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Keratocytes derived from the epidermis of aquatic vertebrates are now widely used for investigation of the mechanism of cell locomotion. One of the main topics under discussion is the question of driving force development and concomitantly subcellular force distribution. Do cells move by actin polymerization-driven extension of the lamella, or is the lamella edge extended at regions of weakness by a flow of cytoplasm generated by hydrostatic pressure? Thus, elasticity changes were followed and the stiffness of the leading front of the lamella was manipulated by local application of phalloidin and cytochalasin D (CD). In scanning acoustic microscopy (SAM), elasticity is revealed from the propagation velocity of longitudinal sound waves (1 GHz). The lateral resolution of SAM is in the micrometer range. Using this method, subcellular tension fields with different stiffnesses (elasticity) can be determined. A typical pattern of subcellular stiffness distribution is related to the direction of migration. Cells forced to change their direction of movement by exposure to DC electric fields of varying polarity alter their pattern of subcellular stiffness in relationship to the new direction. The cells spread into the direction of low stiffness and retract at zones of high stiffness. The pattern of subcellular stiffness distribution reveals force distribution in migrating cells; i.e., if a cell moves exactly in a direction perpendicular to its long axis, then the contractile forces are largest along the long axis and decrease toward the short axis. Locomotion in any angle oblique to this axis requires an asymmetric stiffness distribution. Inhibition of actomyosin contractions by La3+ (2 mM), which inhibits Ca2+ influx, reduces cytoplasmic stiffness accompanied by an immediate cessation of locomotion and a change of cell shape. Local release of CD in front of a progressing lamella activates a cell to follow the CD gradient: The lamella thickens locally and is extended toward the tip of the microcapillary. Release of phalloidin stops extension of the lamella, and the cell turns away from the releasing microcapillary. The response to CD is assumed to be the result of local weakening of the cytoplasm due to severing of the actin fibrils. Phalloidin is supposed to stabilize the leading front by inhibition of F-actin depolymerization. These observations are in favor of the assumption that migration is due to an extension of the cell into the direction of minimum stiffness, and they are consistent with the hypothesis that local release of hydrostatic pressure provides the driving force for the flux of cytoplasm.

Rat hearts hypertrophied by exposure of the animals to low oxygen pressure were perfused by the Langendorff technique. After oxidative stress induced by hypoxia/reoxygenation, functional recovery of the hypertrophied right heart was insufficient when compared to non-hypertrophied controls. Accordingly, mitochondrial membrane potential did not recover sufficiently. There was a positive trend for improvement of the rate-pressure product during reoxygenation in lipoic acid (CAS 1077-28-7; 0.8 mumol/l) treated hearts which was also verified for membrane potential. Adenosine 5'-triphosphate and creatine phosphate contents as well as the ATP/ADP ratio in hypertrophied right ventricle were significantly increased after reoxygenation in hearts treated with lipoic acid. With lipoic acid, there was a significantly higher content of glutathione (oxidized form) after reoxygenation, Ca2+ uptake was significantly increased in mitochondria isolated from hypertrophied right ventricles and treated by 12 nmol/mg protein of lipoic acid. The results reveal a distinct improvement of mitochondrial structure/function by lipoic acid and suggest for therapy a combination with the synergistic free radical scavenging properties of tocopherol (CAS 10191-41-0).

The state of crosslinking of microfilaments and the state of myosin-driven contraction are the main determinants of the mechanical properties of the cell cortex underneath the membrane, which is significant for the mechanism of shaping cells. Therefore, any change in the contractile state of the actomyosin network would alter the mechanical properties and finally result in shape changes. The relationship of microtubules to the mechanical properties of cells is still obscure. The main problem arises because disruption of microtubules enhances acto-myosin-driven contraction. This reaction and its impact on cell shape and elasticity have been investigated in single XTH-2 cells. Microtubule disruption was induced by colcemid, a polymerization inhibitor. The reaction was biphasic: a change in cell shape from a fried egg shape to a convex surface topography was accompanied by an increase in elastic stiffness of the cytoplasm, measured as longitudinal sound velocity revealed by scanning acoustic microscope. Elasticity increases in the cell periphery and reaches its peak after 30 min. Subsequently while the cytoplasm retracts from the periphery, longitudinal sound velocity (elasticity) decreases. Simultaneously, a two- to threefold increase of F-actin and alignment of stress fibers from the cell center to cell-cell junctions in dense cultures are induced, supposedly a consequence of the increased tension.

