The β2-adrenergic receptor/βarrestin complex recruits the clathrin adaptor AP-2 during endocytosis

βarrestins mediate the desensitization of the β2-adrenergic receptor (β2AR) and many other G protein-coupled receptors (GPCRs). Additionally, βarrestins initiate the endocytosis of these receptors via clathrin coated-pits and interact directly with clathrin. Consequently, it has been proposed that βarrestins serve as clathrin adaptors for the GPCR family by linking these receptors to clathrin lattices. AP-2, the heterotetrameric clathrin adaptor protein, has been demonstrated to mediate the internalization of many types of plasma membrane proteins other than GPCRs. AP-2 interacts with the clathrin heavy chain and cytoplasmic domains of receptors such as those for epidermal growth factor and transferrin. In the present study we demonstrate the formation of an agonist-induced multimeric complex containing a GPCR, βarrestin 2, and the β2-adaptin subunit of AP-2. β2-Adaptin binds βarrestin 2 in a yeast two-hybrid assay and coimmunoprecipitates with βarrestins and β2AR in an agonist-dependent manner in HEK-293 cells. Moreover, β2-adaptin translocates from the cytosol to the plasma membrane in response to the β2AR agonist isoproterenol and colocalizes with β2AR in clathrin-coated pits. Finally, expression of βarrestin 2 minigene constructs containing the β2-adaptin interacting region inhibits β2AR endocytosis. These findings point to a role for AP-2 in GPCR endocytosis, and they suggest that AP-2 functions as a clathrin adaptor for the endocytosis of diverse classes of membrane receptors.

Role for G protein-coupled receptor kinase in agonist-specific regulation of μ-opioid receptor responsiveness

The G protein-coupled μ-opioid receptor (μOR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of μOR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate μOR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the μOR. Although both etorphine and morphine effectively activate the μOR, only etorphine elicits robust μOR phosphorylation followed by plasma membrane translocation of β-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause μOR internalization, morphine is unable to either elicit μOR phosphorylation or stimulate β-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce μOR phosphorylation, accompanied by the rescue of β-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote μOR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.

An artificial cell-cycle inhibitor isolated from a combinatorial library

Understanding the genetic networks that operate inside cells will require the dissection of interactions among network members. Here we describe a peptide aptamer isolated from a combinatorial library that distinguishes among such interactions. This aptamer binds to cyclin-dependent kinase 2 (Cdk2) and inhibits its kinase activity. In contrast to naturally occurring inhibitors, such as p21Cip1, which inhibit the activity of Cdk2 on all its substrates, inhibition by pep8 has distinct substrate specificity. We show that the aptamer binds to Cdk2 at or near its active site and that its mode of inhibition is competitive. Expression of pep8 in human cells retards their progression through the G1 phase of the cell cycle. Our results suggest that the aptamer inhibits cell-cycle progression by blocking the activity of Cdk2 on substrates needed for the G1-to-S transition. This work demonstrates the feasibility of selection of artificial proteins to perform functions not developed during evolution. The ability to select proteins that block interactions between a gene product and some partners but not others should make sophisticated genetic manipulations possible in human cells and other currently intractable systems.

Südsee-bilder;

Samoanische Gastfreundschaft.--Kaiser Wilhelmsland und der Bismarck-Archipel.--In Atjeh.--Ein Picknick mit Australnegern.--Beim könig William Barak.--Ein Ausflug von Nouméa nach La Conception und St. Louis.--Auf Meli.--Ein Yangona-fest im Hause des Mbuli von Tavuki.--Ein Besuch der Ngatipahauweras bei Papanui Tamahiki.--Königsgräber auf Tonga.--Der Kilauea.

School of Nursing Class of 1983

Top Row: Barbara A. Fleckenstein, Anne M. Phelan, Julie-Ann Gersin, Laura E. Kemper, Mary Ann McCulloch, Meryl I. Faber, Karen E. Morton, Jennifer S. Miller, Catherine A. Chichester, Dana R. Piper, Harold K. Lohwasser, Michelle A. Lyons, Julia C. Kelly, Deborah L. Rossman, Amy L. Keskey, John F. Nama, Linda Borucki, Michelle M. Bradley, Caroline M. Fischer, Lisa A. Kuhnlein

