Drosophila DBT Autophosphorylation of Its C-Terminal Domain Antagonized by SPAG and Involved in UV-Induced Apoptosis

Drosophila DBT and vertebrate CKIε/δ phosphorylate the period protein (PER) to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. Here, sites of C-terminal autophosphorylation were identified by mass spectrometry and analysis of DBT truncations. Mutation of 6 serines and threonines in the C terminus (DBT^C/ala) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIδ, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBT^C/ala did not affect circadian behavior differently from wild-type DBT (DBT^WT), and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBT^WT protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBT^C/ala did not protect and was not degraded. Finally, we show that the HSP-90 cochaperone spaghetti protein (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.     

EFFECT OF REMOVAL OF CO-OCCURRING SPECIES ON DISTRIBUTION AND ABUNDANCE OF ERYTHRONIUM AMERICANUM (LILIACEAE), A SPRING EPHEMERAL

Growth of spring ephemerals in northern forests is limited to early spring when competition for resources from other species is at a minimum. The abundance of resources during this vernal period suggests that spring ephemerals might grow continuously over a wide range of sites, but distributions tend to be patchy. I hypothesized that co-occurring plants that grow later in the season compete for resources to a limited extent, but that competition from these other species is sufficient to restrict the spread of spring ephemerals into unoccupied sites. Population dynamics of Erythronium americanum were compared on sites at the Hubbard Brook Experimental Forest in New Hampshire subjected to varying removals of cooccurring plants. During the 3-year period after removal of only overstory trees the density and frequency of occurrence of Erythronium increased by 225% and 180%, respectively, and removal of all co-occurring vegetation after 1 year's regrowth had an even more pronounced effect (400% and 195% increases, respectively). The abundance of Erythronium in the undisturbed forest did not change over the 3-year period. Elevational distribution of Erythronium also was directly related to the extent of removal of summer plants. Recruitment was mostly vegetative, but flower production on the tree removal site increased by a factor of six, and some distant sites apparently were colonized by seedlings. For several years following large-scale disturbances that eliminate co-occurring vegetation, the vernal growing season is lengthened and resources are more available, and spring ephemerals such as Erythronium americanum exploit these resource-rich opportunities to expand populations and colonize new sites. It appears that, as forest succession proceeds, the size and survival of newly established colonies are gradually constrained by competition from co-occurring species that grow later in the season.     

Candidate mechanical stimuli for hypertrophy during volume overload.

A myocyte system that senses and responds to mechanical inputs might be activated by any number of features of the time-varying length or force signals experienced by the myocytes. We therefore characterized left ventricular volume and wall stress signals during early volume overload with high spatial and temporal resolution. Left ventricular pressure and volume were measured in open-chest isoflurane-anesthetized male Sprague-Dawley rats 4 and 7 days after surgical creation of an infrarenal arteriovenous fistula or sham operation. Mean wall stresses were calculated by using a simple thick-walled ellipsoidal model. Consistent with previous reports, this surgical model produced a 66% increase in cardiac output and a 10% increase in left ventricular mass by day 7. A number of features of the time-varying volume signal (maximum, mean, amplitude, rates of rise and fall) were significantly altered during early volume overload, whereas many other proposed hypertrophic stimuli, including peak systolic wall stress and diastolic strain, were not. Treating hemodynamic variables more generally as time-varying signals allowed us to identify a wider range of candidate mechanical stimuli for hypertrophy (including some not previously proposed in the literature) than focusing on standard time points in the cardiac cycle. We conclude that features of the time-varying ventricular volume signal and related local deformations may drive hypertrophy during volume overload and propose that those features of the volume signal that also change during pressure overload might be the most interesting candidates for further exploration.