A new method is described for observing and quantifying an aspect of contact inhibition of cell movement that is sometimes called "contact paralysis". Based on the scanning acoustic microscope (SAM), the method can detect changes in the mechanical properties of cells, as well as changes in their motility and may therefore be more sensitive to some dynamic changes than methods based on optical microscopy. With this method intracellular motility of normal and transformed cells of epithelial and fibroblastic origin was investigated. By subtraction of SAM images patterns of motility, domains were detected that changed in a characteristic way among various cell lines. Wave-like and nucleating domains could be distinguished; they were also used for the quantification of motility. Like migration intracellular motility is influenced by cell-cell contacts. In zones where a cell touches its neighbours motility domains change or disappear depending on the cell type. In immortalized epithelial cells (XTH-2 cells) large quiescent zones developed in the region where contact with neighbouring cells was established, whereas in fibroblastic (3T3) cells motility was reduced less and did not change in domain pattern. Epithelial and fibroblastic cells were less motile when in contact with other cells or in confluent cultures than when solitary, i.e. their motility was contact inhibited. Transformed (SV40 3T3) cells, however, did not reduce their motility when in contact to or enclosed by other cells. The molecular basis for motility domains remains to be investigated.

In the present study we investigated the direct effects of potential uremic toxins on the energy metabolism of cultured cardiac myocytes. High-energy phosphates were extracted with perchloric acid and determined by high performance liquid chromatography. Energy charge (calculated from the ratio of [ATP], [ADP] and [AMP] was significantly reduced by 20 mM urea and the combination of creatinine (5 mM) plus urea (200 mM). On the other hand, perfusion with culture media containing clinically relevant amounts of urea (20 mM) or creatinine (1 mM) increased the PCr/ATP ratio. This effect was more pronounced after application of an artificial uremic medium (consisting of uremic serum, urea, creatinine and cytokines) or high amounts of creatinine (5 mM) plus urea (200 mM). As contractility of myocytes is reduced due to application of uremic compounds or uremic serum, we attribute changes in contraction frequency or inotropy to dysregulation of calcium availability within the cell. In fact, the cardiodepressive action of uremic serum (2.5%) could be completely reversed by the calcium agonist, Bay K 8644, thus indicating disturbances in myocardial calcium homeostasis in uremia. Altered calcium regulation by uremic toxins might therefore be responsible for the observed changes in myocardial energy metabolism. These results might contribute to the understanding of the pathogenesis of cardiac damage in end-stage renal disease.

Human melanocytes of the adult skin are slow-cycling cells with a highly dendritic morphology. Nevertheless in vitro proliferation can be achieved using adequate stimulators. Time lapse studies revealed the morphologic changes during melanocyte mitosis: dendrites are drawn back into the cell body, the cell becomes spherical and detaches from the support. Cell division takes place while the cell is suspended. Consecutively the two cells reattach to the support and form new dendrites. About 1% cells per culture are detached from the support and ca. 70% of these cells are viable and putative within mitosis. By every medium change mitotic cells become withdrawn supporting selection of G0-cells, Therefore we recommend centrifugation of exhausted medium in order to add mitotic cells back to the culture.

Eumelanogenesis of human skin melanocytes requires at least three enzymes: tyrosinase, TRP 1, and TRP 2. The regulation of these enzymes on transcriptional level was detected in a semiquantitative attempt. The total RNA of melanocytes was reverse-transcripted and followed by a PCR with degenerated primers for all three enzymes. The amplification products were related to each other densitometrically. We examined five different culture conditions: 1) melanocytes in a popular phorbolester containing F-10-medium, 2) melanocytes in a co-culture medium with EGF, 3) melanocytes in a co-culture medium with high calcium, 4) melanocytes co-cultured with keratinocytes in EGF containing co-culture medium, and 5) melanocytes co-cultured with keratinocytes in co-culture medium with high calcium. Melanocytes cultured in phorbolester containing F-10-medium featured transcripts of tyrosinase, TRP 1, and TRP 2 in the ratio 45:45:10. The same results were obtained for melanocytes co-cultured with keratinocytes under the two different culture conditions. In melanocytes cultured alone in co-culture media only TRP 1-transcripts were present. It is likely that under co-culture conditions a keratinocyte-derived factor supports the transcription of all three enzymes. For melanocytes in the phorbolester-containing melanocyte medium a proteinkinase C dependent regulation of transcription seems possible.

