Accumulation of membranes and photoreceptor degeneration in mouse retina.

Ultrastructural response of photoreceptors to 10 days of treatment with reserpine and bromocryptine, and to 10 days of illumination, was studied. Treatment with isoproterenol was also applied in some experiments with continuous illumination. Treatment with bromocryptine and reserpine induced elongation of rod outer segments and accumulation of membranes in the subretinal space. Increased incidence of apoptosis but not of autophagocytosis was observed in both cases. Continuous illumination with or without treatment with isoproterenol was found to induce intracellular accumulation of membranes, autophagocytosis and apoptosis. Incidence of apoptosis was slightly decreased in the case of isoproterenol application. We conclude that intracellular or extracellular accumulation of membranes can function as the apoptotic trigger in photoreceptors. Triggering of apoptosis was not dependent on autophagocytosis. Possible role of membrane peroxidation in photoreceptor cell death is discussed.

Cytochemical staining of vacuolar compartments in the developing small intestine of mice.

Osmium impregnation, nicotinamide adenine dinucleotide phosphatase (NADPase) and thiamine pyrophosphatase (TPPase) localisation have been applied as a cytochemical tool for the labelling of the endomembranes in the small intestinal cells of mice during embryonic development. Only cisternae of the endoplasmic reticulum were stained by prolonged osmification in the undifferentiated cells. At later developmental stage, Os-black was found in cis Golgi cisternae, small vesicles and also in the endoplasmic reticulum of some epithelial cells. At the early embryonic stage the product of NADPase reaction was visible in medial saccules of the Golgi complex and in lysosomes, while at the later stages positive staining appeared also on the apical cell surface and within the structures probably belonging to endosomal compartment. TPPase activity was constantly present in trans Golgi cisternae of the cells during the studied period of fetal life. These results indicate that the cytochemical markers for Golgi staining can also be found in several other endomembrane systems. They give the insight into various connections between the particular intracellular compartments occurring at different developmental stages.

Analysis of the cell death in the mucoid epithelium of mouse stomach.

The death and phagocytosis of the stomach mucoid epithelium cells in 2-8 day old mice were investigated in relation to the state of cell differentiation and the cell age. The autoradiographic determination of 3H-thymidine and cytochemical demonstration of neutral mucosubstances in secretory granules (TA-UA and PA-TCH-SP methods) were used. During the migration from the proliferation centers to the surface of the mucoid epithelium the cell death occurs in two different ways: by apoptosis and by necrosis. Necrosis is limited to superficial, fully differentiated mucoid cells, which--after the cell death has occurred--are desquamated into the lumen as fragments of irregular shape. The apoptosis is very common and takes place in all the regions: from the proliferation nests up to the surface. It does not depend on the step of differentiation or the age of the cells. In the phagocytosis of apoptotic bodies are included neighbouring mucoid cells. The general occurrence of the apoptosis indicates the important role of this process in the regulation of the cell population during the morphogenesis of the stomach epithelium.

Blood-urine barrier formation in mouse urinary bladder development.

Formation of the blood-urine permeability barrier in differentiating mouse transitional urothelium was studied. It was established that the development of superficial cell barrier is a two-phase process: beginning with formation of the tight junctions, followed by formation of fusiform vesicles and asymmetric apical plasma membranes. Fusiform vesicles differentiate during days 15 and 17 of gestation and fuse with the apical plasmalemma. Thus a thick membrane is formed before the excretion of hypertonic urine into the embryonic bladder. Through some degenerative superficial cells slough between fetal day 17 and the day of birth, the bladder epithelium in mice does not lack an effective permeability barrier.

The role of exocytosis in the apocrine secretion of milk lipid globules in mouse mammary gland during lactogenesis.

