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Physical stimulation of body surface points is known to affect various organ functions. In traditional Chinese medicine, so-called acupoints were defined. These points can be physically stimulated to effectively treat various diseases. Here we describe for the first time the effect of CO(2) laser stimulation at the acupoints Neiguan (PC-6), Quchi (LI-11), Zusanli (ST-36), and Taichong (LR-3) on heart rate and mean arterial blood pressure in anesthetized rats. CO(2) laser stimulation increased the skin surface temperature to 54°C. Our results revealed that the laser stimulation at the left or right PC-6 and LR-3 increased heart rate and mean arterial pressure. There was no response of heart rate and mean arterial pressure during and after stimulation of the left LI-11, but laser stimulation at the right LI-11 slightly increased heart rate and mean arterial pressure. On the other hand, laser stimulation at the left and right ST-36 decreased heart rate and mean arterial pressure. The effects on mean arterial pressure were more pronounced than those on heart rate. After full spinal cord transection, all heart-rate and mean-arterial-pressure responses were attenuated or completely abolished. These results suggest that CO(2) laser stimulation at either the left or right PC-6, ST-36, and LR-3, as well as at the right LI-11 can modulate the cardiovascular functions in anesthetized rats, and its modulatory site might be supraspinal. [ABSTRACT FROM AUTHOR]

Objectives: Mesenchymal–epithelial interactions play a pivotal role in tubular morphogenesis and in maintaining the integrity of the kidney. During renal repair, similar mechanisms may regulate cellular reorganization and differentiation. We have hypothesized that soluble factors from proximal tubular epithelial cells (PTC) induce differentiation of adipose-derived adult mesenchymal stem cells (ASC). This hypothesis has been tested using cultured ASC and PTC. Material and methods: Conditioned medium was prepared from injured PTC and transferred to ASC cultures. ASC proliferation was analysed by a fluorometric and photometric assay. Signal transduction was analysed by phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2). Grade of ASC differentiation was assessed by morphological analysis and cell expression of characteristic markers. Results: Conditioned medium significantly induced proliferation and phosphorylation of ERK1/ERK2 of ASC. After 12 days of incubation, cell morphology changed to an epithelial-like monolayer. Expression of cytokeratin 18 was induced by conditioned medium, while α-smooth muscle actin, CD49a and CD90 expression decreased. These alterations strongly indicate onset of the differentiation process to the epithelial lineage. In summary, soluble factors from PTC induce signal transduction and differentiation of ASC. Conclusions: Our study shows that conditioned medium from renal tubular epithelial cells provides a convenient source of inductive signals to initiate differentiation of ASC towards epithelial lineage. We deduce that these interactions may play an important role during renal repair mechanisms. [ABSTRACT FROM AUTHOR]

Focuses on the effect of intracellular antibodies on the transport of protein toxins. Manifestation of cytotoxic activity of ribosome-inactivating plant proteins (RIPs); Overview of the characteristics of intracellular transport of RIP; Relationship between the resistance of hybridomas to the cytotoxic action of RIP and intracellular interaction between the toxins and the antibodies.