The shape of cells during interphase in sparse cultures often resembles that of fried eggs. XTH-2 cells, which have been derived from tadpole heart endothelia, provide a typical example of this type of shape. To understand the physical basis of this shape, the cytoskeleton of these cells has been investigated in detail. Subcellular elasticity data have been achieved by scanning acoustic microscopy (SAM). Their changes were observed during treatment of the cells with microtubule-disrupting agents (colcemid and low temperature), and shape generation in giant cells produced by electro-fusion was observed with SAM, revealing the role of the nucleus as a force centering organelle. From these observations combined with well-documented observations on cellular dynamics described in the literature, a model is developed explaining the fried-egg shape of cells by means of interacting forces and fluxes (cortical flow, bulk flow of cytoplasm, microtubule-mediated transport of cytoplasm) of cytoplasm. The model also allows the comprehension of the increase of tension in cells treated with colcemid.

XTH-2 cells, a cell line derived from tadpole heart endothelial cells, were blocked at the end of the G1 phase of the cell cycle using desoxyguanosine (dG). Stress fibers are the dominant actin fibril structure in blocked cells. They disappear starting with the release of the dG block until the G2 phase of the cell cycle. In addition, after the release peripheral lamellae and microspikes appear, indicating increased surface motility of the cells. Concomitantly with these changes, net lactic acid production is increased while oxygen consumption remains constant. Increase in the content of F-actin per cell takes place only in subconfluent cultures. Similar morphological changes (organization of F-actin) have been induced by phorbol myristate acetate treatment. These are also accompanied by an increase in lactic acid production and unaltered oxygen consumption. Therefore, the changes in energy metabolism are supposed to result from the rearrangement of the F-actin network: The emanating loose fibrillar pattern provides an increased surface for the association with glycolytic enzymes, which enhances enzyme activity and represents a more motile state. The increased energy demand of the more motile structures is supplied predominantly by ATP derived from glycolysis.

Different models of isolated cardiomyocytes are generally used for biochemical, biophysical, and pharmacological studies. Fetal cardiomyocytes can be easily cultured for several weeks regaining their ability for rhythmical and synchronous contractions. For investigations, differentiated myocytes derived from adult hearts are closer to the in situ situation. Unfortunately, these cells at best exhibit irregular and asynchronous contractions at very low frequencies. Already 1 d after seeding calcium-tolerant rod-shaped adult cardiomyocytes on a suitable substrate, the differentiated cells begin to dedifferentiate forming a confluent monolayer. After 7-10 d their beating activities are like those of fetal cells. Therefore, we tried to combine the advantages of both cell types to achieve fully differentiated cardiomyocytes, rod-shaped and rhythmically beating, isolated from adult hearts. Using contractile fetal cells as a substrate for the adult cardiomyocytes, freshly seeded differentiated adult myocytes are paced by the contraction frequency of the fetal monolayer. As a consequence, the rod-shaped adult cardiomyocytes reach frequencies of more than 140 cycles/min without external electrical stimulation. This model enables us to study cardiomyocytes in a state very similar to the in situ situation with respect to morphology, integrity, and contractile behavior.