Functional relations between exocytotic vesicle membranes, plasmalemma and milk fat globule membranes (MFGM) were studied during the final stages of mouse mammary gland differentiation, in the gland during full lactation and in the postpartum gland in which the synthesis of secretory products was partly inhibited by application of 2-Br-alpha-ergocryptine. Analysis of ultrathin sections, freeze-fracture replicas, scanning electron microscopy and application of a cytochemical marker filipin showed that the apocrine secretion of lipid globules was closely related to the exocytosis of milk proteins. During the last days of gestation the secretion of lipid globules resulted from many exocytotic events of the secretory vesicles that accumulated and fused around the cytoplasmic lipid droplets. Seldom the lipid droplet protruded partly into the gland lumen and a part of its surface became covered with the apical plasmalemma. Although apical plasmalemma became more important in the formation of MFGM in the postpartum period, we could still confirm a direct contribution of secretory vesicle membranes to the final detachment of the lipid globule. The application of 2-Br-alpha-ergocryptine hindered the apocrine secretion of the lipid globules and a situation similar to the situation in the prepartum gland was observed.

Prolonged osmification as an indicator of the differentiation process in the endomembrane system.

The technique of prolonged osmification was used in the analysis of reducing capacity of perinuclear space, endoplasmic reticulum and cis-Golgi cisternae in different epithelial cells during embryonic differentiation and immediately after the birth. Cells of the mouse gastric and intestinal epithelium and of the exocrine pancreas and mammary gland were analyzed. It was shown that endomembrane compartments exhibit high variability in their capacity to reduce OsO4 into lower valency oxides. Typical staining of cis-Golgi cisternae by osmium black does not occur before the cells achieve the developmental state in which production of specific products starts. The changes in stainability occurring from the perinuclear space and endoplasmic reticulum towards the cis-Golgi cisternae indicate a maturation pathway with no direct correlation to the chemical characteristic of the substances produced in different cell types. In the mammary gland the reduction capacity of endoplasmic reticulum disappeared with the intensive synthesis of lipids. Considering our previous results and those of other authors, the possible reasons for the observed dynamics in reducibility in particular segments of endomembraneous space are discussed.

The relationship between synthesis of secretory products and reducing capacity in pancreas and parotid acinar cells.

The secretory products in exocrine pancreas acinar cells in utero were found to reduce osmium tetroxide. This reducing capacity was also exhibited by adult pancreas and parotid glands in different phases of synchronized secretion, and after single or chronic administration of a secretagogue, pilocarpine or isoprenaline. In utero, the reducing capacity appeared in the pancreas concomitantly with the synthesis of secretory products, and was limited to the transitional vesicles on the cis Golgi side. After birth, osmium staining occurred in the cis Golgi vesicles and cisternae of both glands. In the chronically-treated parotid gland, where the occupational programme for secretory proteins had been altered, the reducing capacity was diminished, resembling that in embryonic exocrine pancreas.

Influence of antimicrotubular drugs on the Golgi apparatus of stomach secretory mucoid cells and small intestine absorptive cells.

The effects of vinblastine and colchicine on the Golgi apparatus of stomach surface mucoid and absorptive intestinal cells were compared by cytochemical analysis. The two epithelial cells were chosen because of their different specific functions in the formation of secretory granules, the production of lysosomes and the intensity of membrane traffic in the cytoplasm. For the analysis, adult mice were injected with 1 mg/100 g b.w. of vinblastine and 1 mg/100 g b.w. of colchicine. For the demonstration of cis and trans cisternae of the Golgi apparatus, prolonged osmification, thiamine pyrophosphatase and acid phosphatase activity identification were applied. After treatment with vinblastine or colchicine, polarity of stacks in the Golgi apparatus of surface mucoid cells is preserved although the number of cisternae with thiamine pyrophosphatase or acid phosphatase activity decreases. However, the Golgi apparatus of intestinal absorptive cells completely disintegrates and only a few separated cis or trans cisternae can be identified. The main effect seems to be a reduction of vesicles which can be cytochemically identified as parts of the Golgi apparatus and an accumulation of vesicles which probably originate from budding ER. Communication between the ER and the Golgi apparatus seems to be interrupted.

