Modulation of milk protein synthesis through alteration of the cytoskeleton in mouse mammary epithelial cells cultured on a reconstituted basement membrane

Recent studies indicate that the cytoskeleton may be involved in modulating tissue-specific gene expression in mammalian cells. We have studied the role of the cytoskeleton in regulating milk protein synthesis and secretion by primary mouse mammary epithelial cells cultured on a reconstituted basement membrane that promotes differentiation. After 8 days in culture, cells were treated with cytochalasin D (CD) (0.5-1 micrograms/ml) to alter actin filaments or acrylamide (Ac) (5 mM) to alter intermediate filaments (cytokeratins). CD inhibited synthesis of most proteins in a concentration-dependent manner, with beta-casein being the first affected. In contrast, Ac increased protein synthesis and secretion by 17-31% after a 12 hr treatment. Polyacrylamide gel electrophoresis of total secreted proteins indicates that synthetic rates of most proteins were increased equally by Ac treatment. This increase is apparently controlled at the level of translation, because control and Ac-treated cells contained the same amount of poly-A+ RNA, and neither CD nor Ac altered mRNA levels for beta-casein. There was also no indication that either CD or Ac can induce the expression of milk proteins in quiescent cells cultured on a plastic substratum. In conjunction with the biochemical studies, changes in cytoskeletal morphology caused by the drug treatments were analyzed by immunofluorescence microscopy. As has been observed in other cell types, low concentrations of CD caused cells to round up by disrupting actin filaments. Ac treatment slightly decreased the intensity of actin staining, but no changes in microfilament organization were observed. Ac-treated cells showed slight disorganization of the cytokeratin filaments, with some peripheral interfibrillar bundling, but the cytokeratin network did not collapse and no retraction of cell extensions or breakdown of cell-cell contacts was observed. These results confirm previous reports that the actin cytoskeleton may play a role in regulating tissue-specific protein synthesis. How Ac stimulates protein synthesis is unknown, but it is unlikely that this effect is directly mediated through intermediate filaments.     

Effects of IL-1 and cortisol on beta-adrenergic receptors, cell proliferation, and differentiation in cultured human A549 lung tumor cells

The effects of IL-1 and cortisol, and their interactions on the density of beta-adrenergic receptors (beta AR), cell proliferation, and the adherence of cells to plastic were studied in cultured human A549 lung tumor cells. The density of beta AR, assayed by 125I-pindolol binding, was increased two- to threefold by a 24-h incubation of the cells with IL-1 alpha, IL-1 beta, and TNF-alpha (EC50: 2.7, 8.2, and 24 pM, respectively), although a series of other cytokines and growth factors did not have this effect. Cortisol also increased beta AR density (EC50: 30 nM) and markedly potentiated the effects of IL-1 alpha, IL-1 beta, and TNF-alpha. Neither IL-1 nor cortisol influenced the proportion of cell surface vs internalized beta AR. The IL-1-induced increase in beta AR density was half-maximal after 6 h, was reversible at a similar rate, and was blocked by 1 microM of cycloheximide. The effect of IL-1 on beta AR was specific, as the density of glucocorticoid receptors, measured by 3H-dexamethasone binding, was reduced by IL-1. Both cortisol and IL-1 potentiated the isoproterenol-induced increase in cAMP accumulation. IL-1 inhibited cell proliferation and thymidine uptake, and increased the adherence of A549 cells to the plastic culture flask, as quantified by a cell detachment assay. The effect of IL-1 on cell adherence was not inhibited by cycloheximide. Cortisol decreased cell adherence and prevented the IL-1-induced increase in adherence. The results indicate that multiple effects of IL-1 in a cultured tumor cell line involve different mechanisms, suggesting heterogeneity of IL-1R and/or coupling of IL-1R to distinct, nuclear, and nonnuclear, effector pathways.     

Temporal pattern in swimming activity of two fish species (Danio rerio and Leucaspius delineatus) under chemical stress conditions

Circadian periodicity of swimming activity was investigated in two fish species, the zebrafish (Danio rerio) and the sunbleak (Leucaspius delineatus) under sublethal long-term exposure to the cyanobacteria toxin microcystin-LR (nominal concentrations of 0.5 μg l^- 1, 5 μg l^- 1, 15 μg l^- 1, 50 μg l^- 1) in 15-litre tanks. Swimming activity of fish was monitored continuously by using an automated video-monitoring and object-tracing system over a period of 17 days. Influenced by long-term exposure to microcystin-LR, Leucaspius delineatus reversed their significant diurnal swimming activity and the fish became statistically significant nocturnal. Danio rerio remained diurnal active, but a significant phase shift was registered. In both Danio rerio and Leucaspius delineatus analysis of time series by cosinor regression revealed microcystin-LR induced dose-dependent alterations of the mean of oscillation, amplitude, acrophase and period length in a different extent. For Danio rerio the periodogram analysis revealed a significant circadian component of swimming activity for control as well as exposure groups, whereby the spectral amplitude clearly decreased at microcystin-LR concentrations of 15 and 50 μg l^- 1. For Leucaspius delineatus the amplitude of circadian rhythm was decreased at all exposure concentrations of MC-LR. Furthermore the dominance of circadian rhythm was clearly reduced, whereas the rate of ultradian rhythms increased at elevated MC-LR concentrations of 5 μg l^- 1, 15 μg l^- 1 and 50 μg l^- 1. The studied temporal aspects of behaviour clearly indicated stress symptoms in both fish species, therefore it proved to be a relevant method to characterise the impact of toxic substances in the environment and for biomonitoring.     

A late quarternary lake in the central Namib desert,Southern Africa,and some implications

Lake bed carbonates from Narabeb in the central Namib Desert have been dated by the ²³⁴U/²³⁰Th method at between 210 000 and 240 000 years. Their survival on the ground surface implies that the climate has been arid since this time, and this is interpreted as confirming the hypothesis that the Benguela Current has maintained its influence over the climate of this area throughout the last two glacials.     

Evidence of temperature‐independent metabolic rates in diurnal Namib Desert tenebrionid beetles

To investigate whether the sensitivity to environmental temperature varies between nocturnal and diurnal species of tenebrionid beetle, the metabolic rates of three diurnal species (Onymacris plana Peringuey, Onymacris rugatipennis Haag and Physadesmia globosa Haag) and three nocturnal species (Epiphysa arenicola Penrith, Gonopus sp. and Stips sp.) of beetles from the Namib Desert are measured over a range of temperatures (15–40 °C) that are experienced by these beetles in their natural habitat. The diurnal species O. plana, O. rugatipennis and P. globosa exhibit temperature‐independent metabolic rates (mean Q₁₀ = 1.2) within temperature ranges that are ecologically relevant for diurnal desert beetles (30–40 °C). Onymacris plana, in particular, has a 20–40 °C rate–temperature slope (0.007 log₁₀ mL O₂ h^?1 g^?1 °C^?1; Q₁₀ = 1.1) that is less than half that of the other five beetle species (0.022–0.063 log₁₀ mL O₂ h^?1 g^?1 °C^?1; Q₁₀ ranges from 1.3–1.9), suggesting that O. plana is more metabolically independent of temperature than the other nocturnal and diurnal tenebrionids being investigated. Animals with metabolic rates that are decoupled from body temperature (or ambient temperature) may have an ecological advantage that allows them to exploit thermal and spatial niches during extreme temperature conditions.