Introduction: Cellular rejection mechanisms are characterized by the infiltration of sensitized lymphocytes through the donor endothelium into the transplanted organ. The regulation of cellular adhesion molecules by soluble mediators (cytokines) is thought to play a dominant role in this process. In the present study the kinetics of lymphocyte infiltration were examined using a new established in vitro model and compared to the kinetics of adhesion molecule expression., Materials and Methods: Peripheral blood lymphocytes (PBL) of healthy volunteers were pipetted to allogeneic endothelial cell (EC) monolayers and binding rates evaluated after different incubation times. Adherent PBL could be distinguished from penetrated PBL by means of a combined phase contrast- / reflection interference contrast - microscope., Results: 30-35% of all pipetted PBL adhered to unstimulated EC maximally. Out of these cells < 10% penetrated through the endothelium ( = maximum penetration). The cytokines alpha-, beta-, gamma-interferon (IFN) or IL-1 did not enhance maximum adhesion, but accelerated this process. However, a 2 hrs prestimulus of EC by these cytokines was necessary to induce acceleration. Maximum penetration was enhanced by alpha-, beta-, gamma-IFN, but not by IL-1, irrespective whether PBL were added together with cytokines to unstimulated EC or to already prestimulated EC. Immunocytochemical and fluorometric analyses of adhesion molecule expression revealed a cytokine induced upregulation or de novo expression of the adhesion antigens ICAM-1, ELAM-1 and VCAM-1 on EC membranes. Interestingly, PBL adhered to EC before upregulation or de novo expression of adhesion molecules was detected., Discussion: The results showed that PBL adhesion and penetration processes were regulated differentially by cytokines. The early phase of PBL attachment to EC seemed not to be influenced by adhesion antigens but by an activation of the lymphocyte cytoskeleton.

The effects of 0.1 nmol/l ouabain (CAS 630-60-4) and digitoxin (CAS 71-63-6) on oxygen consumption rate (OCR) and the contractility of cultured chick embryo cardiomyocytes were investigated using a perfusion system which allowed microscopic observation during the experiment. contractility was assessed by a novel image analysis procedure. During substrate free perfusion the OCR was increased on application of 0.1 nmol/l ouabain or digitoxin for 60 min, whilst contractility was not affected. Digitoxin (0.1 nmol/l) elicited no change in the OCR in the presence of glucose, pyruvic acid, lactic acid and caprylic acid. Ouabain (0.1 nmol/l) did not affect the OCR or contractility in the presence of glucose, pyruvic acid and lactic acid, however together with caprylic acid the OCR was increased significantly. These results demonstrate a hitherto unknown stimulation of fatty acid utilization by low concentrations of ouabain.

In the course of the Third Frankfurt Talks, for the first time a congress in dermatology was dedicated exclusively to cell and tissue culture models. The complexity of a whole organ, in this case the skin, could be reduced to single aspects without losing the holistic context. Ways of managing this were discussed on an interdisciplinary level by dermatologists, physiologists, pharmacologists and biologists. The results are also expected to be useful to the clinician. Focus points of in vitro investigations for dermatology are wound closure models and the use of in vitro skin for transplantation in the therapy of non-healing ulcers and vitiligo. As an alternative to animal experiments, cultures of human cells are gaining increasing influence in drug testing. The effect of glucocorticosteroids on normal skin fibroblasts, keratinocytes and permanent cell lines is discussed as an example, and in vitro models of diseases such as psoriasis are established. Additionally, basic events such as differentiation and ageing have been modelled in cell cultures of melanocytes and keratinocytes. Mechanical stress, UV radiation and nicotine are discussed as inductors.

Lymphocyte infiltration through vascular endothelium is one important step in the course of graft rejection. To investigate this process more exactly we established a monolayer invasion assay which enabled us to discriminate between adherent and penetrated cells. Detailed studies of adhesion and penetration kinetics of peripheral blood lymphocytes (PBL) acting on allogeneic human umbilical vein endothelial cells (HUVEC) were carried out by combined phase contrast and reflection interference contrast microscopy. Between 30 and 35% of all PBL attached to HUVEC after 4 hr. Out of these less than 10% penetrated. When HUVEC were prestimulated for 2 hr by interferon (IFN)-alpha,-beta,-gamma or interleukin (IL)-1, PBL adhesion in the early phase of cellular attachment to endothelial cells was accelerated. Overall adhesion however did not increase. Long-term pretreatment of HUVEC for 72 hr with IFN-gamma or IL-1 also modified PBL-HUVEC interactions. However, a 72-hr pretreatment with IFN-alpha or -beta did not influence lymphocyte binding behaviour. PBL penetration was not only accelerated but also enhanced by IFN-alpha,-beta,-gamma, irrespective of whether HUVEC were prestimulated for 2 hr or PBL and cytokines were added simultaneously to HUVEC. On the other hand IL-1 was not able to enhance the amount of penetrated cells but only accelerated the infiltration process. Up-regulation or de novo expression of the adhesion molecules ICAM-1 (intercellular adhesion molecule), ELAM-1 (endothelial leucocyte adhesion molecule) and VCAM-1 (vascular cell adhesion molecule) did not parallel PBL binding kinetics. Therefore an ICAM-, ELAM- and VCAM-independent modulation in the early phase of lymphocyte attachment to endothelium seems likely. The lymphocyte cytoskeleton may have a role in this process.

