Identification of an early endosomal protein regulated by phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinases (PI 3-kinases) have been implicated in membrane trafficking in the secretory and endocytic pathways of yeast and mammalian cells, but the molecular mechanisms by which these lipid kinases operate are not known. Here we identify a protein of 170 kDa that is rapidly released from cell membranes in response to wortmannin, a potent inhibitor of mammalian PI 3-kinases. The amino acid sequence of peptides from p170 reveal its identity to early endosomal antigen (EEA) 1, an endosomal antigen with homology to several yeast proteins genetically implicated in membrane trafficking. Immunofluorescence analysis of 3T3-L1 adipocytes with antisera against p170/EEA1 reveal a punctate peripheral pattern that becomes diffuse in response to wortmannin. In vitro, p170/EEA1 binds specifically to liposomes containing PIns(3)P, suggesting that the effect of wortmannin on cells is due to inhibition of PIns(3)P production. Thus, p170/EEA1 may define a family of proteins that mediate the regulatory effects of 3′-phosphoinositides on membrane trafficking in yeast and mammalian cells.

Dynamin-dependent endocytosis of ionotropic glutamate receptors

Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are removed from the postsynaptic plasma membrane of cultured hippocampal neurons by rapid, ligand-induced endocytosis. Although endocytosis of AMPARs can be induced by high concentrations of AMPA without concomitant activation of N-methyl-d-aspartate (NMDA) receptors (NMDARs), NMDAR activation is required for detectable endocytosis induced by synaptically released glutamate. Activated AMPARs colocalize with AP2, a marker of endocytic coated pits, and endocytosis of AMPARs is blocked by biochemical inhibition of clathrin-coated pit function or overexpression of a dominant-negative mutant form of dynamin. These results establish that ionotropic receptors are regulated by dynamin-dependent endocytosis and suggest an important role of endocytic membrane trafficking in the postsynaptic modulation of neurotransmission.

Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (≈208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a ≈7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.

GIPC and GAIP Form a Complex with TrkA: A Putative Link between G Protein and Receptor Tyrosine Kinase Pathways

NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-γ1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways.

Myosin Vb Is Associated with Plasma Membrane Recycling Systems

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.

rSec6 and rSec8, mammalian homologs of yeast proteins essential for secretion.

Many of the molecules necessary for neurotransmission are homologous to proteins involved in the Golgi-to-plasma membrane stage of the yeast secretory pathway. Of 15 genes known to be essential for the later stages of vesicle trafficking in yeast, 7 have no identified mammalian homologs. These include the yeast SEC6, SEC8, and SEC15 genes, whose products are constituents of a 19.5S particle that interacts with the GTP-binding protein Sec4p. Here we report the sequences of rSec6 and rSec8, rat homologs of Sec6p and Sec8p. The rSec6 cDNA is predicted to encode an 87-kDa protein with 22% amino acid identity to Sec6p, and the rSec8 cDNA is predicted to encode a 110-kDa protein which is 20% identical to Sec8p. Northern blot analysis indicates that rSec6 and rSec8 are expressed in similar tissues. Immunodetection reveals that rSec8 is part of a soluble 17S particle in brain. COS cell cotransfection studies demonstrate that rSec8 colocalizes with the GTP-binding protein Rab3a and syntaxin 1a, two proteins involved in synaptic vesicle docking and fusion at the presynaptic terminal. These data suggest that rSec8 is a component of a high molecular weight complex which may participate in the regulation of vesicle docking and fusion in brain.

Global genetic diversity of human metapneumovirus fusion gene

We analyzed 64 human metapneumovirus strains from eight countries. Phylogenetic analysis identified two groups (A and B, amino acid identity 93%-96%) and four subgroups. Although group A strains predominated, accounting for 69% of all strains, as many B as A strains were found in persons greater than or equal to3 years of age.

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120(ctn), also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.

Down Syndrome Across the Life Span

Beyond the myths: representing people with Down syndrome / Jan Gothard -- Learning in young children with Down syndrome: public perceptions, empirical evidence / Jennifer G. Wishart -- Self-regulation in children and young people with Down syndrome / Sheila Glenn and Cliff Cunningham -- What matters most? A reflection on a quarter century of early childhood intervention / Robin Treloar and Susan Cairns -- Making inclusion work: improving educational outcomes for students with Down syndrome in the regular classroom / Loretta R. Giorcelli -- Cognitive development and education: perspectives on Down syndrome from a twenty-year research programme / Susan Buckley and Gillian Bird -- Broadening approaches to literacy education for young adults with Down syndrome / Christina E. van Kraayenoord ... [et al.] -- Numeracy and money management skills in young adults with Down syndrome / Sandra Bochner ... [et al.] -- Life styles of adults with Down syndrome living at home / Anne Jobling and Monica Cuskelly -- Some studies involving individuals with Down syndrome and their relevance to a quality of life model / Verity Bottroff ... [et al.] -- From autonomy to work placement / Anna Contardi -- An appreciative inquiry about adults with Down syndrome / Susanne Muirhead -- Multiple perspectives of family life / Monica Cuskelly ... [et al.] -- Verbal-motor behaviour in persons with Down syndrome / Brian K.V. Maraj ... [et al.].

