A phosphorylation-regulated brake mechanism controls the initial endocytosis of opioid receptors but is not required for post-endocytic sorting to lysosomes.

The delta-opioid receptor (DOR) can undergo proteolytic down-regulation by endocytosis of receptors followed by sorting of internalized receptors to lysosomes. Although phosphorylation of the receptor is thought to play an important role in controlling receptor down-regulation, previous studies disagree on whether phosphorylation is actually required for the agonist-induced endocytosis of opioid receptors. Furthermore, no previous studies have determined whether phosphorylation is required for subsequent sorting of internalized receptors to lysosomes. We have addressed these questions by examining the endocytic trafficking of a series of mutant versions of DOR expressed in stably transfected HEK 293 cells. Our results confirm that phosphorylation is not required for agonist-induced endocytosis of truncated mutant receptors that lack the distal carboxyl-terminal cytoplasmic domain containing sites of regulatory phosphorylation. However, phosphorylation is required for endocytosis of full-length receptors. Mutation of all serine/threonine residues located in the distal carboxyl-terminal tail domain of the full-length receptor to alanine creates functional mutant receptors that exhibit no detectable agonist-induced endocytosis. Substitution of these residues with aspartate restores the ability of mutant receptors to undergo agonist-induced endocytosis. Studies using green fluorescent protein-tagged versions of arrestin-3 suggest that the distal tail domain, when not phosphorylated, inhibits receptor-mediated recruitment of beta-arrestins to the plasma membrane. Biochemical and radioligand binding studies indicate that, after endocytosis occurs, phosphorylation-defective mutant receptors traffic to lysosomes with similar kinetics as wild type receptors. We conclude that phosphorylation controls endocytic trafficking of opioid receptors primarily by regulating a "brake" mechanism that prevents endocytosis of full-length receptors in the absence of phosphorylation. After endocytosis occurs, subsequent steps of membrane trafficking mediating sorting and transport to lysosomes do not require receptor phosphorylation.     

Life-cycle modification in open oceans accounts for genome variability in a cosmopolitan phytoplankton

Emiliania huxleyi is the most abundant calcifying plankton in modern oceans with substantial intraspecific genome variability and a biphasic life cycle involving sexual alternation between calcified 2N and flagellated 1N cells. We show that high genome content variability in Emiliania relates to erosion of 1N-specific genes and loss of the ability to form flagellated cells. Analysis of 185 E. huxleyi strains isolated from world oceans suggests that loss of flagella occurred independently in lineages inhabiting oligotrophic open oceans over short evolutionary timescales. This environmentally linked physiogenomic change suggests life cycling is not advantageous in very large/diluted populations experiencing low biotic pressure and low ecological variability. Gene loss did not appear to reflect pressure for genome streamlining in oligotrophic oceans as previously observed in picoplankton. Life-cycle modifications might be common in plankton and cause major functional variability to be hidden from traditional taxonomic or molecular markers.     

Effects of temperature on larval fish swimming performance: the importance of physics to physiology

Temperature influences both the physiology offish larvae and the physics of the flow conditions under which they swim. For small larvae in low Reynolds number (Re) hydrodynamic environments dominated by frictional drag, temperature‐induced changes in the physics of water flow have the greatest effect on swimming performance. For larger larvae, in higher Re environments, temperature‐induced changes in physiology become more important as larvae swim faster and changes in swimming patterns and mechanics occur. Physiological rates at different temperatures have been quantified using Q₁₀s with the assumption that temperature only affected physiological variables. Consequently, Q₁₀s that did not consider temperature‐induced changes in viscosity overestimated the effect of temperature on physiology by 58% and 56% in cold‐water herring and cod larvae respectively. In contrast, in warm‐water Danube bleak larvae, Q₁₀s overestimated temperature‐induced effects on physiology by only 5–7%. This may be because in warm water, temperature‐induced changes affect viscosity to a smaller degree than in cold water. Temperature also affects muscle contractility and efficiency and at high swimming velocities, efficiency decreases more rapidly in cold‐exposed than in warm‐exposed muscle fibres. Further experiments are needed to determine whether temperature acts differently on swimming metabolism in different thermal environments. While hydrodynamic factors appear to be very important to larval fish swimming performance in cold water, they appear to lose importance in warm water where temperature effects on physiology dominate. This may suggest that major differences exist among locomotory capacities of larval fish that inhabit cold, temperate waters compared to those that live in warm tropical waters. It is possible that fish larvae may have developed strategies that affect dispersal and recruitment in different aquatic habitats in order to cope not only with temperature‐induced physiological challenges, but physical challenges as well.     

