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[Dark and clear Purkinje's cells in the cerebellum during the postresuscitation period].

Interference microscopy was used to determine dry mass of the nucleus and cytoplasm of light, dark and morphologically changed Purkinje's cells (PC) of the cerebellum of dogs which had suffered clinical death of varying etiology as compared with intact animals. Two weeks after the 12-minute circulatory arrest the area of the nucleus of both light and dark PC sharply increased. Dry mass of the nucleus of dark PC and of the cytoplasm of both PC types also became greater. Two weeks following the 10-minute clinical death because of acute hemorrhage dry mass of the nucleus and cytoplasm of dark PC increased whereas in light PC it was unchanged as compared to the control level. In morphologically changed PC of dogs belonging to the both experimental groups, dry mass of the cytoplasm was essentially higher than in intact animals. It might be assumed that an increase in dry mass of the nucleus and cytoplasm has bearing on intracellular reparative regeneration. Taking into consideration that in intact dogs, dry mass of the cytoplasm and the concentration of dense substances in the nucleus of dark and light PC are similar, it should be suggested that light and dark PC differ in their roles in the maintenance of the population homeostasis rather than in the metabolic rate.

Animals↗

Cholesterol-induced growth stimulation, cell aggregation, and membrane properties of ascites tumor cells in culture.

Ascites tumor cells can be cultivated at a reduced serum concentration if cholesterol (2.50 mg per 100 ml of medium) is added to the culture medium. At serum concentrations of 3%, optimal growth properties are obtained; below 3%, cell cultures usually perish after a few days. Cells grown in the presence of added cholesterol have an elevated content of this molecule per cell as well as in the plasma membrane, and they also show a cholesterol concentration-dependent rate of proliferation. Precursors of the cholesterol-biosynthetic pathway like mevalonic acid, added in mM amounts, or squalene and lanosterol cannot be substituted for cholesterol itself. This is due to the observation that the biosynthetic pathway is blocked at the stage of lanosterol conversion to cholesterol. Cholesterol de novo synthesis from acetate is regulated by the cholesterol content of the cells, which also affects the production of ubiquinone and dolichol. Growth factors such as insulin, prostaglandin F2 alpha, and transferrin added to the medium do not mimic the cholesterol-induced effect. Distribution of DNA during cell cycle and the cell density-dependent reduction in macromolecule synthesis is very similar to the control cells. In contrast, cells without added cholesterol show reduced growth properties accompanied by the accumulation of cells in the mitotic and G2 phase. The cholesterol/phospholipid ratio of the plasma membranes of cholesterol-rich cells is about 15% lower than of the control cells and 40% higher compared to the cholesterol-poor cells, which, however, does not significantly alter the membrane fluidity between the cholesterol-rich and -poor cells as revealed by fluorescence polarization measurements. The most dramatic behavior of the cholesterol-rich cells is their tendency to form aggregates, which is demonstrated either by concanavalin A-induced agglutination or by cell density-dependent aggregation shown by interference microscopy in vivo.

Agglutination↗

Triglycerides, lipid droplets, and lysosomes in aorta smooth muscle cells during the control of cell proliferation with polyunsaturated fatty acids and vitamin E.

The effects of 8,11,14-eicosatrienoic acid [20:3 (n--6)], 5,8,11,14-eicosatetraenoic acid [20:4 (n--6)], and alpha-tocopherol (vitamin E) on the morphology of smooth muscle cells from the guinea pig were studied in tissue culture. Cells were examined by phase and contrast interference microscopy, histochemistry, and transmission electron microscopy for the appearance of lipid droplets and lysosomes. The addition of 120 microM 20:3 (n--6) to the media produces large increases in the number of both lipid droplets and lysosomes. The addition of 10 microM vitamin E to the media has no effect on the morphology of smooth muscle cells. Large increases in the number of both lipid droplets and lysosomes are produced when vitamin E is added together with 20:3 (n--6). Thus, vitamin E has no effects on the morphologic changes induced by 20:3 (n--6). Lipid analyses showed that 80 per cent of the fatty acid taken into the cells is incorporated into triglyceride. Phospholipids incorporated about 17 per cent of the labeled fatty acid while cholesteryl esters, free fatty acids, and a polar neutral lipid fraction each incorporated less than 1 per cent of the labeled fatty acids. Gas liquid chromatography showed that the labeled fatty acid is recovered from the triglyceride fraction with little chain elongation and desaturation. Vitamin E has not effect on the uptake and distribution of labeled fatty acid in smooth muscle cells. 20:3 (n--6) and 20:4 (n--6) are inhibitors of cell proliferation when they are added to cells seeded at low cell density. 20:3 (n--6) has no effect on cell proliferation when it is added to a confluent monolayer which is subsequently split and grown at low cell density. Lipid droplets disappear as these pretreated cells grow. Vitamin E in the presence or absence of fatty acid enhances cell proliferation. The vitamin E and pretreatment studies show that cell proliferation is not related directly to triglyceride accumulation, the formation of lipid droplets, or enhanced lysosomal enzyme activity.

