminated the binding capacity of the immune complex receptors.     

Histochemical demonstration of proteins, lipids and polysaccharides in Japanese monkey oocytes

A histochemical study of proteins, lipids and polysaccharides was carried out in the oocytes of Japanese monkey (Macaca fuscata) during development of follicles. There were a small number of protein granules reactive to acrolein-Schiff in the cytoplasm of oocytes from primordial, secondary and vesicular follicles, while there were no lipid droplets, granules of neutral polysaccharides or acid polysaccharides in the cytoplasm. Proteins reactive to acrolein-Schiff, neutral polysaccharides reactive to periodic acid-Schiff and acid polysaccharides stainable with alcian blue were observed in the zona pellucida of the oocytes of secondary and vesicular follicles. The zona pellucida contained sudanophilic lipids composed of neutral fats and lipoids, besides the proteins and polysaccharides.     

Ultrastructural demonstration of amine granules in the adrenal medullary cells of the rat using acid permanganate fixation.

Potassium permanganate fixative is usually employed at pH 7.0. At this pH the amines in the granules of the adrenal medullary cells do not react with permanganate. When the pH was adjusted to 5.0, electron dense precipitates were seen in the amine granules of part of the medullary cells, probably noradrenalin containing cells.     

Role of pulmonary surfactant components in surface film formation and dynamics

Pulmonary surfactant is a mixture of lipids and proteins which is secreted by the epithelial type II cells into the alveolar space. Its main function is to reduce the surface tension at the air/liquid interface in the lung. This is achieved by forming a surface film that consists of a monolayer which is highly enriched in dipalmitoylphosphatidylcholine and bilayer lipid/protein structures closely attached to it. The molecular mechanisms of film formation and of film adaptation to surface changes during breathing in order to remain a low surface tension at the interface, are unknown. The results of several model systems give indications for the role of the surfactant proteins and lipids in these processes. In this review, we describe and compare the model systems that are used for this purpose and the progress that has been made. Despite some conflicting results using different techniques, we conclude that surfactant protein B (SP-B) plays the major role in adsorption of new material into the interface during inspiration. SP-C's main functions are to exclude non-DPPC lipids from the interface during expiration and to attach the bilayer structures to the lipid monolayer. Surfactant protein A (SP-A) appears to promote most of SP-B's functions. We describe a model proposing that SP-A and SP-B create DPPC enriched domains which can readily be adsorbed to create a DPPC-rich monolayer at the interface. Further enrichment in DPPC is achieved by selective desorption of non-DPPC lipids during repetitive breathing cycles.     

Ultrastructural demonstration of dehydrogenases in rat cerebral cortex

Summary Techniques for the ultrastructural demonstration of dehydrogenases in cerebral cortex are described. The best fixation for good fine structural preservation and retension of LDH and NADH-diaphorase was obtained by perfusion with a mixture of formaldehyde and glutaraldehyde and for SDH by perfusion with formaldehyde. Comparison of incubation conditions showed that consistent results were obtained using enzyme markers NBT and DS-NBT for LDH and NADH-diaphorase; DS-NBT was more satisfactory than NBT and BSPT for SDH. Penetration of incubation media was improved by Triton X-100; DMSO and ultrasonic treatment were less effective. The techniques enabled the first electron cytochemical demonstration of dehydrogenases in different elements of prefixed cerebral cortex. Ultrastructural sites of enzyme activities were localized within cristae and intermembrane spaces of mitochondria in nerve cell cytoplasm and its processes, oligodendrocytes and astrocytes. Authenticity of the ultrastructural sites was confirmed by four different control experiments.     

Ultrastructural demonstration of proteoglycans in adult rat cornea

Proteoglycans in the adult rat cornea were demonstrated at the electron microscope level using two approaches: (a) staining with cuprolinic blue dye in the presence of 0.3 MgCl2, and (b) immunocytochemical localization of glycosaminoglycans with monoclonal antibodies and protein A-gold complexes. In the stroma two kinds of cuprolinic blue-induced filaments were morphologically differentiated and characterized according to their sensitivity to enzymatic degradations as keratan sulphate-rich and chondroitin-dermatan sulphate-rich proteoglycans respectively. Both types were mostly associated with collagen fibres, occupying the whole stroma except in certain areas whose significance is discussed. By immunocytochemistry, anterior and posterior regions of the stroma were found to be richer in chondroitin sulphate than the middle part, whereas keratan sulphate showed an homogeneous distribution throughout the stroma. Glycosaminoglycans were also detected in corneal basement membranes, epithelium and endothelium. The latter localizations are discussed in the light of what is known at present about the production of glycosaminoglycans by corneal cells.