Gene regulation during dedifferentiation in Dictyostelium discoideum

During development of Dictyostelium discoideum, cells acquire the capacity to rapidly recapitulate morphogenesis. Therefore, when cells at the loose aggregate stage are disaggregated and challenged to reaggregate, they do so in a tenth of the original time. If loose aggregate cells are disaggregated and resuspended in buffered dextrose solution (erasure medium), they retain the capacity of rapid recapitulation for 80 min, then completely lose this capacity in a single, synchronous step referred to as the "erasure event." The erasure event sets in motion a program of dedifferentiation during which cells lose developmentally acquired characteristics at different times. The erasure event is inhibited by the addition of 10(-4) M cAMP to erasure medium. The synthesis of 33 growth-associated polypeptides, the synthesis of 53 development-associated polypeptides, and the level of 2 development-associated RNAs have been monitored during the erasure program and in cultures inhibited from erasing by the addition of 10(-4) M cAMP. Growth-associated polypeptides begin to be resynthesized and development-associated polypeptides exhibit dramatic decreases in rate of synthesis at different times throughout the first 240 min in erasure medium. Inhibiting the erasure event with cAMP has no major effect in the resynthesis of the majority of growth-associated polypeptides. Only one growth-associated polypeptide, V28, is completely inhibited by cAMP, suggesting that it may play a role in the erasure process. In contrast, inhibiting the erasure event with cAMP has a marked effect on the synthesis of development-associated polypeptides, causing a dramatic reduction in the rate at which synthesis decreases for 6 polypeptides, and completely inhibits the decrease in the synthetic rate of 8 polypeptides. The two development-associated RNAs, 16G1 and 10C3, exhibit two distinctly different patterns of loss during erasure, but in both cases cAMP added at time zero of the erasure process dramatically retards or inhibits loss. In addition, when cAMP is added just prior to the erasure event, it inhibits the erasure event and stimulates a rapid increase in the level of 16G1 RNA back to the developmental level. The level of 16G1 RNA then remains at this level for at least 400 min. When cAMP is added after the erasure event, it causes a low, transient increase in the level of 16G1 RNA. These results are considered both in relation to the program of erasure, and in relation to the role of cAMP in the expression of developmental genes during the forward program of development.     

A trophic effect of epidermal growth factor (EGF) on rat colonic mucosa in organ culture

The development of an organ-culture system for rat colonic mucosa has enabled a direct assessment of the effect of epidermal growth factor (EGF) on cell division. An augmented mitotic index (AIm) has been employed to identify changes in cell proliferation. Explants of colonic mucosa from four animals were maintained in a medium containing serum for five days. On the fifth day of culture, half of the explants received fresh medium containing EGF (40 ng/ml) and the remainder (controls) fresh medium only. At 6, 12, 24 and 48 hr thereafter groups of both experimental and control explants received the metaphase-arresting drug vincristine (4 micrograms/ml) for 3 hr prior to fixation. The proportions of vincristine-arrested metaphases within the explants were determined. Analysis of the data indicates that when serum is present exogenous EGF exerts a trophic effect which increases with time (P less than 0.001). In a second experiment colonic explants from four animals were maintained for five days in a serum-free medium and were then divided into groups, each of which received one of a range of concentrations of EGF. The AIm was determined for each group after 36 hr. It was found that increasing concentrations of EGF produce a small but significant increase in cell proliferation (P less than 0.01). This effect, however, was less pronounced than that seen when serum was present. These results suggest that EGF has a trophic action on the colon and interacts with additional factors found in serum.     

Loss and resynthesis of a developmentally regulated membrane protein (gp80) during dedifferentiation and redifferentiation in Dictyostelium