Acid Phosphatase↗

[The mechanisms of the recovery of nerve ending functions during the reinnervation of skeletal muscle].

At the experiments on the frog cutaneous-pectoris muscle the nerve terminal functions in course of reinnervation process were investigated by electrophysiological and morphological methods. At the 20-25th days after the nerve crushing the nerve terminal response, which reflect the nerve terminal currents, formed the propagated action potential, were restored and the arising of evoked transmitter secretion occurred. The regenerating terminals are characterized by a low amplitude and altered shape of responses, by small velocity of the excitation propagation and the low level of evoked transmitter release. The 4-aminopyridine effect at the new formed nerve terminals was quite another, than at the intact nerve terminals. This data concluded, that the membrane of regenerating nerve terminal have a low density and a small gradient of sodium channels along the terminal and have not a calcium-activated potassium channels. It was proposed, that different kinds of channels are inserted into the nerve endings membrane at the different periods of the reinnervation process.

4-Aminopyridine↗

[In vitro studies of modification of mucociliary clearance by guinea pig tracheas by exposure to air pollutants of sulfur or nitrogen dioxide].

We studied the effect of sulfur dioxide (SO2) and nitrogen dioxide (NO2) on mucociliary activity (MCA) and ciliary beat frequency (CBF) in 63 guinea pig tracheas. The tracheas were placed in a gas cylinder and exposed for 30 minutes to SO2 concentrations ranging from 2.5 to 12.5 ppm or to NO2 concentrations ranging from 3.0 to 15.0 ppm. Control experiments were performed with exposure of the tracheas to synthetic air. MCA was measured by recording the light reflected from ciliated mucous membranes using an infrared barcode reader and CBF using video-interference microscopy. The exposure to 2.5 ppm SO2 caused a reduction in mean MCA of 63% and no significant changes in CBF. Higher SO2 concentrations caused a further impairment of MCA as well as a dose-dependent decrease in CBF. 10.0 or 12.5 ppm SO2 induced a decrease from baseline values to approximately 20% in MCA and to roughly 30% in mean CBF. The exposure to NO2 at concentrations ranging from 3.0 to 15.0 ppm did not induce any changes in MCA or CBF of the guinea pig tracheas. Our results show that exposure to SO2 for 30 minutes is able to depress the mucociliary clearance of guinea pig tracheas, whereas the exposure to equivalent NO2 concentrations for the same time do not alter the mucociliary transport.

Air Pollutants↗

Presence and localization of vinculin in Giardia.

A requisite element of pathogenicity in Giardia infections is the parasites' ability to adhere to the intestinal epithelial brush border. The presence of vinculin in Giardia was studied because this protein is known to link the cytoskeleton to the plasma membrane and is localized at adhesion foci in many cell-cell and cell-substrate contact sites. Actin, alpha-actinin, and vinculin were identified in Giardia by western blot analysis. Giardia trophozoites attached to glass substrates were examined by interference reflection microscopy (IRM) and immunofluorescence. The IRM defined the lateral crest, bare area, and overlap region of the ventral disk, as well as the ventrolateral flange and lateral shields as close contact areas between parasite and substrate. These close contact regions were then correlated with immunofluorescence localization of actin, alpha-actinin, and vinculin. Actin was seen in the lateral crest, while alpha-actinin was observed in the ventral disc periphery and lateral shields. Vinculin was viewed at the bare and overlap areas of the ventral disc and portions of the lateral crest, as well as the ventrolateral flange and lateral shields. The correspondence of close contact sites with vinculin localization suggests a role for vinculin in Giardia attachment and adherence.

Actinin↗

[In vitro studies of the beat frequency of ciliary cell cultures after short-term exposure to SO2 and NO2].