Segmental body composition determination through a bioelectrical very large scale integration (VLSI) system

The purpose of this investigation was to develop new techniques to generate segmental assessments of body composition based on Segmental Bioelectrical Impedance Analysis (SBIA). An equally important consideration was the design, simulation, development, and the software and hardware integration of the SBIA system. This integration was carried out with a Very Large Scale Integration (VLSI) Field Programmable Gate Array (FPGA) microcontroller that analyzed the measurements obtained from segments of the body, and provided full body and segmental Fat Free Mass (FFM) and Fat Mass (FM) percentages. Also, the issues related to the estimate of the body's composition in persons with spinal cord injury (SCI) were addressed and investigated. This investigation demonstrated that the SBIA methodology provided accurate segmental body composition measurements. Disabled individuals are expected to benefit from these SBIA evaluations, as they are non-invasive methods, suitable for paralyzed individuals. The SBIA VLSI system may replace bulky, non flexible electronic modules attached to human bodies. ^

Peru’s Shining Path: Recent Dynamics and Future Prospects

Although soundly defeated in the early to mid-1990s, Shining Path (Sendero Luminoso) did not disappear. Over the past five years, it has reemerged in a substantially different form, with both a military and a political component. The organization, once again coordinated by jailed leader Abimael Guzmán Reynoso, has eschewed shorter-term military objectives in favor of a longer-term strategy of slowly rebuilding popular support and establishing a party within the Peruvian political system. In so doing, it has also moved from extreme Maoist ideological rigidity to a more pragmatic, though perhaps only tactical, approach. Financial support is derived once again from cocaine production and trafficking in the Upper Huallaga Valley (UHV). Although there may be some individual exceptions, Shining Path is not a narcoterrorist organization. At the same time, Sendero is still very small, in no way a threat to the Peruvian state, and divided. The Lima-based political organization and the military wing in the UHV continue to follow Guzmán‟s leadership, while the Apurímac-Ene Valleys (VRAE) group remains committed to the armed struggle. The Government of Peru (GOP) response to date, both military and civilian, has been inadequate. VRAE military operations are hampered by poor leadership, ill-trained troops, and an outdated strategy. VRAE development resources have been cut, and the long-promised paved road remains in the planning stage. Without significant GOP adjustments, Shining Path is likely to continue to grow.

Selective and specific activation of Rab5 during endocytosis of receptor tyrosine kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

A Systematic Review of Internet-Based Worksite Wellness Approaches for Cardiovascular Disease Risk Management: Outcomes, Challenges & Opportunities

Context The internet is gaining popularity as a means of delivering employee-based cardiovascular (CV) wellness interventions though little is known about the cardiovascular health outcomes of these programs. In this review, we examined the effectiveness of internet-based employee cardiovascular wellness and prevention programs. Evidence Acquisition We conducted a systematic review by searching PubMed, Web of Science and Cochrane library for all published studies on internet-based programs aimed at improving CV health among employees up to November 2012. We grouped the outcomes according to the American Heart Association (AHA) indicators of cardiovascular wellbeing – weight, BP, lipids, smoking, physical activity, diet, and blood glucose. Evidence Synthesis A total of 18 randomized trials and 11 follow-up studies met our inclusion/exclusion criteria. Follow-up duration ranged from 6 – 24 months. There were significant differences in intervention types and number of components in each intervention. Modest improvements were observed in more than half of the studies with weight related outcomes while no improvement was seen in virtually all the studies with physical activity outcome. In general, internet-based programs were more successful if the interventions also included some physical contact and environmental modification, and if they were targeted at specific disease entities such as hypertension. Only a few of the studies were conducted in persons at-risk for CVD, none in blue-collar workers or low-income earners. Conclusion Internet based programs hold promise for improving the cardiovascular wellness among employees however much work is required to fully understand its utility and long term impact especially in special/at-risk populations.

El Norte de Africa y el Sahel y la Estrategia Global de Seguridad de la Unión Europea

El objetivo del artículo es exponer una adecuada y necesaria redefinición de la política de la UE hacia el Norte de África y el Sahel, tanto en términos geográficos como en términos estratégicos de acuerdo con lo expuesto en la nueva Estrategia Global Europea. En este sentido, se revisarán los postulados y la evolución de la antigua Estrategia Europea de Seguridad (EES) y su consiguiente Política de Vecindad (ENP), en comparación con la recién lanzada Estrategia. La nueva Estrategia es concebida como una guía para el desarrollo de la política exterior y de seguridad que la UE ha de llevar a cabo, inter alia, en su periferia. Lamentablemente su concepción de esta zona regional resulta anclada en el pasado.

Self-reported fatigue and associated factors six years after stroke

Several studies have found that fatigue is one of the most commonly reported symptoms after stroke and the most difficult to cope with. The present study aimed to investigate the presence and severity of self-reported fatigue six years after stroke onset and associated factors. The cohort "Life After Stroke Phase I" (n = 349 persons) was invited at six years to report fatigue (Fatigue Severity Scale 7-item version), perceived impact of stroke and global recovery after stroke (Stroke Impact Scale), anxiety and depression (Hospital Anxiety and Depression Scale), life satisfaction (Life Satisfaction Checklist) and participation in everyday social activities (Frenchay Activities Index). At six years 37% of the 102 participants in this cross-sectional study reported fatigue. The results showed that in nearly all SIS domains the odds for post-stroke fatigue were higher in persons with a higher perceived impact. Furthermore, the odds for post-stroke fatigue were higher in those who had experienced a moderate/severe stroke and had signs of depression and anxiety. Fatigue is still present in one-third of persons as long as six years after stroke onset and is perceived to hinder many aspects of functioning in everyday life. There is an urgent need to develop and evaluate interventions to reduce fatigue.