Inhibitory effect of membrane active compounds on the uptake of 14C-alpha-aminoisobutyric acid (AIB) in cultured chick embryo liver cells.

High concentrations of beta-adrenoceptor blocking drugs with membrane active properties and of the membrane active compounds quinidine and lidocaine inhibit the uptake of ∝-aminoisobutyric acid by chick embryo liver cells in culture. Beta-adrenoceptor blockers without membrane active properties were without effect. These results are in accordance with previous findings which showed partial inhibition of incorporation of amino acids into proteins caused by membrane active drugs in this system.     

Immunofluorescent studies of epidermal protein during UV induced carcinogenesis.

Previous studies with agar diffusion technique demonstrated that antibodies produced in rabbits by injection of urea extractable proteins of rat cornfied cells cross react with proteins extracted from normal epidermis of hairless mice using the same technique. In the present study we investigated by indirect immunofluorescence microscopy the immunoreactivity of epidermal proteins in normal and ultraviolet light (UVB) induced hyperplasia and malignant transformation. Reactivity to the antibody was seen over the entire epidermis of nontreated skin and hypertrophied epidermis which occurred at 6-8 weeks after initiation of UVB irradiation. However, the reactivity diminished when malignant changes took place in the epidermal cells. Almost complete disappearance of the immunoresponse was observed in squamous cell carcinoma produced by further UVB radiation. These results suggest that the reactivity of this urea extractable protein serves as an additional immunologic marker for normal epidermal cells. Alterations in the immunoreactivity parallels UVB induced carcinogenesis.     

Ontogenetic changes in adenylate cyclase, cyclic AMP phosphodiesterase and calmodulin in chick ventricular myocardium.

The activities of cyclic AMP phosphodiesterase (3',5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) and adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and calmodulin content during development of chick ventricular myocardium were determined. The specific activity of cyclic AMP phosphodiesterase was relatively low in early embryos, increased during embryogenesis by about 4-fold to reach highest values just before hatching, and then decreased by approx. 30% within 1 week after hatching. In contrast, adenylate cyclase did not change during embryonic development, but increased by approx. 50% within 1 week after hatching. Calmodulin content remained constant at 9 micrograms/g wet wt. during embryonic development and decreased to 6 micrograms/g wet wt. by 1 week after hatching. DEAE-Sephacel chromatography of chick ventricular supernatant revealed a single major form of cyclic nucleotide phosphodiesterase activity in early embryonic (9-day E) and hatched (6-day H) chicks. This enzyme form was eluted at approx. 0.27 M-sodium acetate, hydrolysed both cyclic AMP and cyclic GMP, and was sensitive to stimulation by Ca2+-calmodulin, with an apparent Km for calmodulin of approx. 1 nM. In contrast, ventricular supernatant from late-embryonic (18-day E) chicks contained two forms of phosphodiesterase separable on DEAE-Sephacel: the same form as that seen at other ages, plus a cyclic AMP-specific form which was eluted at approx. 0.65 M-sodium acetate and was insensitive to stimulation by Ca2+-calmodulin. The ontogenetic changes in cyclic AMP phosphodiesterase activity in chick ventricular myocardium are consistent with reported ontogenetic changes in the steady-state contents of cyclic AMP in this tissue and suggest that this enzyme may be responsible for the changes that occur in this nucleotide during development of chick myocardium.     

The highly modified membrane of cornified cells in stratified squamous epithelia: A comparison of heterogenous deposits in keratinized and nonkeratinized epithelia

Summary The chemical nature of the thickened plasma membrane of cornified cells in stratified squamous epithelium was investigated in comparison with that in noncornified epithelium. Localizations of transglutaminase, molecular weight 92000 daltons, and detection of epidermal cysteine proteinase inhibitor were effected with a monoclonal antibody and a monospecific rabbit anti-inhibitor immunoglobulin, respectively, directed to the antigens. N-(7-dimethylamino-4-methylcoumarinyl) maleimide was used to demonstrate S-S cross-linking. In all keratinizing epithelia, the enzyme and inhibitor were deposited on membranes of granular cells. S-S bonds were formed in cornification with the appearance of electron-dense material by the inner leaflet. Both enzyme and inhibitors occurred on the corneal epithelium, but S-S linkage and the thickened plasma membrane did not form even at the last stage of maturation. On the other hand, the internal vaginal epithelium in the proestrous stage without keratinization contained the enzyme, but neither inhibitor nor S-S linkage. Both antigens and S-S bonds were detected when keratinization proceeded during estrus. The staining patterns in the epithelium near the vaginal introitus were identical to those in the skin. Cuboidal and simple epithelia exhibited none of those constituents. The findings indicated that heterogenous components contribute to modification of the plasma membrane of cornified cells, but S-S cross-linkages are associated exclusively with formation of the ultrastructurally unique membrane structure. In addition, findings suggested hormonal regulation in the chemical modification of the membrane in estrogen-sensitive internal vaginal epithelium.     