When developing cultures of Dictyostelium discoideum are disaggregated and resuspended in nutrient medium, they lose the capacity to rapidly reaggregate after 90 min, in a rapid and synchronous step referred to as the "erasure event." They then proceed to lose remaining developmentally acquired functions in a program of dedifferentiation culuminating with the loss of EDTA-resistant cohesion roughly 5 hr later. Immediately following the erasure event, cells can be stimulated to reenter the developmental program even though they still possess a number of developmentally acquired functions. These cells therefore appear to undergo dedifferentiation and redifferentiation simultaneously (D. R. Soll and L. H. Mitchell, 1982, Dev. Biol. 91, 183-190). In this report, we have employed an antiserum made against a developmentally acquired membrane glycoprotein, gp80, to examine whether gp80 is lost during dedifferentiation and whether it is either reutilized or resynthesized during redifferentiation. Results are presented which demonstrate that (1) when 9-hr developing cells are disaggregated and resuspended in nutrient medium, gp80 continues to accumulate for several hours after the erasure event, then is lost at roughly the same time as EDTA-resistant cohesion; (2) when cells are stimulated to reenter the developmental program immediately after the erasure event, both gp80 and EDTA-resistant cohesion are still lost according to the program of dedifferentiation, but are then reacquired soon afterwards according to the program of redifferentiation; (3) during redifferentiation, cells do not reutilize gp80 which had been synthesized during initial development; rather they synthesize gp80 de novo; and (4) developing cells of a dedifferentiation-defective variant, HI4, when disaggregated and resuspended in nutrient medium, retain gp80, EDTA-resistant cohesion, and the capacity to rapidly reinitiate aggregation for at least 12 hr. This last result indicates that the loss of gp80 is regulated by the dedifferentiation process and is not an independent response to disaggregation or the reintroduction of nutrients. Together, these results reinforce the conclusion that dedifferentiation and redifferentiation can function independently and simultaneously in the same cells.     

Leuprolide acetate can reversibly prevent egg laying in cockatiels (Nymphicus hollandicus)

Leuprolide acetate acts as a superactive gonadotropin-releasing hormone (GnRH) agonist in mammals. Its administration to humans in a depot formulation (Lupron Depot 3.75 mg; TAP Pharmaceuticals, Deerfield, IL) consisting of microspheres suspended in a diluent of carboxymethylcellulose and other elements leads to an initial increase in serum gonadotropin levels followed by a prolonged suppression of ～ 1 month's duration. To test whether it might act in cockatiels to prevent egg laying, we administered Lupron Depot to groups of pairs (at least 6 pairs/group) stimulated to reproduce by provision of nest boxes and exposure to sexually stimulatory daylengths (15:9 L:D). A single intramuscular injection of Lupron Depot, calculated to achieve a daily release rate of 0 (control; diluent only), 17, 52, or 156 μg/kg/day of leuprolide acetate, was administered on day 0, when birds received nest boxes and after daylength had been stepwise increased. Egg production began on day 12 in the 0 and 17 μg dose groups, and on day 31 in the groups receiving the higher doses. Number of eggs per clutch, candled fertility, and percent hatchability were not significantly different among the groups. In a separate experiment in which leuprolide was administered prior to photostimulation and nest box presentation, nest-inspection behavior was not prevented. We conclude that a single injection of Lupron Depot is effective in reversibly preventing egg laying in cockatiels. © 1994 Wiley-Liss, Inc.     

Optical measurement of isolated canine lung filtration coefficients at normal hematocrits

Klaesner, Joseph W., N. Adrienne Pou, Richard E. Parker,Charlene Finney, and Robert J. Roselli. Optical measurement ofisolated canine lung filtration coefficients at normal hematocrits. J. Appl. Physiol. 83(6):1976-1985, 1997.In this study, lung filtration coefficient(K_fc) valueswere measured in eight isolated canine lung preparations at normalhematocrit values using three methods: gravimetric, blood-correctedgravimetric, and optical. The lungs were kept in zone 3 conditions andsubjected to an average venous pressure increase of 10.24 ± 0.27 (SE) cmH₂O. The resulting K_fc(ml · min¹ · cmH₂O¹ · 100 g dry lung wt¹) measuredwith the gravimetric technique was 0.420 ± 0.017, which wasstatistically different from theK_fc measured bythe blood-corrected gravimetric method (0.273 ± 0.018) or theproduct of the reflection coefficient(_f) andK_fc measuredoptically (0.272 ± 0.018). The optical method involved the use of aCellco filter cartridge to separate red blood cells from plasma, whichallowed measurement of the concentration of the tracer in plasma atnormal hematocrits (34 ± 1.5). The permeability-surface areaproduct was measured using radioactive multiple indicator-dilutionmethods before, during, and after venous pressure elevations. Resultsshowed that the surface area of the lung did not change significantlyduring the measurement ofK_fc. Thesestudies suggest that_fK_fccan be measured optically at normal hematocrits, that this measurement is not influenced by blood volume changes that occur during the measurement, and that the optical_fK_fcagrees with theK_fc obtained viathe blood-corrected gravimetric method.