Phylogenetics and evolution of the eastern Asian-eastern North American disjunct aphid tribe,Hormaphidini (Hemiptera: Aphididae)

A conspicuous biogeographic pattern of the Northern Hemisphere is the disjunct occurrence of related taxa on different continents. Perhaps best studied in plants, this pattern includes disjunct distributions of genera in eastern Asia and eastern North America. Such continental disjunctions are thought to be the remnants of a mostly continuously distributed, mixed mesophytic forest dating to the Miocene, which subsequently became fragmented by geological and climatic changes. Some highly host-specific insects, namely aphids, live on descendants of the mixed mesophytic forest taxa and exhibit the same disjunct distributions as that of their host plants. We estimated the phylogeny of Hormaphidini aphids, which host-alternate between witch-hazel (Hamamelis; an eastern Asian-eastern North American disjunct genus) and birch (Betula). Based on partial nuclear elongation factor 1alpha and mitochondrial tRNA leucine/cytochrome oxidase II sequences, trees inferred from maximum-parsimony and maximum-likelihood showed strong support for two monophyletic genera (Hamamelistes and Hormaphis), each containing a clade of Japanese and a clade of North American species. The estimated divergence dates of Asian and North American clades in both genera was 20-30 million years ago, consistent with the idea that aphids may have experienced the same vicariance events as those of their host plants.     

Levels and determinants of inflammatory biomarkers in a Swiss population-based sample (CoLaus study)

Objective

to assess the levels and determinants of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) in a healthy Caucasian population.

Methods

population sample of 2884 men and 3201 women aged 35 to 75. IL-1β, IL-6 and TNF-α were assessed by a multiplexed particle-based flow cytometric assay and CRP by an immunometric assay.

Results

Spearman rank correlations between duplicate cytokine measurements (N = 80) ranged between 0.89 and 0.96; intra-class correlation coefficients ranged between 0.94 and 0.97, indicating good reproducibility. Among the 6085 participants, 2289 (37.6%), 451 (7.4%) and 43 (0.7%) had IL-1β, IL-6 and TNF-α levels below detection limits, respectively. Median (interquartile range) for participants with detectable values were 1.17 (0.48–3.90) pg/ml for IL-1β; 1.47 (0.71–3.53) pg/ml for IL-6; 2.89 (1.82–4.53) pg/ml for TNF-α and 1.3 (0.6–2.7) ng/ml for CRP. On multivariate analysis, greater age was the only factor inversely associated with IL-1β levels. Male sex, increased BMI and smoking were associated with greater IL-6 levels, while no relationship was found for age and leisure-time PA. Male sex, greater age, increased BMI and current smoking were associated with greater TNF-α levels, while no relationship was found with leisure-time PA. CRP levels were positively related to age, BMI and smoking, and inversely to male sex and physical activity.

Conclusion

Population-based levels of several cytokines were established. Increased age and BMI, and to a lesser degree sex and smoking, significantly and differentially impact cytokine levels, while leisure-time physical activity has little effect.     

Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors.

Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.     

Multivariate statistical approach to the temporal and spatial patterns of selected bioindicators observed in the North Sea during the years 1995–1997

A comprehensive database, containing biological and chemical information, collected in the framework of the bilateral interdisciplinary MARS project (”biological indicators of natural and man-made changes in marine and coastal waters”) during the years 1995–1997 in the coastal environment of the North Sea, was subjected to a multivariate statistical evaluation. The MARS project was designated to combine a variety of approaches and to develop a set of methods for the employment of biological indicators in pollution monitoring and environmental quality assessment. In total, nine ship cruises to four coastal sampling sites were conducted; 765 fish and 384 mussel samples were analysed for biological and chemical parameters. Additional information on the chemical background at the sampling sites was derived from sediment samples, collected at each of the four sampling sites. Based on the available chemical data in sediments and black mussel (Mytilus edulis) a pollution gradient between the selected sites, was established. The chemical body burden of flounder (Platichthys flesus) from these sites, though, did not reflect this gradient equally clear. In contrast, the biological information derived from measurements in fish samples displayed significant a regional as well as a temporal pattern. A multivariate bioindicator data matrix was evaluated employing a factor analysis model to identify relations between selected biological indicators, and to improve the understanding of a regional and temporal component in the parameter response. In a second approach, applying the k-means algorithm on the data matrix, two significantly different clusters of samples, characterised by the current health status of the fish, were extracted. Using this classification a temporal, and in the second order, a less pronounced spatial effect was evident. In particular, during July 1996, a clear sign of deteriorating environmental conditions was extracted from the biological data matrix. Received: 20 June 1999 / Received in revised form: 8 November 1999 / Accepted: 8 November 1999