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Summary. Out of 32 embryos flown (16 @ E2 + 16 @ E9) for 5 days, 16 survived. All sixteen E2 were dead at landing. Eight were opened and eight were incubated at 1.0G. Autopsy showed that 4 E2 survived over 24 hours in space. Eight E14 hatched without anatomical malformations, and 8 E14 were fixed. The height of the macular epithelia was (mean 31 µm) in control and 26 µm in flight chicks. The cross-sectional area of macular nuclei of control was 17 µm2 for hair cells and 14 µm2 in supporting cells. In flight, cross-sectional area was 17 µm2 in hair cells and 15 µm2 in supporting cells (n=250). The shape factor of cartilage cells (1.0 = perfect circle) between control (mean = 0.70) and flight (mean = 0.72), and the area of cartilaginous cells between controls (mean = 9 µm2) and flight (mean = 9 µm2) did not differ (n=300). The nuclei of support cells were closer to the basement membrane in flight than in control chicks. The immunoreactivity of otoconia with anti keratan, fibronectin or chrondroitin sulfate was not different between flight and control ears. There were more afferent fibers inside the macular epithelia of flight (p=0.05) than control. Three of 8 flight animals had elevated vestibular thresholds (VT), with normal mean response amplitudes and latencies. Modified afferent innervation patterns requiring weeks to compensate are sufficient to elevate VT, and should be investigated further. Other reversible (sublethal) microgravity effects on sensory epithelia (vacuoles, swelling, etc) require quantification.
Cochlear outer hair cells (OHC) are commonly assumed to function as mechanical effectors as well as sensory receptors in the organ of Corti (OC) of the inner ear. OHC in vitro and in organ explants exhibit mechanical responses to electrical, chemical or mechanical stimulation which may represent an aspect of their in vivo effector activity. A detailed description, however, of OHC effector operation in situ is still missing. Specifically, little is known, how motility of isolated OHCs in response to electrical stimulation and to K+ glyconate is probably under voltage control and causes depolarization (shortening) and hyperpolarization (elongation). This work was undertaken to investigate if the movements that were observed in isolated OHC, and that are induced by ionic stimulation, could change the geometry of the OC. A synchronized depolarization of OHC was induced in guinea pig cochleae by exposing the entire OC to artificial endolymph (K+). Subsequent morphometry of mid-modiolar sections from these cochleae revealed that the distance between the basilar membrane (BM) and the reticular lamina (RL) had decreased considerably. Furthermore, in the three upper turns OHC had significantly shortened in all rows. The results suggest that OHC can change their length in the OC thus deforming the geometry of the OC. The experiments reveal a tonic force generation within the OC, that may change the position of RL and/or BM, contribute to damping, modulate the BM-RL-distance and control the operating points of the RL and sensory hair bundles. Thus, the results suggest active self-adjustments of cochelar mechanics by slow OHC length changes. Such mechanical adjustments have recenlty been postulated to correstpond to timing elements of animal communication, speech or music.
Protein kinase C (PKC) has been implicated in the signal transduction pathways for the biological effects of both interleukin-3 (IL-3) and erythropoietin (EPO) in hematopoietic target cells. The goal of this stury was to identify specific classical isoforms of PKC and their localization in hematopoietic cells in response to the growth factor, IL-3 or EPO. In addition to murine fetal liver cells as a source of normal erythroid progenitor cells, we have utilized the B6SDUt.EP cell line, a non-tranformed hematopoietic cells line that requires IL-3 for proliferation, but for which EPO can substitute as a growth factor. With polyclonal antibodies prepared against peptide sequences specific for the alpha, Beta I, II and Y isoforms of PKC, we have idenfitied beta I and II as the prdominante nuclear isoforms in target cells that proliferate in response to IL-3 or EPO.
