These data indicate that hypoxia stimulates VEGF-dependent signaling not only by upregulation of VEGF ligand but also by functional upregulation of a specific signaling receptor. Therefore, these data provide evidence that the endothelium plays an active role in hypoxia-induced angiogenesis.
In the newborn several situations of hyperinsulinism can be associated with myocardial hypertrophy and increased contractility. Insulin and the insulin-like growth factors (IGF) are derived from a common ancestral molecule. Insulin exerts mainly metabolic action, whereas the IGFs promote cell multiplication and differentiation. Using an assay system of cultured neonatal myocardial cells the stimulatory action of insulin and the insulin-like growth factors I and II on myocardial cell contractility was investigated. Spontaneously beating aggregates of myocardial cells were synchronized by an electric impulse generator. Contractility was measured via the amplitude of contraction by an optoelectronic system. Insulin at a concentration of 6,250 and 12,500 microU/ml increased the contractility by 11 and 18%; IGF-I at a concentration of 12 and 25 ng/ml, and IGF-II at a concentration of 25 and 50 ng/ml increased the contractility by 16 and 22%, and 13 and 18%, respectively. Lower concentrations did not provoke a significant increase in contractility. Insulin only in supraphysiological doses increases the contractility of neonatal myocardial rat cells, whereas both insulin-like growth factors act in physiological concentrations. Therefore, during hyperinsulinism insulin may increase myocardial contractility via the IGF receptor and not via the insulin receptor.
SummaryThis article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness.As opposed to the uniform excitation field in conventional fluorescence microscopes an SWFM uses a standing-wave pattern of laser light. This pattern consists of alternating planar nodes and antinodes. By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished. Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope. An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light. Otherwise several planes are illuminated simultaneously and their separation is hardly feasible.The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness. Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing. Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique.The instrument developed uses a green-emitting heliumneon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope. Each beam is passed on to the specimen by a planoconvex lens and an objective lens. The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter. Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces.The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats. The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength. Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods.To obtain futher information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al. (Proc. SPIE, 2655. To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen.A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.
Myoballs were cultured from neonatal rat skeletal muscle without the use of antimitotic drugs. Electron microscopic investigation showed that 7-day-old myoballs are multinucleated syncytia in a state of differentiation where filaments are abundant and already in hexagonal arrays. The resting potential of 142 myoballs kept at 20 degrees C was not correlated with the cell size. Its mean value was -64 mV. Cells with a high resting potential were capable of generating action potentials with a threshold of -51 mV, an overshoot of +31 mV, and a rate of rise of 100 V/s. The steady-state current-voltage relation showed inward rectification on hyperpolarization and outward rectification on depolarization. The dynamic sodium and potassium currents were investigated at 37 degrees C with the whole-cell-recording technique. The sodium current had its maximum at -20 mV. The potassium current showed delayed activation and a very slow and incomplete inactivation. The electrophysiological results from these cultured cells are very similar to those obtained from adult cells.
SUMMARY A multipurpose chamber is described for growing and testing cultured cells. The chamber can be converted from a perfusion chamber to an ‘Open’ or ‘closed’ culture system. The chamber provides optimum conditions for microscopy using all common objectives and condensers for different microscopic methods.
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