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Biomedical subjects

J Bereiter-Hahn

Publications and source records attributed to J Bereiter-Hahn.

At least 55 records · Page 3Linked to original sources
PubMed1992

Microcompartmentation of glycolytic enzymes in cultured cells.

The microcompartmentation of aldolase and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was investigated in four different cell types (3T3 cells, SV 40 transformed 3T3 cells, mouse fibroblasts, chick embryo cardiomyocytes) combining cell permeabilization and indirect immunofluorescence technique. Permeabilization of the cells prior to fixation released the soluble fractions, whilst the total amount of enzymes was preserved in nonpermeabilized cells. Both enzymes exist in a soluble as well as in a structure-bound form. The soluble fraction of aldolase and GAPDH is distributed homogeneously throughout the cytoplasm, excluding the nucleus and vesicles. The permeabilization-resistant form is associated with the actin cytoskeleton. A considerable amount of both enzymes is located in the perinuclear region and cannot be attributed to a definite structure. Comparing the staining patterns of aldolase and GAPDH in four different cell types we found that the distribution of the enzymes corresponds with diverse forms of actin cytoskeletal organization of these cells. The codistribution is maintained in cells treated with cytochalasin D.

3T3 Cells↗
PubMed1992

Comparative study on effects of cytochalasins B and D on F-actin content in different cell lines and different culture conditions.

Effects of cytochalasins on actin polymerization state in living cells were measured using fluorimetry of TRITC-phalloidin bound to F-actin. Normal (3T3) and tumour (SV-3T3, B16 melanoma, and Ehrlich ascites) cells were treated with cytochalasin B and cytochalasin D (1 microgram/ml). Three effects of cytochalasins were demonstrated--depolymerization of F-actin, promotion of polymerization, and redistribution of actin without change in polymerization state. Occurrence of a given effect was dependent on cell type, cell density, cytochalasin concentration and type. This indicates that cells from different lines, and even the same cells in different culture conditions may differ significantly in their state of actin polymerization, which we suppose is the cause of their different reactions to cytochalasins. Accordingly, caution should be taken in generalizing the results concerning the effect of cytochalasis on the polymerization state of actin.

3T3 Cells↗
PubMed1991

Dynamic morphology of metastatic mouse T-lymphoma cells invading through monolayers of 10T1/2 cells.

We have used an in vitro model system to analyze cytomechanical aspects of tissue infiltration by T-lymphocytes. The interaction of metastatic T-lymphoma cells with a precultured monolayer of 10T1/2 fibroblast-like cells was recorded in time-lapse video with alternating phase contrast and reflection interference contrast microscopy. Sectioning of embedded specimens as well as cytoskeletal stainings have been performed on matching cocultures. The lymphoma cells did not strongly attach or spread on the dorsal surface of the monolayer cells. Invasion started with the protrusion of a pseudopodium through a narrow gap, and conspicuous constriction of the invading cell's body and nucleus was a consistent feature during the later steps. Overt retraction of the target cells was not seen, but the invading lymphoma cells elevated the fibroblasts over relatively large areas, thereby creating dome-shaped open spaces, allowing for further migration under the monolayer with minimal resistance. Invasion was not unidirectional but was readily reversible at any stage. Due to this wavering character, an invasion event could take more than 1 hour, although the shape alterations involved were fast. Even after the invasion process had been completed, the lymphoma cells could come out from below the monolayer again. Therefore we propose that invasion in this model should be considered as a dynamic equilibrium. Invading T-lymphoma cells displayed diffuse F-actin staining and a well-organized microtubular complex with the centrosomes behind the nucleus in the uropod, which also contained most vesicular organelles.

Actins↗
PubMed1991

Emigration of bilayered epidermal cell sheets from tadpole tails (Xenopus laevis).

Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which then also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly lifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.

Animals↗
PubMed1991

Acoustic microscopy of cultured cells. Distribution of forces and cytoskeletal elements.