The otoconial matrix (OM) of chicks (Gallus domesticus) inner ear was analyzed. Histochemically the OM was reacted with phosphotungstic acid (PTA) and immuno-histochemically with the monoclonal antibody anti-keratan sulfate (anti-KS). The OM was digested with the enzyme Endo-ß-galactosidase (E-ß-Galase) or separated by 1-D and 2-D gel electrophoresis. PTA which reacts with glycoproteins precipitated the OM, suggesting that the OM contains glycoproteins. A central core in each crystal had no PTA staining, suggesting that the core lacked glycoproteins. Anti KS antibody stained the OM with increased density in older embryos as determined by color thresholding. E-ß-Galase, which cleave the lactosamine repeating units in KS, decreased the immunostain by 30% in the OM and by 20% in the cartilage. The OM from the utricle, saccule and macula lagena contained similar molecular weight bands. Five dense bands in the OM were less dense in tissue and blood controls, suggesting that such bands are enriched in the OM. Isoelectric focusing (2-D gels) of the OM showed a negatively charged high molecular weight smear not present in blood and faint in tissue controls. The high affinity of the OM for the cationic PTA stain, the strong immunohistochemical reaction of the OM with anti KS antibody and high molecular weight negative smear in the 2-D gels taken together suggest that: a) the OM contains large amounts of glycoproteins and glycans, one of which is keratan sulfate, because its immuno stain by anti-KS antibody was decreased by the enzyme E-ß-Galase, b) the utricle, saccule and macula lagena may have similar composition, and c) the concentration of KS may increase gradually until complete mineralization of the OM is reached.
We evaluated anti-S100ß expression in the chick (Gallus domesticus) inner ear and determined that: 1) the monomer anti-S100ß is expressed differentially in the vestibular and auditory perikarya; 2) expression of S100ß in the afferent nerve terminals is time-related to synapse and myelin formation; 3) the expression of dimer anti-S100aabb and S100ß overlaps in most inner ear cell types. Most S100aabb positive cells express S100ß, but S100ß positive cells do not always express S100aabb. 4) the expression of S100ß is diffused over the perikaryal cytoplasm and nuclei of the acoustic ganglia but is concentrated over the nuclei of the vestibular perikarya. 6) S100ß is expressed in secretory cells, and it is co-localized with GABA in sensory cells. 7) Color thresholding objective quantitation indicates that the amount of S100ß was higher (mean 22, SD±4) at E19 than at E9 (mean 34, SD±3) in afferent axons. 8) Morevoer, S100ß was unchanged between E11-E19 in the perikaryal cytoplasm, but did change over the nuclei. At E9, 74%, and at E21, 5% of vestibular perikarya were positive. The data suggest that S100ß may be physically associated with neuronal and ionic controlling cells of the vertebrate inner ear, where it could provide a dual ionic and neurotrophic modulatory function.
This article reviews the basic aspects of video imaging as it relates to measurements of histological and anatomical features, with particular emphasis on the advantages and disadvantages of color and black and white imaging modes. In black and white imaging, calculations are based on the manipulation of picture elements (pixels) that contain 0-255 levels of information. Black is represented by the absence of light (0) and white by 255 grades of light. In color imaging, the pixels contain variation of hues for the primary (red, green and blue = RGB) and secondary (magenta, yellow, cyan, pink) colors. Manipulation of pixels with color information is more computer intense than manipulation of black and white pixels, because there are over sixteen million possible combinations of color in a system with a 24 bit resolution. The narrow 128 possible grades of separation in black and white often makes distinction between pixels with overlapping intensities difficult. Such difficulty is greatly reduced by color thresholding of systems that base the representation of color on a combination of hue-saturation-intensity format (HSI).
Elliptical cells (E-P) are present at the perilymphatic interface lumen (PIL) of the lagena. The E-P cells often separate from the tegmentum vasculosum (TV), and have touching processes that form a monolayer between the K+ rich perilymph and the Na+ rich endolymph, similar to the mammalian Reissner¹s membrane. We examined the TV of chicks (Gallus domesticus) and quantitated the expression of anti-S100aabb & S100ß. There was a 30% increase of S100ß saturation in the light cells facing the PIL when compared to other TV light cells. We show that: 1) The dimer anti-S100aabb and the monomer anti-S100ß are expressed preferentially in the light cells and the E-P cells of TV; 2) Expression of S100ß is higher in light cells facing the PIL than in adjacent cells; 3) The expression of the dimer S100aabb and monomer S100ß overlaps in most inner ear cell types, including the cells of the TV. Most S100aabb positive cells express S100ß, but S100ß positive cells do not always express S100aabb. 4) The S100ß expression in light cells, the abundant Na+ K+ ATPase on dark cells of the TV, and previously demonstrated co-localization of S100ß/GABA in sensory cells suggest that S100ß could have, in the inner ear, a dual neurotrophic-ionic modulating function.
Apoptosis is a process of cell death character ized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the numbers of cultured CD4+ T lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to Gamma irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in Gammairradiated RH9 cells.
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