Mitochondria are semi-autonomous organelles which are endowed with the ability to change their shape (e.g., by elongation, shortening, branching, buckling, swelling) and their location inside a living cell. In addition they may fuse or divide. These dynamics are discussed. Dislocation of mitochondria may result from their interaction with elements of the cytoskeleton, with microtubules in particular, and from processes intrinsic to the mitochondria themselves. Morphological criteria and differences in the fate of some mitochondria argue for the presence of more than one mitochondrial population in some animal cells. Whether these reflect genetic differences remains obscure. Emphasis is laid on the methods for visualizing mitochondria in cells and following their behaviour. Fluorescence methods provide unique possibilities because of their high resolving power and because some of the mitochondria-specific fluorochromes can be used to reveal the membrane potential. Fusion and fission often occur in short time intervals within the same group of mitochondria. At sites of fusion of two mitochondria material of the inner membrane, the matrix compartment seems to accumulate. The original arrangement of the fusion partners is maintained for some minutes. Fission is a dynamic event which, like fusion, in most cases observed in vertebrate cell cultures is not a straight forward process but rather requires several "trials" until the division finally occurs. Regarding fusion and fission hitherto unpublished phase contrast micrographs, and electron micrographs have been included.

The mode of topical action of minoxidil (U-10,858, CAS 38304-91-5) ist not entirely clear. The influence of minoxidil on the ultimate differentiation of keratinocytes was investigated. Human keratinocytes (HaCaT-cells) were incubated with minoxidil containing culture medium (10-250 micrograms/ml). Minoxidil inhibited in a concentration dependent manner cell mobility whereas the adhesion area and the cell circumference were increased. The minoxidil treated cultures had a higher desmosome density compared to parallel control cultures and they expressed the suprabasal keratins 1 and 10 (indicating progress in differentiation) earlier and to a larger extent than the controls. Keratins were revealed immunohistochemically and by two-dimensional polyacrylamide gel electrophoresis. The results suggest that minoxidil supports the differentiation of human keratinocytes.

Scanning acoustic microscopy (SAM) observes all mechanical properties of living cells. Subtraction of the SAM images (SubSAM) of live cells was developed as a method for investigating minimal changes in cellular topography and elasticity. The image formation in the SubSAM takes into account the motion of cell mass as well as the changes of tension. High spatial and temporal resolution of the SubSAM revealed the structure of motile processes that develops at increasing time intervals, thus allowing the arising complexity of motion to be registered and investigated. Independent spots of activity emerge on a quiescent background as motility domains; they may change position, divide, merge, or disappear after a long time interval. In addition, zones of quiescence were identified over central parts of cytoplasmic lamellae. Nonmalignant (Ep: tadpole epidermal cells, XTH2: endothelial cells from tadpole hearts, 3T3 cells) and neoplastic cells (K2 cells of rat fibrosarcoma, A870N cells selected from K2) were investigated with the SubSAM. Three types of domains of subcellular cytoplasmic motility were identified in time series of two-dimensional SubSAM iamges in normal and neoplastic cells. Of them only the wave-like domain is self-evident, being derived from ruffling and protruding activity at the cell margin. Two other domains wait for detailed analysis. The oscillating domain is a visualization of tension within the cell(s), and the nucleating domain indicates intracellular processes possibly preceding locomotion. Differences in motile domains were found between low K2 and high A870N metastatic cells. The dynamics of motility domains of the A870N cells resembled that of the highly motile Ep cells. Cell morphotype and motile activity of the A870N cells are significantly influenced by the pH of the medium. It became evident that identification of the otherwise invisible motile domains in living cells by SubSAM opens a new approach to a characterization of cell motility in vitro and to an understanding of early cellular reactions to various stimuli.

