Subtype-specific regulation of receptor internalization and recycling by the carboxyl-terminal domains of the human A1 and rat A3 adenosine receptors: consequences for agonist-stimulated translocation of arrestin3

In this study, we have characterized the differential effects on inhibitory adenosine receptor (AR) trafficking of disrupting predicted sites for palmitoylation and phosphorylation within each receptor's carboxyl terminus. While a Cys(302,305)Ala-mutated rat A(3)AR mutant internalizes significantly faster than the wild-type (WT) receptor in response to agonist exposure, analogous mutation of the human A(1)AR (Cys(309)Ala) had no effect on receptor internalization. Moreover, unlike the WT A(3)AR, the entire pool of internalized mutant A(3)AR is able to recycle back to the plasma membrane following agonist removal. These properties do not reflect utilization of an alternative trafficking pathway, as internalized WT and mutant A(3)ARs both accumulate into transferrin receptor-positive endosomal compartments. However, receptor accumulation into endosomes is dependent upon prior G-protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptor's carboxyl terminus, as replacement of the carboxyl-terminal domain of the human A(1)AR with the 14 GRK-phosphorylated amino acids of the rat A(3)AR confers rapid agonist-mediated endosomal accumulation of the resulting chimeric A(1)CT3AR. Sensitivity to GRK-mediated phosphorylation also dictates the distinct redistribution of arrestin3 observed upon agonist exposure. Thus, while the nonphosphorylated A(1)AR redistributes arrestin3 from the cytoplasm to punctate clusters at the plasma membrane, GRK-phosphorylated WT and Cys(302,305)Ala-mutated A(3)ARs, as well as the A(1)CT3AR chimera, each induce the redistribution of arrestin3 into punctate accumulations both at the plasma membrane and within the cytoplasm. Neither the human A(1)AR nor the rat A(3)AR colocalized with arrestin3 under basal or agonist-stimulated conditions. Together, these results demonstrate that inhibitory AR-mediated changes in arrestin3 distribution are subtype-specific, with specificity correlating with the sensitivity of the receptor's carboxyl-terminal domain to GRK phosphorylation. In the case of the rat A(3)AR, sensitivity to GRK-mediated internalization appears to be regulated in part by the integrity of putative palmitate attachment sites upstream of its GRK phosphoacceptor sites.     

In Vitro CuIture of the Tropical Sponge <Emphasis Type="Italic">Axinella corrugata</Emphasis> (Demospongiae): Effect of Food Cell Concentration on Growth,Clearance Rate,and Biosynthesis of Stevensine

In vitro culture is one possible method for supplying sponge metabolites for pharmaceutical applications, but appropriate feeding regimens that maximize both growth and metabolite biosynthesis are largely unknown. According to the natural concentration (NC) of cells 1 to 50 µm in size that are available to wild Axinella corrugata, we fed explants a multispecific diet of bacteria, microalgae, and yeast at 4 different concentrations: 1NC, 3NC, 5NC, and 5+1NC (the last consisted of 5 NC of bacteria and 1 NC of microalgae and yeast). Explants fed a 3NC diet had the best culture response, growing on average from 8.5 g to 10.3 g in 8 weeks, and showing a 110% increase in concentration (milligrams per gram of dry weight) of the antitumor compound stevensine. Stevensine production in 3NC explants, representing the total milligrams of metabolite per explant, increased by 157% over the study. Explants fed at 1NC had relatively stable weights, indicating that the diet met metabolic costs only. Explants fed at the two highest concentrations lost weight after 4 weeks, possibly because long-term high cell concentration blocked their aquiferous system, reducing their ability to feed efficiently. Stevensine production in explants fed the 1NC, 5NC, or 5+1NC diets were similar, and varied little from the initial amount. A separate experiment showed that the clearance rate for A. corrugata is similar between the examined food types and cell concentrations over 5 hours, averaging 766 ml h^–1 g DW^–1.Overall, this study demonstrates that relatively small changes in food abundance can greatly affect both sponge growth and metabolite biosynthesis. The good growth and increased production of the target metabolite stevensine for A. corrugata explants fed a 3NC diet suggests that in vitro culture is a viable method of supplying some sponge metabolites.     