The scanning acoustic microscope (SAM) allows one to measure mechanical parameters of living cells with high lateral resolution. By analyzing single acoustic images' sound attenuation and sound velocity, the latter corresponding to stiffness (elasticity) of the cortical cytoplasm can be determined. In this study, measurements of stiffness distribution in XTH-2 cells were compared with the organization of F-actin and microtubules. Single XTH-2 cells exhibit relatively high stiffness at the free margins; toward the cell center this value decreases and reaches a sudden minimum where the slope of the surface topography enlargens at the margin of the dome-shaped cell center. The steepness of the increase in slope is linearly related to the decrease in sound velocity at this site. Thus, a significant determinant of cell shape is paralleled by an alteration of stiffness. In the most central parts, no interferences could be distinguished, therefore, this region had to be excluded from the calculations. Stiffness distribution roughly coincided with the distribution of F-actin, but no correlation to microtubule arrangement was found. Following the treatment of XTH-2 cells with ionomycin in the presence of calcium (in the culture medium), the cell cortex first contracted as indicated by shape changes and by a marked increase in stiffness (deduced from sound velocity). This contraction phase was followed by a phase of microtubule and F-actin disassembly. Concomittantly, sound velocity decreased considerably, indicating the loss of elasticity in the cell cortex. No structural equivalent to sound attenuation has been identified.

Acoustics↗
PubMed1991

Demonstration of calcium in dermal melanocytes of Xenopus laevis and Poecilia reticulata with electron energy-loss spectroscopy and electron spectroscopic imaging.

The subcellular distribution of calcium in dermal melanocytes of Xenopus laevis and Poecilia reticulata has been analysed. Using two cytochemical methods, phosphate precipitation and a combined oxalate-pyroantimonate technique, electron energy-loss spectroscopy and electron spectroscopic imaging have been applied for elemental analysis. Both precipitation techniques revealed a high calcium content in the melanosomes of both species. Calcium was also located in the vicinity of collagen fibrils and in the plasma membrane.

Animals↗
PubMed1991

Effects of ouabain and digitoxin on the respiration of chick embryo cardiomyocytes in culture.

The effect of digitoxin (CAS 71-63-6) and ouabain (g-strophantin, CAS 630-60-4) on respiration, morphology and beating activity of cardiomyocytes in culture derived from embryonic chick hearts has been investigated. The drugs were applied in a perfusion system using a protein- and substrate-free perfusion medium (BSS) at two concentration of K+ (5.4 and 4 mmol/l). In either K+ concentration oxygen consumption was 0.13 +/- 0.05 nmol O2 h-1 per 1000 cells. During 3 h of perfusion with BSS oxygen consumption declines only slightly to 79 +/- 15% of the initial value. No relation was found between beating frequency and oxygen consumption. Increase in respiration ranged from 5 to 45% and lasted between 5 and 120 min. At concentrations being inhibitory to the Na+/K(+)-ATPase (greater than or equal to 1 mumol/l) ouabain stimulated respiration by about 20% at 4 mmol/l K+ and 10% at 5.4 mmol/l K+ while digitoxin was effective at 5.4 mmol/l only (a transient increase of 20%). At 0.1 nmol/l, (a concentration below the KD of the high affinity binding site of the Na+/K(+)-ATPase) ouabain caused a long lasting activation of respiration by about 30%, digitoxin induced a transient rise of up to 20% at 5.4 mmol/l K+. At 4 mmol/l K+ digitoxin did not affect respiration while ouabain caused a transient increase. The lowest concentration of ouabain inducing a reproducible activation of oxygen consumption was 0.1 pmol/l. At this concentration digitoxin was no longer effective. At 1 fmol/l respiration was stimulated only occasionally.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

The distribution of Tyr- and Glu-microtubules during fish scale regeneration.

Cyclic rearrangements of the microtubules (Mts) occur in the hyposquamal scleroblasts which synthesize the highly ordered three-dimensional collagen network forming the basal plate of the fish scale. The distribution of Mts containing tyrosinated and detyrosinated alpha-tubulin (Tyr-Mts and Glu-Mts, respectively) was analyzed in relation to the frequency of Mt reorganization in the teleostean elasmoid scale during collagen deposition using two specific monoclonal antibodies. In the very flat hyposquamal scleroblasts of fully developed scales (with a very low synthetic activity) two microtubule populations were identified. Most contain Tyr-tubulin while Glu-tubulin is found in some "stable" Mts. In the tall prismatic scleroblasts of regenerating scales (active in collagen synthesis) only Tyr-Mts have been revealed. In late stage of regeneration, when the synthetic activity decreases and the scleroblasts flatten, an increasing centriole and Mt labeling with Glu-tubulin antibody was observed. The intimate relationship of intracellular microtubule arrangement and extracellular collagen fibril pattern reported earlier (Zylberberg et al., Cell Tissue Res. 253, 597-603 (1988], together with the results presented here, support the hypothesis that a changing Mt pattern is involved in the generation of the collagen plywood.