Cytochemical localization of carbohydrates in intercalated duct and acinar cells of mouse parotid gland.

The glycoconjugate composition of mouse intercalated duct and acinar cells of parotid gland has been compared. Mucins containing 1,2-glycols were demonstrated by the tannic acid-uranyl acetate technique. Hexose residues of glycoconjugates were identified using ferritin conjugated with Canavalia ensiformis agglutinin (Con A), Triticum vulgare or wheat germ agglutinin (WGA), Ricinus communis I agglutinin (RCA-I), Phaseolus vulgaris agglutinin (PHA-E) and Arachis hypogaea agglutinin (PNA). Whereas qualitative and quantitative differences were observed in sugar residues of secretory granules in intercalated duct and acinar cells, apical plasmalemmae were labelled sparsely and similarly. This indicates that the glycocalyx composition of apical plasmalemmae in the parotid acinar and intercalated duct cells is little influenced by secretory granule composition.

Prolonged osmification of stomach epithelium.

Osmium impregnation was used upon the mice stomach epithelium to show possible differences in staining during differentiation. In the cells of the stratified gastric epithelium of 14-day-old mice embryos Os black was completely lacking in the Golgi complex. In some but not all cells the staining appears in the perinuclear space and in the endoplasmic reticulum. In the mucoid cells 1 and 8 days after the birth the osmiophility is not uniformly distributed throughout the endomembrane segments, except the cis face of the Golgi complex which is heavily stained. Our results indicate on the variability of the reduction capacity of particular endomembrane segments during differentiation and among the cells at the definite developmental stage.

Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse.

Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained. These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated and treated acinar cells.

Cytochemical and scanning electron-microscopic analysis of apoptotic cells and their phagocytosis in mucoid epithelium of the mouse stomach.

The formation of apoptotic cells and their phagocytosis by viable neighbouring cells in the gastric epithelium of 2- to 6-day-old mice was analysed. In order to observe the topographic relationship between apoptotic and normal epithelial cells using scanning electron microscope, the critical-point dried tissues was cracked before coating with gold. Cytochemical methods for the identification of surface carbohydrates and different tracers for apical and lateral cell membranes were applied for the analysis using the transmission electron microscope. Apoptotic cells were found on apical and lateral surfaces; this indicates the presence of tight connections with viable cells at some points. Ruthenium red strongly stained all accessible surfaces of normal cells and of apoptotic bodies. The quantity of neutral mucosubstances, as revealed by staining with tannic acid-uranyl acetate, seemed to decrease in the glycocalyx of apoptotic cells. The scanning and transmission electron-microscopic results suggest that the phagocytotic vacuoles arise at the lateral side of the cells. The phagocytotic activity is not dependent upon a definite differentiation step of the mucoid cell.

Lectin-binding pattern in parotid acinar cells. The fracture-labelling method and post-embedding staining.

The localization of complex carbohydrates in the Golgi apparatus, secretory granules and plasmalemma of mouse parotid acinar cells was studied using the fracture-labelling method. The hexose residues of glycoconjugates were identified using ferritin conjugated with Wheat Germ Agglutinin (WGA-), Ricinnus Communis Agglutinin II (RCA-II-), Phaseolus Vulgaris Agglutinin (PHA-) and Limulus Polyphemus Agglutinin (LPA-). We found that the fracture-labelling method allows not only the labelling of membrane faces but also analysis of the compartment's content that is exposed during the fracturing of the tissue. Our results revealed differences in the hexose residues located in the Golgi apparatus, secretory granules and the apical and lateral plasmalemma. Numerous binding sites for WGA-, PHA- and RCA-II-ferritin were demonstrable in the Golgi apparatus. In secretory granules, the WGA- and RCA-II-ferritin binding sites were most numerous, while LPA-ferritin binding sites were very rare. The density of the binding sites for PHA-ferritin showed considerable variation in secretory granules. The apical plasmalemma exhibited a high density of binding sites for all of the lectins used. In the lateral plasmalemma, LPA-ferritin was not bound, and there were fewer binding sites for WGA-, RCA-II- and PHA-ferritin.