Nicotine is rapidly taken up by human keratinocytes (HaCaT cells) and after 3 h the uptake is approximately 50% of maximum. Cotinine, a metabolite of nicotine, was detected, thus demonstrating the metabolism of nicotine in HaCaT cells. Low nicotine concentrations (0.1-200 micrograms/ml) did not influence the incorporation rate of thymidine into DNA or amino acids into proteins. Inhibition of DNA and protein synthesis was only observed at concentrations > 200 micrograms/ml. After application of 400 micrograms/ml nicotine, the cells were vacuolated. This process was reversed after nicotine withdrawal. At low nicotine concentrations, no changes in microtubules and actin filaments could be detected. However, in the presence of nicotine (1-10 micrograms/ml), keratin filaments showed a more orderly pattern that controls, and the expression of the suprabasal keratins 1 and 10/11 was induced and increased according to the concentration of nicotine. The number of cornified envelopes also increased markedly. Nicotine concentrations > 100 micrograms/ml led to a disarrangement of keratin filaments and to a decrease in keratin expression and cornified envelope formation. Our results suggest that nicotine at concentrations up to 100 micrograms/ml is not an irritant but may induce cornification of the skin.

Benzamide (10mM) in Ca-free solution anesthetizes small cold blooded animals such as tadpoles of Rana temporaria, Xenopus laevis and an aquarium fish, guppy (Lebistes [Poecilia] reticulatus). Trypsinization of tails of tadpoles in Ca-free, benzamide-supplemented solution permits isolation of keratinocytes (epitheliocytes) maintaining polygonal shapes and prevents cell rounding. Such cells quickly attach to glass, spread and commence locomotion in contrast to those cells which assume spherical shape after standard trypsinization.

In density-inhibited 3T3 fibroblast cultures F-actin is localized almost exclusively at the cell-to-cell borders. Depolymerization of microtubules results in a rearrangement of the F-actin pattern. Numerous actin bundles are formed, most of them are anchored to the cell borders. This rearrangement is preceded by a transient rise in F-actin content.

Cardiac impairment is a major complication in immunotherapy. In order to investigate whether the cardiac complications are due to directly cardiodepressant cytokine effects, the contractility of heart cells in culture under immune mediator exposition was studied. The beat frequency and inotropy of spontaneously contracting cultured cardiac myocytes was determined in a microscope perfusion system equipped with computer image analysis devices. Under control conditions, the perfused cardiac myocytes were rhythmically and synchronously beating for many hours. Perfusion with the cytokines (IL-1, IL-2, IL-3, TNF) induced arrhythmias resulting in a complete cessation of spontaneous contractions and a severe loss of myocyte inotropy. The effects were concentration-dependent and reversible. Our results indicate a direct toxic interference of the interleukins and TNF with the contractile function of cultured cardiac myocytes.

Cardiovascular complications determine the prognosis of patients with chronic renal failure. The contribution of compounds retained during uremia to specific myocardial lesions is controversial. We investigated the contractility of spontaneously beating mouse cardiac myocytes in culture under perfusion with sera derived from patients on maintenance hemodialysis and test solutions containing possible toxins. Cellular contractility under defined environmental conditions is determined by a computer-assisted digital image analysis. 'Uremic sera', creatinine, urea, and combinations of these compounds reduce inotropy of the cultured heart cells, induce arrhythmias or asynchronies in a concentration-dependent manner. We propose the myocyte perfusion technique as an in vitro approach to identify cardiotoxins in the body fluids of chronically uremic patients.

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.