Animals↗
PubMed1990

A new model of epidermal differentiation: induction by mechanical stimulation.

In vivo, epidermal cells are committed to terminal differentiation in which they undergo a series of morphological and biochemical changes. In vitro, keratinocytes are able to undergo some steps of this differentiation process only. In view of the fact that in vivo skin is continuously subjected to mechanical stress, we investigated the stimulation of differentiation of transformed keratinocytes by mechanical stimulation. The cells, grown in plastic culture dishes, were periodically treated with weights exerting a pressure of 0.015 Ncm-2. This stimulation lasted from 1 to 4 days. Then keratinocytes were examined using indirect immunofluorescence, 3H-thymidine and 14C-amino acid incorporation, SDS polyacrylamide gel electrophoresis, and Western blotting. Following pressure treatment, the previously monolayered keratinocytes locally grew up to several layers, the number of horny scales increased and, after 4 days, the pattern of cytokeratin was modified. The total amount of keratin increased, forming granular accumulations, while the proliferation rate of the cells decreased. Both the 67 kDa and 49.5 kDa keratin subunits increased in stimulated cells. Moreover, a weak keratin band of 44 kDa appeared that was not present in controls. The results demonstrate that cyclic pressure promotes differentiation of cultivated epidermal cells.

Amino Acids↗
PubMed1990

Measurements of cells in culture by scanning acoustic microscopy.

A novel method allowing the determination of thickness, attenuation and impedance of both living and fixed cells in culture using a single image recorded with a scanning acoustic microscope is described. The method is based on the recording of the image data locating the surface of the cell far below the geometric focus. In this way only the normal incident acoustic wave is used for image formation. Higher values of impedance and attenuation coefficients were found in the cell periphery than in the central part of the cell. This phenomenon is suggested to be due to the different organization of cytoskeletal elements.

Animals↗
PubMed1990

Spreading of trypsinized cells: cytoskeletal dynamics and energy requirements.

The spreading of trypsinized XTH-2 cells (a line derived from Xenopus laevis tadpole heart endothelia) on glass was investigated. Three phases can be distinguished: (1) blebbing of rounded cells, first attachment to a solid substratum and formation of a broad smooth contact area; (2) organization of a peripheral zone of actin fibrils and reinforcement of the basal cytoplasm by a stress fibre-like pattern; (3) extension of lamellae. The first phase seems to be independent of a supply of metabolic energy, while the others clearly depend on it. This is concluded from the close relationship between cellular projection area and energization of mitochondria as revealed by (a) the fluorescence intensity of cells vitally stained with the mitochondria-specific fluorochrome DASPMI (2-4-(dimethylamino)-styryl-1-methylpyridinium-iodine); (b) the degree of spreading in the presence of inhibitors of respiration; (c) effective amelioration of spreading (phases (2) and (3] under conditions of high ATP content. In phase (2) the extension of the central part of the cells becomes stabilized, the cell body settles on the basal cytoplasmic layer and further expansion of the projection area is achieved by lamella formation (phase (3]; motile and stabile regions of the cells become separated. This sequence of events is interpreted as a self-organizing process based on the development of internal hydraulic pressure, actin polymerization and contraction of the newly developed actomyosin network. During trypsinization, depolymerization of actin does not occur but rather on addition of Ca2(+)-containing media. Cellular ATP content drops as well on trypsinization, as on addition of Ca2+. Manganese promotes spreading by decreasing F-actin disassembly and maintaining a high level of cytosolic ATP, most probably because it is not accepted by the calcium pumps. Regarding the association of glycolytic enzymes with F-actin and their influence on actin assembly, lactate dehydrogenase has been inhibited with oxamic acid. This treatment improves the correlation between F-actin content and the degree of spreading; however, the total amount of F-actin remains smaller and the cells spread more.

Actins↗
PubMed1990

[Antiproliferative activity of a highly purified coal tar preparation in comparison with clobetasol-17-propionate].