The carbohydrates of secretory granules and the glycocalyx in developing mucoid cells.

Complex carbohydrates in secretory granules and at the apical cell surface of mouse gastric mucoid cells were studied during embryogenesis and in the early postnatal period by various cytochemical methods; the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) and tannic acid-uranyl acetate (TA-UA) procedures made neutral mucosubstances (NMS) visible, whereas the hexose residues of glycoconjugates were identified using WGA-, RCA II- and ConA-ferritin. The glycocalyx was stained with ruthenium red (RR). During differentiation of the embryonic mucoid cells the number of secretory granules increased in parallel to the increase in their carbohydrate component. NMS-stainable parts in secretory granules also had binding sites for the conjugates RCA II- and WGA-ferritin, but the binding of ConA could not be identified. The increasing quantity of NMS in secretory granules was correlated with the increased amount of PA-TCH-SP and TA-UA positive substances in the apical glycocalyx only in 14- and 18-day-old embryos. The observed uniform affinity for RR and lectin conjugates in all analysed developmental stages remains to be explained.

[Fibromyxoma of the left ventricle as a cause of heart aneurysm and recurrent thromboembolism]. / Fibromiksom levega prekata kot vzrok srcne anevrizme in ponavljajocih se tromboembolij.

Primary heart tumours are a rarity but no more an academic curiosity. When recognised they may be successfully surgically treated. A Cardiac tumour is to be suspected in thromboembolism of young people especially in the absence of cardiac symptoms. A short review of possible symptoms and diagnostic methods available in the present is given. A successfully operated case of fibromyxoma of the left ventricle with consecutive cardiac aneurysm and peripheral thromboembolism is presented. Selective coronary angiography and cine-cardioangiography were the deciding investigations. The nature of the tumour is shown by means of classical histology as with the aid of electron microscopy.

Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice.

The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2-6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.

Nafenopin-induced proliferation of peroxisomes in the small intestine of mice.

The effect of nafenopin on the epithelial cells of the small intestine of mice was studied. After 17 days the control and nafenopin-treated groups were sacrificed. The tissues were incubated in alkaline DAB medium. Ultra-thin sections of small intestinal tissue from both groups were examined by electron microscopy. Electron micrographs were prepared and examined stereologically so that any morphologic differences in the epithelial cell peroxisomes and mitochondria between the experimental and control groups could be evaluated quantitatively. In the nafenopin-treated group proliferation of peroxisomes occurred, as indicated by significant increases in volume, and surface and numerical density of these structures compared with controls. No such alterations were found in the mitochondria. Our results show that the response of small intestinal epithelial cells to nafenopin is analogous to that produced in hepatocytes by the same drug. Hepatocyte peroxisomes are supposed to be involved in lipid metabolism and it seems that small intestinal epithelial peroxisomes play a similar role.

The development of microperoxisomes in the cells of the proximal tubules of the kidney and epithelium of the small intestine during the embryonic development and postnatal period.

Microperoxisomes were identified in the cells of proximal tubules of the kidney and small intestine of mice from 13-19 days old embryos, in suckling period and adult animals. Examination of both tissues after incubation in diaminobenzidine medium revealed anucleoid microperoxisomes closely related to the granular endoplasmic reticulum and occasionally found in close relation to it. The number of microperoxisomes within the cells considerably increases with the advanced stage of differentiation of both studies tissues. In the cells of proximal tubules the changes in shape and size of microperoxisomes are also observed with a higher stage of differentiation. These studies indicate that the formation and augmentation of microperoxisomes passes strictly parallelly with the differentiation, i.c. with the ability of cells to perform their specific functions.