In this paper, a mathematical formulation is presented to compute the V(z) of a tapering layered solid and applying this formulation to the determination of acoustic properties of biological cells and tissues. The formulation is adopted in the simplex inversion algorithm to obtain the acoustic properties of a tapering cell from its V(z) values. The influence of two parameters had been considered: The tapering angle and the presence of a thin liquid layer present between cells and the substratum to which they adhere. Up to a tapering angle less than 10 degrees, it can be safely neglected. However, if a larger angle is neglected, then the acoustic wave velocity in the cell is overestimated. Cell thickness estimation is not affected significantly when the tapering angle is ignored. The calculations of acoustic properties of cells are considerably influenced by the introduction of a thin fluid layer between the solid substratum and the overlying cell, neglecting the presence of at least a very thin layer (20-30 nm), in general, results in a considerable overestimation of sound velocity. The reliability of the data calculated from V(z) values was ascertained using an independent method to determine cell thickness by calculating it from the interference fringe pattern obtained with the reflection-interference light microscope. The shape of the glutaraldehyde-fixed cells was similar to fried eggs. The highest sound velocities were found close to the periphery of the dome-shaped cell center. In the very center and over most of the area of the thin periphery, sound velocity was close to that in saline.

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.

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.

In this paper a new technique is proposed to determine the acoustic properties as well as the thickness (and volume) of biological cells. Variations of thickness, density, acoustic wave velocity, stiffness, and attenuation coefficient of a living or dead cell are obtained by scanning the cell by an acoustic microscope. The distance between the cell and the microscope lens is varied and several voltage curves are thus obtained. These curves are then inverted by simplex optimization technique to obtain the cell parameters. The spatial resolution of the method is limited to the resolution of the scanning acoustic microscope. It allows to take advantage of the full range of frequencies and amplification of the microscope. Characteristic distributions of stiffness are exemplified with an endothelial cell in culture. The main part of the thin, lamellar cytoplasm has high stiffness, which drops close to the lamella/cell body transition region and only slightly increases again through the central part of the cell. Acoustic attenuation seems to be related to two factors, cytoplasm accumulation (in the lamellar parts) and scattering in the central part rich in organelles.

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.

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)

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.

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.

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.

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.

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.

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.

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.

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.

The physiological significance of the association of glycolytic enzymes with actin fibrils was investigated in cell culture. Cytochalasin D (CD) was used to induce the known actin-based sequence of events in a culture of an endothelial-cell line (XTH-2) derived from hearts from tadpoles of Xenopus laevis. 1 min following addition of CD, ruptures in the cortical fibrillar meshwork and in stress fibres are seen. At the same time the cellular ATP level decreases by ca. 25%. This and the following reactions resulting in a kind of arborization depend on a continuous supply with metabolic energy. As shown by measurements of oxygen consumption, cells with intact energy metabolism provide the ATP needed from glycolysis; ATP produced by oxidative phosphorylation is not utilized as long as lactate dehydrogenase (LDH) reoxidizes NADH2. After inhibition of LDH, respiration in XTH-2 cells doubles. CD treatment induces a transient increase in oxygen consumption, indicating an increased energy supply by respiration. From these results we conclude: The energy needed by the actomyosin system is - under normal metabolic conditions -supplied from ATP phosphorylated in glycolysis. The processes of energy metabolism seem to be highly compartmentalized; ATP is not a parameter that is kept constant in time intervals of minutes up to one hour.

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.

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.

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.

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.

In perfused rat hearts alterations of aortic flow and mitochondrial membrane potential resulting from uncoupling of oxidative phosphorylation, hypoxia and treatment with a cardioprotective drug (2-mercaptopropionylglycine (MPG) have been studied. Mitochondrial membrane potential was followed by surface fluorimetry on DASPMI stained hearts. This fluorochrome specifically stains mitochondria in living cells; fluorescence intensity is related to the electrochemical gradient. Aortic flow turned out to be a much more sensitive indicator of heart function than ventricular pressure or mitochondrial membrane potential. No direct relationship exists between mitochondrial membrane potential and ATP production under the different metabolic conditions. Two phases of hypoxic mitochondrial damage have been deduced: the first results in derangement of ATP synthases while membrane potential is maintained, the second in irreversible damage of mitochondrial membranes with loss of membrane potential.