The effects of androgens on the β-glands of the tokay (Gekko gecko): Modification of an hypothesis

Study of the posterior abdominal epidermis in hypophysectomized/thyroidectomized male and female tokays following surgery, and subsequent androgen therapy, indicates that, contrary to a previous model, all aspects of β-gland differentiation are under direct androgenic control. On the other hand, another epidermal specialization, the digital foot-pad, shows a pattern of histogenesis directly comparable to that of β-glands, but is unaffected by androgens. These data are discussed with respect to the evolution of glandular epidermal specializations in gekkonid lizards and the possible role of androgens in modifying the control of cell differentiation in lizard epidermis.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

Differentiation properties of pure populations of human dystrophic muscle cells

The interpretation of the majority of studies of Duchenne muscular dystrophy (DMD) has been complicated by the heterogeneous composition of the cultures used. In addition to muscle cells, muscle tissue contains adipocytes and fibroblasts and the proportion of these cell types varies, especially in disease states. To overcome this problem we developed culture conditions which permitted isolation and characterization of pure populations of clonally derived human muscle cells [1, 2]. Here we report the successful application of these methods to muscle cells from biopsies of individuals with diagnosed DMD. The normal and mutant human muscle cells were used in experiments of muscle differentiation in the same manner as cell lines. Frozen-stored cells were thawed, plated in a series of replicate plates, and allowed to differentiate under similar culture conditions. Yet, in contrast with cell lines, the cells were karyotypically normal, not altered by adaptation to long-term culture, and had a finite lifespan. We have systematically analysed specific properties of the normal and DMD muscle cells which differentiated in culture. The kinetics and extent of myoblast fusion, myotube morphology, and the accumulation and distribution of membrane acetylcholine receptors were monitored. In addition, the isozyme composition of creatine kinase and its intracellular and extracellular distribution were determined. Our results indicate that DMD muscle cells are fully capable of initiating myogenesis in culture and do not differ from normal muscle in several important parameters of differentiation.     

Accelerated transbilayer movement of phosphatidylcholine in sickled erythrocytes. A reversible process

The transbilayer mobility of phosphatidylcholine (PC) molecules in the membrane of homozygous reversible sickle cells (RSCs) was studied using a PC-specific exchange protein from beef liver. In deoxygenated RSCs, all of the PC present in the membrane of the intact cell is rapidly available for exchange, mediated by this protein. Since a substantial amount of the PC is present in the inner membrane leaflet of these cells, this observation implies that the PC molecules in their membranes do experience rapid transbilayer movements. To determine the actual rate of transbilayer movement of the PC, radioactive PC was introduced into the outer monolayer of oxygenated RSCs using the PC-specific exchange protein. Subsequently, the cells were incubated at 37 degrees C under oxy- and deoxygenating conditions to enable the PC to equilibrate within the bilayer. At various time intervals, samples were taken and treated with phospholipase A2, which selectively degrades the PC in the outer monolayer. Analysis of the specific radioactivities of the lyso-PC thus produced, as well as of the residual PC, enabled us to follow the fate of the radioactive PC previously introduced into the outer membrane layer. The half-time value for transbilayer equilibration of the PC in deoxygenated RSCs was determined to be 3.5 h, which is about four times lower than that for oxygenated RSCs. This increased transbilayer mobility of PC, observed in deoxygenated RSCs, is immediately restored to the normal low rate upon reoxygenation of the cells, indicating a complete reversibility of this phenomenon.     

Tolerance to diazepam and methyl-beta-carboline-3-carboxylate measured in substantia nigra of benzodiazepine tolerant rats

The spontaneous activity of neurons in the pars reticulata of substantia nigra (SNpr) was studied in chloral hydrate anesthetized rats. As a function of dose, intravenous diazepam decreased, and methyl-beta-carboline-3-carboxylate (beta CCM) increased discharge frequency. Two days after terminating a one week treatment with flurazepam (FZP), both diazepam and beta CCM showed decreased ability to alter SNpr neuronal activity. Neither residual FZP nor down-regulation of benzodiazepine receptors can account for these results. In contrast, behavioral testing revealed no change in the ability of i.v. beta CCM to cause convulsions, suggesting that sites other than the SNpr are of prime importance in expressing the convulsant actions of systemically injected beta CCM.     

Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography.

The biotin-binding protein streptavidin was crystallized as two-dimensional periodic arrays on biotinylated phospholipid monolayers. Electron diffraction patterns and images of the arrays embedded in vitreous ice were recorded to near-atomic resolution. Amplitudes and phases of structure factors were computed and combined to produce a 3 A projection density map. The reliability of the map was verified by comparing it to the available x-ray atomic model of the molecule. Projection densities from beta-strands and some amino acid side chains were identified from the electron cryomicroscopy map. These results demonstrate the first near-atomic image of this type of protein periodic array by electron crystallography, which has a great potential to aid in the structural characterization of molecular arrays engineered on a monolayer for various basic or biotechnological applications.