Mucociliary transport is an important defense mechanism of the respiratory tract. The aim of this study was to investigate the effect of SO2 and NO2 at different concentrations on ciliary beat frequency (ZSF). Single ciliated cells were obtained from 25 volunteers by nose brush. ZSF was quantified using video-interference-microscopy. The cells were placed on a polycarbonate membrane, which was in contact with the surface of a reservoir filled with RPMI medium (bicarbonate buffered) or electrolyte solution (Ringer), allowing the cells to be supplied by capillarity. In an exposure chamber the cells were exposed for 30 to 120 min to SO2 2.5 to 15.0 ppm at 37 degrees C. SO2 induced a dose dependent decrease in ZSF of the cells, supported by Ringer solution. 2.5 ppm SO2 caused a 42.8%, 12.5 ppm a nearly 100% decrease (8.10 +/- 0.24 vs. 0.28 +/- 0.20 Hz). ZSF of cells cultured in RPMI medium was reduced moderately after 12.5 ppm SO2 exposure (7.90 +/- 0.26 vs. 6.66 +/- 0.31 Hz). In Ringer solution we observed a decrease of pH after 30 min SO2 exposure with 12.5 ppm to a minimum value of 3.6. In marked contrast, the pH of RPMI medium remained constant at 7.5 under identical conditions. After adding RPMI medium to Ringer solution, ZSF increased in parallel to the pH (5.0 ppm: 2.77 +/- 0.37 to 7.97 +/- 0.49 Hz). After an initial increase in ZSF, 120 min NO2 exposure to 15.0 ppm yielded a decrease in ZSF of 23.3% under conditions of constant pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Essential role of filopodia in chemotropic turning of nerve growth cone induced by a glutamate gradient.

Pathfinding of growing neurites depends on turning of the growth cone in response to extracellular cues. Motile filopodia of the growth cone are known to be critical for mediating contact-dependent guidance of the growth cone. However, whether filopodia also play an essential role in growth cone turning response induced by a diffusible chemotropic substance is unclear. Growth cones of cultured Xenopus spinal neurons exhibited chemotropic turning responses in a gradient of glutamate within a limited range of concentrations. This turning response depends on the activation of the NMDA subtype of glutamate receptors and requires the presence of extracellular Ca2+. Time-lapse differential interference contrast microscopy with quantitative analysis of filopodia dynamics showed a close correlation between an increased number of filopodia on the side of the growth cone facing the glutamate source and the turning. Such filopodia asymmetry was observed within minutes after the onset of the glutamate gradient, before any detectable turning of the growth cone. In Ca(2+)-free medium, no filopodia asymmetry was induced by the glutamate gradient, and no growth cone turning was observed. Furthermore, elimination of filopodia with a low concentration of cytochalasin B completely abolished the turning response without substantially affecting neurite extension. Thus, filopodia may be required for chemotropic guidance of the growth cone, and an asymmetry in filopodia distribution may be an early cellular event responsible for determining the direction the growth cone advances.

Actins↗

Combined exposures of human ciliated cells to different concentrations of sulfur dioxide and nitrogen dioxide.

In the present study we investigated the influence of two common air pollutants, sulfur dioxide (SO2) and nitrogen dioxide (NO2) on ciliary beat frequency (CBF). Ciliated cells were obtained by nose brush from 12 healthy volunteers and placed on a polycarbonate membrane which was in contact with Ringer's electrolyte solution. This allowed the supply of the cells by capillarity in parallel to the reaction of the pollutants with the cell surfaces. In an exposure chamber the cells were exposed for 30 min. at 37 degrees C either to SO2 (2.5-12.5 ppm) or to NO2 (3.0-15.0 ppm), or to a mixture of NO2 (12.0 ppm) and SO2 (2.5 or 5.0 ppm). CBF was measured by video-interference-microscopy. With SO2 we observed a dose-dependent decrease in CBF with Ringer's solution. 2.5 ppm SO2 caused a 42.8% decrease and 12.5 ppm a decline of approximately 100% (8.10 +/- 0.24 Hz vs. 0.28 +/- 0.20 Hz). In parallel, we observed a decrease in the pH-value from 7.4 to 3.6. 30 min. NO2 exposure (3.0-15.0 ppm) induced a significant dose dependent increase in CBF from 8.4 +/- 0.34 Hz to 9.4 +/- 0.44 Hz. Exposure to a mixture of SO2 and NO2 with Ringer's solution revealed that SO2 exerts a stronger influence on CBF than NO2. Exposure to both pollutants resulted in the same as exposure to SO2 alone. Our findings demonstrate a strong correlation between SO2-modified pH values and CBF. Exposure to a combination of two pollutants revealed the dominant influence of SO2 on CBF while the augmented effect of exposure to NO2 alone might be due to the oxidative potential of this gas.

Adult↗