Row 2: Karen M. Pardo, Laura L. Price, Mollie A. McDonald, Jan M. Grable, Janna S. Nichols, Laura A. Quain, Patricia M. Battel, Claudia J. Koch, Maureen G. D'hondt, Trudy J. Tervo, Linda A. Walz, Cheryl K. Ebling, Patricia A. Merte, Lauri R. Klock, Maria A. Lomibao, Mary E. Eisenhauer, Ellen B. Malvern, Josephine A. Polesnak

Row 3: Yvonne D. Krisel, Rosemary T. Coyne, Janey A. Porterfield, Deborah A. Mulawa, Janet E. Lovelace, Susan P. O'brien, Margaret T. Perrone, Brenda K. Luckhardt, Terry A. Layher, Sharon A. Potonac, Susan K. Watson, Janet A. Servatowski

Row 4: Vivian A. Reeves, Tracey A. Weeks, Marilyn K. Morgan, Terrilynn Phillips, Susan S. Kirk, Robert J. Ziola, Fred Roberts, Karen S. Myron, Pamela M. Przybylski, Mary Jo F. Lafata, Janet A. Scapini, Mary J. Swails

Row 5: Julie E. Reitz, Julie A. Symons, Ave M. Reagor, Catherine A. Regan, Marsha A. Glass, Susan M. Derubeis, Judy L. Goode, Jennifer P. Wylie, Janet L. Nowak, Karen M. Ulfig, Cynthia E. West, Carol A. Czarnecki, Gloria J. Verdi, Lisa D. Singleton

Row 6: Cynthia Wiggins, Monica L. Babyak, Gail M. Ray, Karen S. Desloover, Ladonna L. Christian-Combs, Deborah J. Dunnaback, Deborah A. Cecchini, Nancy A. Neville, Julia H. Grove, Wendy A. Weinfurtner, Susan M. Twigg, Jolynne Vanotteren, Lori A. Clark, Susan T. Savidge

Row 7: Marianne Ojeda, Ann M. Tucker, Lisa A. Valiquette, Sharon J. Bergmann, Elizabeth A. Rice, Marjorie R. Hovis, Laura I. Berry, Janice B. Lindberg, Rhetaugh G. Dumas, Susan B. Steckel, Helen L. Erickson, Kathleen M. Oshea, Tricia A. Richardson, Cheryl L. Sanders, Ann L. Shcoene, Anita M. Bargardi, Constance S. Siler, Anne L. Scott

Row 8: Gassenie Thomas, Victoria L. cadagin, Sheryl A. Strace, Joyce I. Sourbeck, Mary S. Donald, Cindy Tollis, Miriam L. Allis, Julie J. Watson, Patricia A. Shefferly, Nina M. Squire, Carol J. Debrodt, Jennifer A. Dreps, Cynthia B. Stone, Martha A. House, Elizabeth A. Hull, Laurie J. Bommarito, Erin A. Swain, Lisa D. Davis

Row 9: Lisa W. Barak, Charlotte L. Allport, Karen J. Baker, Julie M. Sweet, Pamela R. Armfield, Kathleen A. Hornick, Marcianna M. Davis, Joann L. Holdridge, Barbara A. Black, Scott L. Baker, Lawrene S. Gardipee, Julie A. Hemsteger, Mary Ann Barz, Carla L. Arnett, Danielle L. Bonam, Janice S. Brady, Karen L. Eischer, Amy A. Hing, Marcia L. Hassig, Heidi G. Henn

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Individual Palmitoyl Residues Serve Distinct Roles in H-ras Trafficking, Microlocalization, and Signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopallmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cbolesterol-dependent and cholesterol-independent microdomains. In contrast, monopallmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-pahnitate is weaker. Thus, membrane affinity of a pallmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopahnitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

Examining the process of regime change in the North Atlantic Treaty Organization: The divide between rhetoric and reality

The North Atlantic Treaty Organization (NATO) is a product of the Cold War through which its members organized their military forces for the purpose of collective defense against the common threat of Soviet-backed aggression. Employing the terminology of regime theory, the creation of NATO can be viewed as the introduction of an international security regime. Throughout the Cold War, NATO member states preserved their commitment to mutual defense while increasingly engaging in activities aimed at overcoming the division of Europe and promoting regional stability. The end of the Cold War has served as the catalyst for a new period of regime change as the Alliance introduced elements of a collective security regime by expanding its mandate to address new security challenges and reorganizing both its political and military organizational structures. ^ This research involves an interpretive analysis of NATO's evolution applying ideal theoretical constructs associated with distinct approaches to regime analysis. The process of regime change is investigated over several periods throughout the history of the Alliance in an effort to understand the Alliance's changing commitment to collective security. This research involves a review of regime theory literature, consisting of an examination of primary source documentation, including official documents and treaties, as well as a review of numerous secondary sources. This review is organized around a typology of power-based, organization-based, and norm-based approaches to regime analysis. This dissertation argues that the process of regime change within NATO is best understood by examining factors associated with multiple theoretical constructs. Relevant factors provide insights into the practice of collective security among NATO member states within Europe, while accounting for the inability of the NATO allies to build on the experience gained within Europe to play a more central role in operations outside of this region. This research contributes to a greater understanding of the nature of international regimes and the process of regime change, while offering recommendations aimed at increasing NATO's viability as a source of greater security and more meaningful international cooperation.^

