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Biomedical subjects

J Bereiter-Hahn

Publications and source records attributed to J Bereiter-Hahn.

At least 91 records · Page 5Linked to original sources
PubMed1984

Interaction of metabolic inhibitors with actin fibrils.

The dependence of the arrangement of fibrillar actin in cultured endothelial cells on metabolic conditions was investigated with cellular elements derived from the heart of Xenopus laevis tadpoles. Either primary culture or an established cell line (XTH-2) were used in these studies. The metabolic stage of the cells was influenced by inhibiting respiration and lactate production. The actin pattern was revealed either by indirect immunofluorescence or by tetramethylrhodaminyl (TRITC)-phalloidin fluorescence. Total block of energy supply causes in all cases a distinct loss of actin fibrils, while inhibition of respiration alone increases the variability of actin organization. In primary XTH cells but not in XTH-2 cells cyanide disintegrates most of the actin fibres during 3 h of treatment. This effect is independent of the inhibition of respiration, since actin gels prepared from skeletal muscle also undergo destruction in the presence of cyanide. It is concluded that the actin fibrils of the primary cells and the established line behave differently to changing metabolic conditions and to application of KCN.

Actins↗
PubMed1984

Control of cell shape and locomotion by external calcium.

Dependence of locomotion of Xenopus laevis epidermal cells on calcium influx from the external medium was investigated. Inhibition of Ca2+ influx by 2 mM La3+ or 4 mM Tb3+ in the culture medium causes an immediate stop to locomotion and a loss of motion at the outer margin of the lamella; microcolliculi disappear and the entire lamella becomes flat and very thin. The cell body region enlarges by spreading into the lamella to an extent approximately coincident with the distribution of myosin. The increase in thickness of this area is the result. The cytoskeletal elements actin, alpha-actinin and myosin become homogeneously distributed throughout the cell and a great number of straight microtubules extend to the margin after 20 min in La3+-containing media. Prekeratin distribution does not change. Reduction of calcium concentration in the external medium by EGTA leads to cessation of cell locomotion. Sr2+ (1-4 mM) is also able to replace calcium for triggering locomotion. These findings point to a control of Ca2+-activated contractions of actomyosin by influx of external Ca2+. According to our model of cell locomotion [14] the contractions generate a hydrostatic pressure extending the lamella by flow of hyaloplasm towards the margin. Small swellings (microcolliculi) appearing thereby will be dislocated by a calcium-dependent sol-gel transformation in this area, which contains actin but not myosin.

Animals↗
PubMed1983

Fluorimetry of mitochondria in cells vitally stained with DASPMI or rhodamine 6 GO.

The fluorescent dyes DASPMI and rhodamine 6 GO specifically stain mitochondria in living cells. Dye concentrations from 10(-8) to 5 X 10(-6) mole l-1 can be used. Excitation and emission spectra, and quantum efficiency of DASPMI depend on solvent characteristics. Spectra of mitochondria in living cells correspond to those in phospholipids (excitation around 470 nm, emission 560-570 nm). Fluorescence intensity of DASPMI is a measure for the energization of mitochondria, as revealed by in vitro studies. In living cells uptake of the dye is strongly influenced by inhibitors of oxidative phosphorylation (i.e. by oligomycin, FCCP). Distribution of fluorescence intensity indicates an intracellular gradient in energy load of endothelial cells. Single mitochondria exhibit oscillations in fluorescence. Mitochondria loaded with DASPMI release the dye suddenly into the cytoplasm on treatment with FCCP. Cyanide and antimycin however, do not diminish fluorescence in vivo under optimal nutritional conditions, while they do so in mitochondrial suspension, indicating different mitochondrial behaviour in vivo and in suspension.

Animals↗
PubMed1983

A microscope perfusion respirometer for continuous respiration measurement of cultured cells during microscopic observation.

The construction of a microscope perfusion respirometer (MPR) for simultaneous recording of cellular respiration and microscopic morphology is described. All light microscope techniques for living cells (e.g. phase contrast, differential interference contrast (DIC), fluorimetry) can be applied to the monolayer cells grown on a coverslip. The main constituents of the MPR are a) a precision operating perfusion pump (constant volume output), b) a modified Dvorak-Stotler perfusion chamber, c) a special holder for the Clark-type oxygen probe, d) gas-tight connections of stainless steel tubing with dead volume-free fittings, and e) a temperature control unit. The cell material, established XTH (Xenopus laevis tadpole heart) cell is characterized. Examples of operation are presented, concerning a) normal respiration, b) respiration during uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and c) lactate production under anoxia. The corresponding mitochondrial in situ-morphology is demonstrated on photomicrographs. Details of construction and application are discussed. This new technique is supposed to extend the use of cell cultures instead of animal experiments in pharmaceutical routine tests.

Anaerobiosis↗
PubMed1982

Dynamics of the cytoskeleton of epidermal cells in situ and in culture.

