Myoblasts transferred once a month for six months failed to improve strength in patients with Duchenne's muscular dystrophy. The value of exon-specific peptide antibodies in the interpretation of myoblast-transfer results was demonstrated in a patient with Duchenne's muscular dystrophy who had a high percentage of donor-derived dystrophin. Specific variables affecting the efficiency of myoblast transfer need to be identified in order to improve upon this technique.

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Cytomegalovirus (CMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in CMV infection is well documented. Traditionally the virus has been propagated in fibroblasts, however this process may alter CMV's characteristics, thereby limiting the fibroblast model's utility as a research tool. In our efforts to develop a more accurate in vitro model of CMV/endothelial cell interaction, we have propagated a recent isolate (CMV VHL) through multiple passages in human umbilical vein endothelial cells (HUVE) and, collaterally in neonatal human dermal fibroblasts (NHDF). Infection of HUVE inoculated with either sub-strain of the virus was confirmed by CMV-specific in situ hybridization and by immunocytochemical staining for CMV antigens. Whereas infection of HUVE by substrain VHL/E (endothelial-raised) was accompanied by dramatic cytopathology resembling that observed clinically, the endothelial cytopathic potential of VHL/F (fibroblast-raised) was lost by its 20th passage in NHDF. Similarly, the ability of VHL/F to initiate sustained productive infection in HUVE was severely attenuated; plaque assay of culture supernatants and infected cell fractions, as well as virus-specific DNA polymerase assay of cell lysates, demonstrated progressive viral reproductive activity in VHL/E-inoculated HUVE, whereas VHL/F reproduction was barely detectable. Since properties of VHL/F bear strong resemblance to those of the fibroblast-raised AD169, these studies suggest that while the fibroblast adaptation process commonly employed in the propagation of CMV restricts the host range of the virus and attenuates its spectrum of cytopathic potential, endothelial-based propagation preserves the natural endothelial cytopathogenicity of the original isolate.

Abstract. The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57-and 65-kD bands present in the sensory epithelium and the cytoskeleton.It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57-and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125/~ transverse striping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.

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The alkaline microgel electrophoresis technique was modified to achieve a substantial increase in sensitivity for the detection of radiation-induced DNA damage in human lymphocytes. This increased sensitivity was achieved through: (1) the addition of free radical scavengers to the electrophoresis solution to reduce DNA damage generated during alkaline unwinding and electrophoresis; (2) the modification of the electrophoresis unit to achieve a more uniform electric field; (3) the use of YOYO-1, a DNA dye, producing fluorescence 500-fold more intense than ethidium bromide; and (4) the introduction of an image analysis system for the quantitation of DNA migration. In addition to increasing sensitivity, these modifications have increased the speed with which observations can be quantified, and improved reproducibility from experiment to experiment. In human lymphocytes, these modifications have resulted in an increased sensitivity of several fold, allowing the detection of DNA damage in the range of 50 mGy. This increased sensitivity for the detection of DNA damage should extend the utility of this technique.

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