Coal tar and glucocorticosteroids are among the preparations used for the treatment of hyperproliferative inflammatory dermatosis. Coal tar generally seems to have a better effect on psoriasis capillitii than betamethasone-17-valerate. This finding is confirmed by investigations with human skin fibroblasts. The coal tar preparation Berniter has a strongly inhibitory effect on cell proliferation. The action of the preparation with tar can be clearly distinguished from the effect of the vehicle. At the ED50 concentration of the substance with tar, the vehicle without tar causes no inhibition. Based on weight equivalents Berniter has a greater inhibitory effect on proliferation of human skin fibroblasts than clobetasol-17-propionate. In the case of both substances protein biosynthesis is only affected at concentrations higher than those needed for the inhibition of proliferation. Berniter and the vehicle without tar have an equally strong effect on protein synthesis. Berniter affects the nucleoside triphosphatase bound to the nuclear membrane, and thus influences the nucleocytoplasmic transport of mRNA. The effect of the vehicle and the preparation containing tar on this parameter are very similar. Berniter has a positive effect on cell mobility. In preconfluent cultures the mobility of the cell is slightly increased and in confluent cultures significantly. Our results show that Berniter inhibits proliferation in cell cultures of human skin fibroblasts to a higher degree than clobetasol-17-propionate. It is to be assumed that neither the inhibition of protein synthesis nor the inhibition of mRNA transport play a causal role in the antiproliferative effect of coal tar. Inhibition of proliferation as a result of general damage to the cell can also be practically ruled out.

Adenosine Triphosphatases↗
PubMed1989

Quantitation of the volume of liquid injected into cells by means of pressure.

The amount of TRITC-labeled bovine serum albumin (TRITC-BSA) released from the tip of standardized microcapillaries at different injection pressures and times was investigated. Three test systems were used: (a) formation of droplets of the test solution (TRITC-BSA) in paraffin oil, (b) injection of the test solution into buffer droplets suspended in paraffin oil, and (c) injection into living 3T3 cells. The amount of test substance released was determined by scanning fluorometry. The first procedure (a) allows the fluorometrically determined amount of TRITC-BSA to be related to the volume of released test solution. For this rather large pressures (about 700 hPa) are required to overcome the surface forces counteracting droplet formation. The volume of the spheres was evaluated from photomicrographs of the droplets. The values obtained correlate very well with those determined by measuring the fluorescence emitted by the droplets. Injection into preformed droplets of buffer (b) can be performed with pressures in the range used for injecting cells (50-400 hPa). High reproducibility in the volume released is obtained with a single microcapillary; however, large variations exist between different capillaries, although these should theoretically be of equal diameter. The volume injected into living cells (c) under a given condition may vary by a factor of 5 or more. This variation may be due to viscosity differences of cytoplasm. We recommend, therefore, injection of fluorescent marker substances together with the test substance, enabling the injected volume to be determined by scanning fluorometry or by image analysis.

Animals↗
PubMed1989

Enzyme release from chick myocytes during hypoxia and reoxygenation: dependence on pH.

On reoxygenation of ischemic or hypoxic hearts a sudden release of cytosolic enzymes coupled with hypercontraction and cell injury occurs, which has been termed the "oxygen paradox". We have attempted to imitate this phenomenon in cultured chick myocytes to try to find the cause of this sudden enzyme release. During 4 hours of normoxic perfusion (pH 7.4) monolayer cultures of chick embryonic myocytes retain their normal morphology, beat rhythmically, and show no release of creatine kinase (CK) into the perfusate. Hypoxic perfusion (O2 less than or equal to 0.25 microliter/ml) stops cell contraction (15-20 min) and causes "blebbing" of the sarcolemma (20-30 min). Membrane blebs increase in size and number with continuing hypoxia and eventually the cells become irreversibly damaged. Perfusion at pH 7.4 leads to a release of CK shortly after membrane damage occurs (30-40 min), with peak enzyme levels at 60-90 min. Reoxygenation after 120 min hypoxia does not exacerbate release. Hypoxic perfusion at pH 7.0 suppresses the release of CK from the cells despite extensive membrane blebbing. Normoxic perfusion at pH 7.4 after 100 min hypoxia (pH 7.0) causes an efflux of enzyme from the irreversibly injured cells. This can be prevented by reoxygenating the cells at pH 7.0 and stimulated by raising the pH of the hypoxic perfusate to 7.4. Shorter hypoxic periods (30 mins) at pH 7.0 followed by normoxic perfusion at pH 7.4 lead to a sudden large efflux of CK, arrhythmic contractions and hypercontraction of myofilaments, i.e. the typical symptoms of the "oxygen paradox". Thus changes in external pH can influence the release of intracellular enzymes during hypoxia and reoxygenation.

Animals↗