Observations of Lan Bo Chen’s group using a mitochondria-selective fluorochrome 5,5’,6,6’- tetrachloro- 1,1’,3,3’- tetraethylbenzimidazolocarbocyanine iodide (JC-1) indicate that mitochondria in situ may have zones of different electrochemical potential along their length. This was indicated by the formation of J-aggregates of this dye at distinct sites along a single mitochondrion. Also, intensity variations along single mitochondria were found with diamino-styryl-pyridinium methiodide (DASPMI), another fluorochrome that selectively stains mitochondria depending on their electrochemical potential. DASPMI exchanges easily with the cytoplasm and changes its quantum yield when bound to mitochondrial membranes. Therefore, fluorescence intensity is primarily controlled by the membrane environment rather than by mass accumulation. Two possible explanations of intramitochondrial fluorescence intensity variations have to be discussed: variations in the amount of mitochondrial inner membrane per unit of projection area (or voxel), and differences in the electrochemical gradient. This problem has been approached by comparing fluoro-micrographs of mitochondria in endothelial cells stained with either JC-1 or DASPMI with electron micrographs of the same mitochondria after fixation with glutardialdehyde and osmium tetroxide and ultrathin sectioning. JC-1 red fluorescence (revealing J-aggregate formation) as well as high-intensity staining with DASPMI correlate roughly with the local thickness of mitochondria; no differences in the crista organization are revealed for those areas or mitochondria exhibiting red JC-1 fluorescence and those with green fluorescence. The distance between red fluorescing areas in a single mitochondrion seem to be caused by competition for dye molecules placed in between centres of JC-1 aggregation. Isolated mitochondria are of uniform small size and spherical shape; therefore, no differences in shape interfere with JC-1 staining. Thus JC-1 may be an appropriate indicator of membrane potential in isolated mitochondria. In living cells mitochondria often are large and elongated, and thus the situation is not straightforward to interpret. However, evidence is provided that there are submitochondrial zones, which differ in membrane potential from one adjacent area to another, because DASPMI staining of intramitochondrial zones reveals differences in fluorescence intensity and preferred photodamage of these areas. In some cases separation of the zones of higher membrane potential by cristae traversing the whole diameter of a mitochondrion has been observed. Local photobleaching of stained mitochondria results in a loss of fluorescence along the total length of a mitochondrion. However, this type of bleaching develops over tens of seconds, not in the very short time range (e.g. ms) expected from the discharge of all the membranes if they were electrically coupled. [ABSTRACT FROM AUTHOR]

A pressure device has been used to obtain information about the forces involved in the maintenance of the aggregated state of melanophores of the angelfish, Pterophyllum scalare. Single aggregated melanophores of isolated scales were submitted to mechanical compression with forces ranging from 50-320 μp. As a function of the gradually increasing force melanophores disperse their pigment, the degree of dispersion being proportional to the intensity of the force. When microtubules are destroyed by treatment with 0.3 mM vinblastine in KCl solution, pigment dispersion in response to the external force is similar to that observed in KCl alone. After changing the medium to NaCl solution, melanin granules remain concentrated in the cell center; the force-induced melanosome dispersion, however, is significantly enhanced. Distinctly lower forces are required to produce an expansion equivalent to that observed in KCl solution. When the medium is changed to vinblastine-KCl again, the dispersion in response to the external force resembles that obtained before NaCl treatment. Removal of Ca and Mg ions by treatment with 2 mM EDTA orEGTA in Ringer's solution containing 0.1 mM adrenalin produces a remarkable enhancement of melanosome dispersion in response to increasing external force. This effect of EDTA or EGTA is completely reversible. When the medium is changed to normal Ca-Ringer's, the force/dispersion curve resembles that obtained before EDTA or EGTA treatment. It is concluded that a state of equilibrium exists between the external force and an opposing force produced by the melanophore. The differences in the opposing force under different experimental conditions may be due to a 'contractile component'. This component seems to be independent of microtubules, as indicated by vinblastine experiments. It 'contracts' under aggregating stimulus and 'relaxes' under dispersing stimulus. From the data presented in this paper, the order of magnitude of the pressure developed by the contractile component in the completely aggregated state was calculated as between 5-7 p/cm in the relaxed state and about 20 p/cm during contraction. These values are comparable to those observed in other nonmuscular cells. [ABSTRACT FROM AUTHOR]

