Identification of RhoGAP22 as an Akt-dependent regulator of cell motility in response to insulin

Insulin exerts many of its metabolic actions via the canonical phosphatidylinositide 3 kinase (PI3K)/Akt pathway, leading to phosphorylation and 14-3-3 binding of key metabolic targets. We previously identified a GTPase-activating protein (GAP) for Rac1 called RhoGAP22 as an insulin-responsive 14-3-3 binding protein. Insulin increased 14-3-3 binding to RhoGAP22 fourfold, and this effect was PI3K dependent. We identified two insulin-responsive 14-3-3 binding sites (pSer(16) and pSer(395)) within RhoGAP22, and mutagenesis studies revealed a complex interplay between the phosphorylation at these two sites. Mutating Ser(16) to alanine blocked 14-3-3 binding to RhoGAP22 in vivo, and phosphorylation at Ser(16) was mediated by the kinase Akt. Overexpression of a mutant RhoGAP22 that was unable to bind 14-3-3 reduced cell motility in NIH-3T3 fibroblasts, and this effect was dependent on a functional GAP domain. Mutation of the catalytic arginine of the GAP domain of RhoGAP22 potentiated growth factor-stimulated Rac1 GTP loading. We propose that insulin and possibly growth factors such as platelet-derived growth factor may play a novel role in regulating cell migration and motility via the Akt-dependent phosphorylation of RhoGAP22, leading to modulation of Rac1 activity.     

The structure of MSK1 reveals a novel autoinhibitory conformation for a dual kinase protein

Mitogen and stress-activated kinase-1 (MSK1) is a serine/threonine protein kinase that is activated by either p38 or p42ERK MAPKs in response to stress or mitogenic extracellular stimuli. MSK1 belongs to a family of protein kinases that contain two distinct kinase domains in one polypeptide chain. We report the 1.8 A crystal structure of the N-terminal kinase domain of MSK1. The crystal structure reveals a unique inactive conformation with the ATP binding site blocked by the nucleotide binding loop. This inactive conformation is stabilized by the formation of a new three-stranded beta sheet on the N lobe of the kinase domain. The three beta strands come from residues at the N terminus of the kinase domain, what would be the alphaB helix in the active conformation, and the activation loop. The new three-stranded beta sheet occupies a position equivalent to the N terminus of the alphaC helix in active protein kinases.     

Effect of metal and sampling rate on accuracy of Flock of Birds electromagnetic tracking system

Electromagnetic tracking devices are used in many biomechanics applications. Previous studies have shown that metal located within the working field of direct current electromagnetic tracking devices produces significant errors. However, the effect of sampling rate on the errors produced in a metallic environment has never been studied. In this study, the accuracy of Ascension Technologies' Flock of Birds was evaluated at sampling rates of 20, 60, 100, and 140 Hz, in the presence of both aluminum and steel. Aluminum interference caused an increase in measurement error as the sampling rate increased. Conversely, steel interference caused a decrease in measurement error as the sampling rate increased. We concluded that the accuracy of the Flock of Birds tracking system can be optimized in the presence of metal by careful choice in sampling rate.     

Techniques for analysis and purification in high-throughput chemistry

The success of combinatorial chemistry, and the increased emphasis on single well-characterised compounds of high purity, has had a significant impact on analytical and purification technologies. The requirement for ever-increasing throughput has led to the automation and parallelisation of these techniques. Advances have also been made in developing faster methods to augment throughput further.     

Low-copy-number human transgene is recognized as an X inactivation center in mouse ES cells, but fails to induce cis-inactivation in chimeric mice

X chromosome inactivation is initiated from a segment of the mammalian X chromosome called the X inactivation center. Transgenes from this region of the murine X chromosome are providing the means to identify the DNA needed for cis inactivation in mice. We recently showed that chimeric mice carrying transgenes from the human X inactivation center (XIC) region also provide a functional assay for human XIC activity; approximately 6 copies of a 480-kb human transgene (ES-10) were sufficient to initiate random X inactivation in cells of male chimeric mice (Migeon et al., 1999, Genomics, 59, 113-121). Now, we report studies of another human transgene (ES-5), which contains less than 300 kb of the human XIC region on Xq13.2 including an intact XIST locus and which has inserted in one or two copies into mouse chromosome 6. The ES-5 transgene is recognized as an X inactivation center in mouse embryonic stem cells, but is not sufficient to induce random X inactivation in somatic cells of highly chimeric mice. Human transgenes in chimeric mice provide a means to uncouple the key steps in this complex pathway and facilitate the search for essential components of the human XIC region.     

Being in the thick of things: context-dependent network centrality in a captive flock of American flamingos

We test the hypothesis that the relationship between networks resulting from unresolved agonistic interactions (URI) and social dominance was context-dependent in a captive group of American Flamingos (Phoenicopterus ruber ruber). URI formed networks that differed substantially from the dominance network and depended on the context in which the interactions occurred. Betweenness centrality in the URI network on the region (“the island”) where nesting took place was strongly correlated with dominance score. This correlation was particularly strong in the case of males, but, in both sexes, the dominant individual in a dyad was likely to have a higher betweenness centrality in the island URI. On the other hand, betweenness centrality in URI networks at the feeder did not show any relationship with dominance, but was associated with observed visits to the feeder. Thus, centrality in both the island URI network and the feeder URI network was associated with access to a resource over which individuals competed. Along with other results suggesting that non-dominance interactions may form social networks distinct from, but related to, dominance networks, our results support the hypothesis that relationships within animal social groups can be modeled as a series of distinct but inter-related networks.     

Boron neutron capture synovectomy (BNCS) as a potential therapy for rheumatoid arthritis: boron biodistribution study in a model of antigen-induced arthritis in rabbits

Boron neutron capture synovectomy (BNCS) is explored for the treatment of rheumatoid arthritis (RA). The aim of the present study was to perform boron biodistribution studies in a model of antigen-induced arthritis (AIA) in female New Zealand rabbits, with the boron carriers boronophenylalanine (BPA) and sodium decahydrodecaborate (GB-10) to assess the potential feasibility of BNCS for RA. Rabbits in chronic phase of AIA were used for biodistribution studies employing the following protocols: intra-articular (ia) (a) BPA-f 0.14 M (0.7 mg ¹⁰B), (b) GB-10 (5 mg ¹⁰B), (c) GB-10 (50 mg ¹⁰B) and intravenous (iv), (d) BPA-f 0.14 M (15.5 mg ¹⁰B/kg), (e) GB-10 (50 mg ¹⁰B/kg), and (f) BPA-f (15.5 mg ¹⁰B/kg) + GB-10 (50 mg ¹⁰B/kg). At different post-administration times (13–85 min for ia and 3 h for iv), samples of blood, pathological synovium (target tissue), cartilage, tendon, muscle, and skin were taken for boron measurement by inductively coupled plasma mass spectrometry. The intra-articular administration protocols at <40 min post-administration both for BPA-f and GB-10, and intravenous administration protocols for GB-10 and [GB-10 + BPA-f] exhibited therapeutically useful boron concentrations (>20 ppm) in the pathological synovium. Dosimetric estimations suggest that BNCS would be able to achieve a therapeutically useful dose in pathological synovium without exceeding the radiotolerance of normal tissues in the treatment volume, employing boron carriers approved for use in humans. Radiobiological in vivo studies will be necessary to determine the actual therapeutic efficacy of BNCS to treat RA in an experimental model.