The cytoskeleton of primary tissue-culture cells from the epidermis of Xenopus laevis tadpoles was investigated by phase-contrast, immunofluorescence, and electron microscopy. The connection between the arrangement of different types of filaments and the mechanical properties of the epidermis is discussed. The bilayered epidermis attains stability from thick bundles of tonofilaments interconnecting the basal desmosomes. Twisting of tonofilaments around each other can explain the occurrence of elastic filamentous curls forming a meshwork braced between rows of "small desmosomes" in the apical region of the epidermis. Actin is arranged as a diffuse meshwork and sometimes forms bundles intermingling with tonofilament bundles. Surface membranes and rows of "small desmosomes" are delineated by actin and contain alpha-actinin. Actin raises the tension for rounding and spreading of cells. Microtubules stabilize already well-developed lamellae.

Animals↗
PubMed1981

Interferometric studies of endothelial cells in primary culture.

A population of endothelial cells grown from Xenopus laevis tadpole hearts was investigated by microinterferometry. The resulting interferograms were evaluated by an automatic image analyzer. The mean values of dry mass were 778 +/- 340 pg (10(-15) g) for whole cells, 648 +/- 309 pg for cytoplasm, 116 +/- 45 pg for nuclei, and 19 +/- 10 pg for nucleoli. Two subpopulations of cells were identified, an actively growing one and a less active one. The density (dry mass per microm2) of the nuclei and nucleoli of less active cells was greater than that of the nuclei and nucleoli of actively growing cells. In addition, the inactive cells were always large and possessed a considerable amount of cytoplasm. The entrance of cells into S-phase could not be detected by microinterferometry; and no differences were apparent between cells possessing one nucleolus and those containing two nucleoli. The values obtained in these amphibian cells were compared with those derived from mammalian cells.

Animals↗
PubMed1981

Locomotion of Xenopus epidermis cells in primary culture.

The locomotion of single epidermis cells, grown out from Xenopus laevis tadpole tails has been investigated by time-lapse cinemicrography using phase-contrast and reflection-contrast optics. The cells develop a large, mostly 200-250 nm thick, lamella, which adheres homogeneously to the supporting coverglass and exceeds the projection area of the cell body. From the comparison of RIC-pictures taken at high (1.06) and low (0.62) numerical aperture of illumination (I.N.A.) we deduce that at low I.N.A. the embossment of the medium-facing side of the lamella is visualized. By this method microcolliculi are demonstrated, which form at the edge of the lamellipodium and move backward. They resemble ruffles, but are flatter and no membrane flow towards the perinuclear region is observed. Indirect immunofluorescence reveals an enhanced staining for actin and alpha-actinin in the lamellipodium and in the transition region of cell body and lamella. Tonofilaments do not participate in lamella formation, the relatively few microtubules seem to be oriented in the direction of cytoplasmic flow. Electron micrographs demonstrate the course of fibrils in the cell body and a meshwork of actin filaments and membranous tubules in the lamella. Based on these findings a model for cell locomotion is presented: the motive force is generated by the cell body causing a flow of cytoplasm towards the periphery and extension of the lamella at its edge. The activity of the lamellipodium has to ensure the flat form of the advanced edge; microcolliculi are assumed to represent a small membrane store for the extension of the lamella. The lamellipodium is not involved in the production of motive force. The cell body is anchored to the lamella by radiating fibrils and the fibrillar meshwork is inserted at the 'dorsal' membrane of the lamella and the basal filament cortex of the cell body. This anchorage provides the structural basis for the uptake of lamella material into the cell body in the transition region.

Animals↗
PubMed1981

Evaluation of reflection interference contrast microscope images of living cells.

Reflection contrast microscope methods are generally used for studies of those portions of the cell that are turned towards the glass coverslip, to comprehend the structure of the cytoskeleton and the dynamics of cell movement, as well as formation of cell-glass contacts. In incident illumination only reflected light contributes to picture formation. The intensity of which in the case of observation of unstained cells is small because of small refraction differences. To overcome this problem a reflection contrast system was developed by Leitz according to Ploem [49], in which by using contrast preserving measures the reflection becomes prominent in comparison with the lens reflexes. The emerging pictures are a result of interferences of reflections at glass-cell, cell-culture medium and culture medium-cell interfaces. According to Fresnel's equations the reflected intensity depends on the differences of the particular refractive indices and the thickness of the layers, which determine the phase of interfering beams. In idealized systems of thin films the reflected intensity is a measure for their optical constants. Relative reflection measurements from glass-cell areas is comparison with the known glass-medium reflection, can therefore be revealing as far as refraction index, cell-glass distance or cell thickness are concerned. The estimates by Bereiter-Hahn et al. [15] were made in the assumption of vertical illumination neglecting its actual conical shape: the comparison of two Fresnel functions of cytological relevant measurements show - in accordance with Gingell and Todd [24] - that this is only permitted under certain conditions, depending on the required accuracy of the measurements; an incidence angle of about 30 degrees leads to an error of about 10%, an angle of 50 degrees to more than 50%.

Animals↗
PubMed1979

Quantitative reflection contrast microscopy of living cells.

Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium.

Animals↗
PubMed1979

Metabolic state dependent preservation of cells by fixatives for electron microscopy.

The preservation of mitochondria, cytoplasmic vacuoles and cytoplasm by various fixatives after various pretreatments of ethothelial heart cells from Xenopus laevis tadpoles in tissue culture was investigated. The study was based on phase contrast cinemicrographic recordings and on qualitative and quantitative observations with the electron microscope. Three fixatives were used: 3% glutaraldehyde in phosphate buffer, followed by 1% osmium tetroxide postfixation, fixation only with 1% osmium tetroxide in phosphate buffer and the fixing medium according to Dalton. Cells were either not treated or pretreated for 20 min: 10 microM FCCP (Carbonylcyanide-p-trifluoromethoxy-phenylhydrazone) or 4 mM KCN. The superiority of glutaraldehyde was exemplified by its very rapid action, good preservation of cytoplasm, vacuoles, and mitochondria. It was the only medium which maintained an electron density of the mithochondria matrix. In both of the other fixatives swelling of mitochondria and coagulated appearance of cytoplasm (in phase contrast) was more pronounced in cells pretreated with metabolic inhibitors than in controls. Observations with the light microscope have been confirmed by morphometry of electron micrographs of mitochondria. The relation of matrix space to intracristal space is changed in opposite directions after glutaraldehyde and after the Dalton-type fixation. The results indicate a higher sensitivity against fixation artifacts in cells under pathological conditions than normal cells.

Animals↗
PubMed1978

Transient occurrence of chloride cells in the abdominal epidermis of the guppy, Poecilia reticulata Peters, adapted to sea water.

Chloride cells are characterized by a great number of mitochondria and a uniquely proliferated agranular endoplasmic reticulum through which ions are transported to an apical pit for secretion to the environment. Chloride cells are extremely rare in the epidermis of the guppy, Poecilia reticulata Peters, adapted to fresh water. They are frequent in the epidermis of guppies adapted to sea water three weeks after adaptation, but have disappeared in fish that were kept in sea water for approximately one year. The fine structure of epidermal chloride cells and the influence of prolactin treatment on their number are described. The significance of these cells with respect to the possible roles of the epidermis and the pituitary hormone prolactin in teleost osmoregulation is discussed.

Abdomen↗
PubMed1978

Intracellular motility of mitochondria: role of the inner compartment in migration and shape changes of mitochondria in XTH-cells.

Mitochondrial movements have been followed by phase-contrast microscopy in living XTH-cells (Xenopus laevis tadpole-heart cells) in tissue culture. The same organelles have been viewed subsequently in electron micrographs. Locomotion of mitochondria proceeds at velocities up to 100 micrometer/min. Formation of branches of mitochondria and other shape changes may occur with the same speed. Mitochondrial motility can be classified into 4 types: (I) Alternating extension and contraction at the two ends of rod-shaped mitochondria. (2) Lateral branching. (3) Alternate stretching and contraction of arbitrary parts of a mitochondrion amounting to a kind of peristaltic action. (4) Transverse wave propagation along the organelle. Types I to 3 can be reduced to the same underlying principle; they cause locomotion. Formation of mitochondrial extensions is due to elongation of cristae. The observations are discussed in terms of 4 specific proposals. (I) Intracellular locomotion of mitochondria is caused by local enlargements and contractions of the organelles. (2) The shape changes are correlated with alterations in the arrangement of the cristae. (3) Such arrangements are not associated with overall swelling or shrinkage of the mitochondrion; they are local features. (4) Estimates of the time required for rearrangement of the inner compartment amount to less than 0.3 s for single crista arrangements during the fastest shape changes, and less than 1-3 s during slower alterations. This high velocity is in good accord with the hypothesis of energy conservation by conformational events during oxidative phosphorylation.

Animals↗
PubMed1977

[Inhibition of cell viability by cellular debris. Model experiments to study tissue damage (author's transl)].

Some aspects of tissue damage as it may accompany shock are investigated in an in vitro model system. Some effects of cell debris obtained by sonication of BHK 21 cells on viable cultures of freshly dissociated cells of the same line are evaluated. After 24 h incubation on a coverglass, the area covered by cell nuclei per square millimeter was determined. Spreading of viable cells is inhibited by the cell debris. In this way, growth and viability are also impeded. After 24 h the mitotic index in controls and treated cell-cultures is the same. The inhibition is exhibited as well by the supernatant as the pellet of a centrifuged suspension of sonicated cells. The growth retardation is counteracted by the polyvalent protease inhibitor Trasylol (50 KIE/ml), when the whole suspension or its pellet are added to the culture, but not when the supernatant affects the cells. These results indicate that the injurious substance is not a protease. The protective action of Trasylol may be due to a stabilisation of lysosomes. Tissue injury is often accompanied by alterations in cellular adhesiveness to substrate and in growth rate. The system used here offers a simple model to study the influence of necrotic tissue on cell viability as well as its pharmacological susceptibility.

Animals↗