Cyclic adenosine monophosphate (cAMP) has been shown to cause pigment dispersion in amphibian and fish melanophores. Since pigment displacements in melanophores of Pterophyllum scalare are known to be accompanied by assembly and disassembly of microtubules, the effect of cAMP on this process was investigated. Melanophores of isolated scales were treated with cAMP in the presence of vinblastine, a potent antimicrotubular agent. During the initial phase of vinblastine action, cAMP as well as its dibutyryl derivative are capable of counteracting the inhibitory effects of vinblastine on pigment dispersion. In addition, cAMP retains the velocity of pigment dispersion at about the maximum level during 1 hour experiments. Pigment aggregation was unaffected by cAMP. Since pigment dispersion in Pterophyllum-melanophores is accompanied by assembly of microtubules, it is concluded that cAMP influences, at least in part, melanosome dispersion through facilitation of microtubule assembly. [ABSTRACT FROM AUTHOR]

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)-α, -β, -γ 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-γ or IL-1 also modified PBL-HUVEC interactions. However, a 72-hr pretreatment with IFN-α or -β did not influence lymphocyte binding behaviour. PBL penetration was not only accelerated but also enhanced by IFN-α, -β, -γ, 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. [ABSTRACT FROM AUTHOR]

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 the 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 fifted 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. [ABSTRACT FROM AUTHOR]

Immunofluorescence and electron microscopy were used to analyze the relationships between the organization of collagen fibrils in elasmoid scales, and the orientation of microtubules and actin microfilaments in the scleroblasts producing this collagenous stroma. Attention was focused on the basal plate of the scales because of the highly ordered three-dimensional arrangement of the collagen fibrils in superimposed plies forming an acellular plywood-like structure. The collagen fibrils are synthesized by the scleroblasts forming a monolayered pseudo-epithelium, the hyposquama, at the lowest surface of the scale. Fully developed scales with a low collagen deposition rate were compared with regenerating scales active in fibrillogenesis. When an ordered array of the collagen fibrils is found, the innermost collagen fibrils are coaligned with microtubules and actin microfilaments. Thus, because of this coalignment, microtubules and actin microfilaments of the hyposquamal scleroblasts are subjected to consecutive alterations during the formation of the plies of the basal plate. The sequence of events when the collagen fibrils change their direction from one ply to the other in the basal plate is deduced from immunofluorescence and phase-contrast-microscopic observations. During the formation of the orthogonal plywood-like structure in the regenerating scales, first microtubules may change their curse with a rotating angle of about 90°; then, actin microfilaments are disorganized and reorganized by interacting mechanically with the microtubules with which they are coaligned. Collagen fibrils are synthesized in a direction that is roughly perpendicular to that of the preceding ply. The unknown signals inducing the change in direction of the cytoskeleton may be transmitted throughout the hyposquama via gap junctions. [ABSTRACT FROM AUTHOR]

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. This stimulation lasted from 1 to 4 days. Then keratinocytes were examined using indirect immunofluorescence, H-thymidine and C-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. [ABSTRACT FROM AUTHOR]

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°, 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human polymorphonuclear leucocytes and epitheliocytes isolated from tadpole tails of Xenopus laevis were used to observe the responses of cells to mechanical stimulation with a microneedle. Biphasic responses were observed: a retraction phase lasting 1-3 s was followed by an extension phase lasting 10-40s. Weak stimulation evoked a local response whilst on strong stimulation the whole cells rounded up. Spreading after induced rounding was at least one order of magnitude faster (it lasted less than 1-2min) than cell spreading after chemical dissociation of cell cultures. Local or extended loss of cell attachment to the substratum (observed with reflection interference contrast microscopy) preceded changes in cell morphology, visible with phase contrast microscopy. Repeated weak stimulation of one cell side induced extension and locomotion of the cell in this direction. The reported biphasic responses of cells to mechanical stimulation highlight the significance of exact timing when following any cell response to external stimuli. [ABSTRACT FROM AUTHOR]

FITC-Con A fluorescence was used to visualize rER arrangement of endothelial cells derived from Xenopus laevis tadpole hearts. In particular determinants of rER organization, intracellular localization and the interrelationships with other organelles were analysed. rER occurs in association with nucleus, mitochondria and microtubules. The structure of rER is strongly affected by energy metabolism and by microtubules. In order to elucidate the interdependence of structure and function we examined the influence of cellular respiration, net lactate production and protein synthesis on rER morphology, as well as the relationship between energy metabolism and protein synthesis. ER morphology is determined primarily by energy consuming intracellular transport mechanisms. Energy needed for protein synthesis is supplied by the respiratory chain while ATP from aerobic glycolysis only compensates when respiration is disturbed. [ABSTRACT FROM AUTHOR]