Spatial and temporal effects of free-air CO2 enrichment (POPFACE) on leaf growth,cell expansion,and cell production in a closed canopy of poplar

Leaf expansion in the fast-growing tree, Populus x euramericana was stimulated by elevated [CO(2)] in a closed-canopy forest plantation, exposed using a free air CO(2) enrichment technique enabling long-term experimentation in field conditions. The effects of elevated [CO(2)] over time were characterized and related to the leaf plastochron index (LPI), and showed that leaf expansion was stimulated at very early (LPI, 0-3) and late (LPI, 6-8) stages in development. Early and late effects of elevated [CO(2)] were largely the result of increased cell expansion and increased cell production, respectively. Spatial effects of elevated [CO(2)] were also marked and increased final leaf size resulted from an effect on leaf area, but not leaf length, demonstrating changed leaf shape in response to [CO(2)]. Leaves exhibited a basipetal gradient of leaf development, investigated by defining seven interveinal areas, with growth ceasing first at the leaf tip. Interestingly, and in contrast to other reports, no spatial differences in epidermal cell size were apparent across the lamina, whereas a clear basipetal gradient in cell production rate was found. These data suggest that the rate and timing of cell production was more important in determining leaf shape, given the constant cell size across the leaf lamina. The effect of elevated [CO(2)] imposed on this developmental gradient suggested that leaf cell production continued longer in elevated [CO(2)] and that basal increases in cell production rate were also more important than altered cell expansion for increased final leaf size and altered leaf shape in elevated [CO(2)].     

Formyl-Met-Leu-Phe induces calcium-dependent tyrosine phosphorylation of Rel-1 in neutrophils

Chemoattractant priming and activation of PMNs results in changes in cytosolic Ca²⁺ concentration, tyrosine kinase activity, and gene expression. We hypothesize that the initial signaling for the activation of a 105 kDa protein (Rel-1) requires Ca²⁺-dependent tyrosine phosphorylation. A rapid and time-dependent tyrosine phosphorylation of Rel-1 occurred following formyl-Met-Leu-Phe (fMLP) stimulation of human PMNs at concentrations that primed or activated the NADPH oxidase (10⁻⁹ to 10⁻⁶ M), becoming maximal after 30 s. Pretreatment with pertussis toxin (Ptx) or tyrosine kinase inhibitors abrogated this phosphorylation and inhibited fMLP activation of the oxidase. The fMLP concentrations employed also caused a rapid increase in cytosolic Ca²⁺ but chelation negated the effects, including the cytosolic Ca²⁺ flux, oxidase activation, and the tyrosine phosphorylation of Rel-1. Conversely, chelation of extracellular Ca²⁺ decreased the fMLP-mediated Ca²⁺ flux, had no affect on the oxidase, and augmented tyrosine phosphorylation of Rel-1. Phosphorylation of Rel-1 was inhibited when PMNs were preincubated with a p38 MAP kinase (MAPK) inhibitor (SB203580). In addition, fMLP elicited rapid activation of p38 MAPK which was abrogated by chelation of cytosolic Ca²⁺. Thus, fMLP concentrations that prime or activate the oxidase cause a rapid Ca²⁺-dependent tyrosine phosphorylation of Rel-1 involving p38 MAPK activation.     

On meta-analytic assessment of surrogate outcomes

We discuss the strengths and weaknesses of the meta-analytic approach to estimating the effect of a new treatment on a true clinical outcome measure, T, from the effect of treatment on a surrogate response, S. The meta-analytic approach (see Daniels and Hughes (1997) 16, 1965-1982) uses data from a series of previous studies of interventions similar to the new treatment. The data are used to estimate relationships between summary measures of treatment effects on T and S that can be used to infer the magnitude of the effect of the new treatment on T from its effects on S. We extend the class of models to cover a broad range of applications in which the parameters define features of the marginal distribution of (T, S). We present a new bootstrap procedure to allow for the variability in estimating the distribution that governs the between-study variation. Ignoring this variability can lead to confidence intervals that are much too narrow. The meta-analytic approach relies on quite different data and assumptions than procedures that depend, for example, on the conditional independence, at the individual level, of treatment and T, given S (see Prentice (1989) 8, 431-440). Meta-analytic calculations in this paper can be used to determine whether a new study, based only on S, will yield estimates of the treatment effect on T that are precise enough to be useful. Compared to direct measurement on T, the meta-analytic approach has a number of limitations, including likely serious loss of precision and difficulties in defining the class of previous studies to be used to predict the effects on T for a new intervention.     

Gravity independence of seed-to-seed cycling in Brassica rapa

 Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity. Received: 3 August 1999 / Accepted: 27 August 1999