Global distributions of diazotrophs abundance and biomass - Depth integrated values computed from a collection of source datasets - Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types

The MAREDAT atlas covers 11 types of plankton, ranging in size from bacteria to jellyfish. Together, these plankton groups determine the health and productivity of the global ocean and play a vital role in the global carbon cycle. Working within a uniform and consistent spatial and depth grid (map) of the global ocean, the researchers compiled thousands and tens of thousands of data points to identify regions of plankton abundance and scarcity as well as areas of data abundance and scarcity. At many of the grid points, the MAREDAT team accomplished the difficult conversion from abundance (numbers of organisms) to biomass (carbon mass of organisms). The MAREDAT atlas provides an unprecedented global data set for ecological and biochemical analysis and modeling as well as a clear mandate for compiling additional existing data and for focusing future data gathering efforts on key groups in key areas of the ocean. The present data set presents depth integrated values of diazotrophs abundance and biomass, computed from a collection of source data sets.

Global distributions of diazotrophs nitrogen fixation rates - Depth integrated values computed from a collection of source datasets - Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types

The MAREDAT atlas covers 11 types of plankton, ranging in size from bacteria to jellyfish. Together, these plankton groups determine the health and productivity of the global ocean and play a vital role in the global carbon cycle. Working within a uniform and consistent spatial and depth grid (map) of the global ocean, the researchers compiled thousands and tens of thousands of data points to identify regions of plankton abundance and scarcity as well as areas of data abundance and scarcity. At many of the grid points, the MAREDAT team accomplished the difficult conversion from abundance (numbers of organisms) to biomass (carbon mass of organisms). The MAREDAT atlas provides an unprecedented global data set for ecological and biochemical analysis and modeling as well as a clear mandate for compiling additional existing data and for focusing future data gathering efforts on key groups in key areas of the ocean. The present data set presents depth integrated values of diazotrophs nitrogen fixation rates, computed from a collection of source data sets.

Activity concentrations of radionuclides in 5 short cores taken in 2007-2008 measured by ICP-MS and HPGe gamma spectrometry

Natural radionuclides and man-made 137Cs were analyzed in five short sediment cores taken in northern part of the Gulf of Eilat (Gulf of Aqaba) in order to provide information on sedimentation and mixing rates and sediment sources. The maximum estimates of sedimentation rates based on excess 210Pb were found to vary between 0.105 ± 0.020 and 0.35 ± 0.23 cm · year**-1. Even the lowest estimates are significantly higher than those expected from dust deposition, suggesting other sources and processes being responsible for most of the allochthonous material accumulation, including periodical floods following heavy rain events, internal erosion or triggers, like earthquakes. In 137Cs depth profiles no 1963 related nuclear weapon test maxima were found; instead, the activities decrease monotonically, suggesting that a major process leading to radionuclides' depth distribution might be mixing. The mixing rates calculated from 137Cs, excess 210Pb and excess 228Th reach values up to 2.18 ± 0.69 cm**2 · year**-1.

Questioning the merits and demerits of electronic communication in Social Work

This paper examines the social dynamics of electronic exchanges in the human services, particularly in social work. It focuses on the observable effects that email and texting have on the linguistic, relational and clinical rather than managerial aspects of the profession. It highlights how electronic communication is affecting professionals in their practice and learners as they become acculturated to social work. What are the gains and losses of the broad use of electronic devices in daily lay and professional, verbal and non-verbal communication? Will our current situation be seriously detrimental to the demeanor of future practitioners, their use of language, and their ability to establish close personal relationships? The paper analyzes social work linguistic and behavioral changes in light of the growth of electronic communication and offers a summary of merits and demerits viewed through a prism emerging from Baron’s (2000) analysis of human communication.