A method is described for the determination of cellular F-actin content using fluorometry of TRITC-phalloidin at very high dilution. In this case no saturation of all binding sites available is reached, however the staining is highly specific and the specificity is not affected by the preparative procedure as may be the case at high concentrations of TRITC-phalloidin. The method is based on calculation of fluorescence intensity at equilibrium conditions from measurements at two different numbers of exchange of the staining solution by using Lineweaver-Burk plotting. The relative content of F-actin has been determined for three established cell lines, an amphibian cell line XTH-2 of endothelial origin, 3T3 cells and SV 40 transformed 3T3 cells. In the two non transformed lines F-actin decreases with increasing cell density starting with the onset of confluency of the culture. SV 40 transformed 3T3 cells generally contain less F-actin and do not show any significant point of change. The decrease in F-actin with increasing cell density is accompanied by a disappearance of stress fibres. SV 40 3T3 cells generally are devoid of stress fibres. The observations are discussed considerung a possible involvement of F-actin in growth control. [ABSTRACT FROM AUTHOR]

The scanning of fluorescence of a cell culture along a path several milimeters long, gives a series of signals, which allow calculation of the fluorescence emitted per cell. The emission at 450±10 nm, excited at 360 nm, provides a measure of NADH (and NADPH) per cell. Combination of this method with modifications in energy metabolism, allows determination in the same sample of total NAD content, of NAD redox state, content of NAD in the mitochondria and content of NAD plus NADP in the cytoplasm. The method can be extended to measure other cellular constituents by labelling with fluorescent markers, e.g. antibodies. [ABSTRACT FROM AUTHOR]

FITC-Con A-fluorescence and electron microscopy have been used in parallel for the visualization of rough endoplasmic reticulum in Xenopus laevis tadpole heart primary tissue culture cells. rER distribution pattern as revealed by both methods is compared by superimposition of images of the same cell. Coincidence of rER distribution is established. [ABSTRACT FROM AUTHOR]

A population of endothelial cells growth 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 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 μm) 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. [ABSTRACT FROM AUTHOR]

The antimitotic drugs colchicine and vinblastine were applied to the melanophores of isolated scales of Pterophyllum scalare. The time course of inhibition of pigment migrations was studied quantitatively with the photoelectric method. Within one hour of treatment with 5 mM colchicine, a partial and reversible inhibition of pigment movements is obtained. During this time, the number of microtubules per cell process does not differ significantly from controls when pigment is dispersed. However, their number increases concomitantly with the decrease in rate of aggregation induced by KCl up to about the same value as in dispersed pigment cells. This indicates a close connection between pigment movements and the microtubule turnover known to occur in angelfish melanophores. When applied in a concentration of 0,2 mM, vinblastine has a total and irreversible effect on pigment movements, with an especially rapid and profound inhibition of pigment dispersion. The fine structure of vinblastine-treated melanophores is characterized by an almost complete loss of microtubules and the appearance of crystalloid bodies, which differ in fine structure from vinblastine-induced crystals of all other cell types so far examined. Possibly their peculiar structure is related to special properties of melanophore microtubules, such as rapid turnover and relatively high resistance to colchicine. [ABSTRACT FROM AUTHOR]

A method for evaluation of microtubule content in cultured cells has been developed. The method is based on scanning microfluorometric measurement of immunofluorescently labelled microtubules. The method has been applied to the comparison of microtubule content in epithelial XTH-2 cells grown in culture at various cell densities. The results have shown that the microtubule content in the cells is not dependent on their proliferative state rather than it depends on cellular contacts. [ABSTRACT FROM AUTHOR]

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