Transfer of granulomatous inflammation with nonviable preparations of schistosome granulomas in naive mice

Hepatic granulomas of euthymic (nu/+) mice infected with Schistosoma mansoni were freeze-dried or freeze-thawed 3 times and transplanted subcutaneously into naive nu/+ and athymic (nu/nu) mice. The grafted sites, studied histologically, showed formation of organized granulomas in nu/+ mice similar to donor granulomas as observed after grafting of freshly isolated granulomas. On the other hand, in nu/nu mice, the nonviable transplants elicited small and disorganized granulomas, like hepatic granulomas in nu/nu mice with schistosomiasis, but different from fresh nu/+ transplants in nu/nu skin. The findings indicate viable cells are not required for transfer of granulomatous reactions, but T cells are needed for full expression.     

Modulation of myosin assembly

Myosin self-assembly is generally considered to be the major process in thick filament formation within striated muscles. The biological assembly of myosin into thick filaments is being analysed by genetic dissection as well as biochemical and morphological experiments in the nematode Caenorhabditis elegans. This work shows that the assembly of myosin is modulated by its biosynthesis and interaction with non-myosin proteins. Assemblages which generate multiple nascent thick filaments may play a central role in a catalytic cycle of myosin assembly.     

Subpopulation analysis of drug-induced cell-cycle delay in human tumor cells using 90 degrees light scatter

A mitotic cell subset has been identified with nuclear light scatter. Colcemid-treated T-47D human breast cancer cells were permeabilised, stained with ethidium bromide, and analysed by flow cytometry. Cells with G2M DNA content exhibited a unimodal distribution for DNA fluorescence and forward scatter, but two peaks were discernible with 90 degrees light scatter. A discrete low-scattering cell cluster could be distinguished from the G2 cell subset on two-dimensional contour plots of 90 degrees light scatter vs. DNA fluorescence; this cluster was reproduced by mitotic shake-off experiments and varied quantitatively with mitotic indices determined either by microscopy or by stathmokinetic cell-cycle analysis of DNA fluorescence. Cell sorting confirmed that the low-scattering cell cluster comprised predominantly metaphase and anaphase cells. Identification of mitotic cells with this one-step technique enables rapid analysis of drug-induced cell-cycle delay in cell populations with different rates of cell-cycle traverse. Hence, vincristine-induced cytostasis is shown to arise in part because of premitotic G2 arrest, whereas etoposide is shown to affect cycling cells with equal sensitivity in quiescent and activated cell populations. The use of light scatter to discriminate mitotic cells in this way facilitates analysis of drug-induced cell-cycle delay and supplements the information obtainable by conventional cell-cycle analysis.     

Bone graft survival in expanded skin

The effect of tissue expansion on iliac bone graft (onlay) survival was studied on the skulls of 35 New Zealand white rabbits. Wet bone weights at the time of grafting and at sacrifice in control animals (group I) were compared to three experimental groups. Histologic sections of the developing and resolving pseudosheath and skin envelope were performed. A self-inflating 5-mil-thick silicone expander was used for soft-tissue expansion over the rabbit snout. Bone grafts were subsequently placed in this site. Elliptical snout excision without expansion (group II) demonstrated no statistically significant difference in bone graft survival when compared to controls (group I) (p = 0.350). Full tissue expansion followed by immediate bone grafting (group III) within the pseudosheath cavity likewise demonstrated no statistically significant difference in bone graft survival when compared to controls (group I) (p = 0.500); however, when full tissue expansion was followed by delayed (2 weeks) bone grafting to allow for resolution of the giant cell inflammatory reaction of the pseudosheath (group IV), a statistically significant increased bone graft survival was achieved (p less than 0.001). The study demonstrates that the increased vascularity in the pseudosheath and in the expanded soft-tissue envelope significantly increased bone graft survival only when bone grafting was delayed.