The ADP-ribosylation factor (ARF) 6 small GTPase regulates vesicle trafficking and cytoskeletal actin reorganization. The GTPase-activating proteins (GAPs) catalyze the formation of inactive ARF6 GDP . Centaurin-␣ 1 contains an ARF GAP and two pleckstrin homology (PH) domains, which bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). Here, we show that centaurin-␣ 1 specifically inhibits in vivo GTP loading of ARF6 and redistribution of ARF6 from the endosomal compartment to the plasma membrane, which are indicative of its activation. Centaurin-␣ 1 also inhibited cortical actin formation in a PIP 3 -dependent manner. Moreover, the constitutively active mutant of ARF6, but not that of ARF1, reverses the inhibition of cortical actin formation by centaurin-␣ 1 . An artificially plasma membrane-targeted centaurin-␣ 1 bypasses the requirement of PIP 3 for its involvement in ARF6 inactivation, suggesting that PIP 3 is required for recruitment of centaurin-␣ 1 to the plasma membrane but not for its activity. Together, these data suggest that centaurin-␣ 1 negatively regulates ARF6 activity by functioning as an in vivo PIP 3 -dependent ARF6 GAP. Phosphatidylinositol (PI)1 3-kinases phosphorylate the 3Ј position of the inositol ring of PI and its derivatives. The 3-phosphorylated PIs such as PIP 3 function as second messengers in the regulation of many cellular functions, including cell migration and vesicle transport (1). PIP 3 is localized in the cytosolic leaflet of the plasma membrane and acts as a site-specific signal for recruitment and/or activation of cytosolic proteins required for the formation of functional complexes at the plasma membrane. A large number of down stream targets have been identified for this lipid and used to characterize agonist activated PI 3-kinase associated cellular pathways. These include ARF regulators such as cytohesins, ARAP3 (ARF GAP, Rho GAP, ankyrin repeat, PH protein 3) and centaurin-␣ 1 (2).ARF family of small GTPases regulate vesicle trafficking by shuttling between an inactive GDP-and an active GTP-bound form (3). Among the known six-mammalian ARF isoforms (ARFs 1-6), ARF1 and ARF6 are the most distantly related and the best characterized. ARF1 localizes to the cytosol in GDP-bound form and to the Golgi membrane in GTP-bound form and regulates transport from the Golgi complex. In contrast, ARF6 localizes to endosomes in GDP-bound form and to the plasma membrane in GTP-bound form and regulates transport between these two organelles and cortical actin re-arrangements at the plasma membrane, which are vital for many cellular functions such as endocytosis, chemotaxis, and focal adhesion (4). ARF guanine-nucleotide exchange factors (GEFs) activate ARFs by catalyzing the release of ARF-bound GDP and permitting the subsequent binding of GTP. In contrast, ARF GAPs stimulate intrinsic ARF GTPase activity, resulting in the hydrolysis of ARF-bound GTP to GDP. Cytohesin 1-3 ARF GEFs recruit to the plasma membrane in agonist stimulated cells by binding PIP 3 and then...

GTPase activating proteins (GAPs) of the centaurin family regulate the actin cytoskeleton and vesicle trafficking through inactivation of the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. We report the functional characterisation of centaurin-␣ 2 , which is structurally related to the centaurin-␣ 1 ARF6 GAP. Centaurin-␣ 2 contains an N-terminal GAP domain followed by two pleckstrin homology (PH) domains (N-PH and C-PH). In vitro, GFP-centaurin-␣ 2 specifically binds the phosphatidylinositol (PI) 3-kinase lipid products, PI 3,4-P 2 and PI 3,4,5-P 3 (PIP 3 ), through its C-terminal PH domain. In agreement with this observation, GFPcentaurin-␣ 2 was recruited to the plasma membrane from the cytosol in EGF-stimulated cells in a PI-3-kinasedependent manner. Moreover, the C-PH domain is sufficient and necessary for membrane recruitment of centaurin-␣ 2 . Centaurin-␣ 2 shows sustained kinetics of PI-3-kinase-mediated membrane recruitment in EGFstimulated cells, owing to its binding to PI 3,4-P 2 . Centaurin-␣ 2 prevents ARF6 translocation to, and cortical actin formation at, the plasma membrane, which are phenotypic indications for ARF6 activation in EGFstimulated cells. Moreover, the constitutively active mutant of ARF6 reverses the effect of centaurin-␣ 2 on cortical actin formation. The membrane targeted centaurin-␣ 2 is constitutively active. Together, these studies indicate that centaurin-␣ 2 is recruited in a sustained manner to the plasma membrane through binding to PI 3,4-P 2 and thereby regulates actin reorganisation via ARF6.

ObjectiveNew medical graduates are the front-line staff in many hospital settings and manage patients with diabetes frequently. Prescribing is an area of concern for junior doctors, however, with insulin prescribing reported as a particular weakness. This study aimed to produce an educational intervention which aimed to improve preparedness to manage patients with diabetes and evaluate it using a mixed methods approach.Research design and methodsAn e-resource (http://www.diabetesscenariosforjuniordoctors.co.uk) was created to contain commonplace and authentic diabetes decision-making scenarios. –32 junior doctors (n=20) and year 5 students (n=12) in South West England worked through the scenarios while ‘thinking aloud’ and then undertook a semistructured interview. Qualitative data were transcribed verbatim and analyzed thematically. Participant confidence to manage patients with diabetes before, immediately after, and 6 weeks after the educational intervention was also measured using a self-rating scale.ResultsParticipants reported that patients with diabetes were daunting to manage because of the wide array of insulin products, their lack of confidence with chronic disease management and the difficulty of applying theory to practice. The e-resource was described as authentic, practical, and appropriate for the target audience. Junior doctors’ self-rated confidence to manage patients with diabetes increased from 4.7 (of 10) before using the e-resource, to 6.4 immediately afterwards, and 6.8 6 weeks later. Medical students’ confidence increased from 5.1 before, to 6.4 immediately afterwards, and 6.4 6 weeks later.ConclusionsProviding opportunities to work with authentic scenarios in a safe environment can help to ameliorate junior doctors’ lack of confidence to manage patients with diabetes.

Childhood spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by mutations in the survival motor neuron (SMN) gene. The severity of the disease is dictated by the copy number of a second copy of the gene, known as SMN2, with higher copy numbers associated with milder forms of SMA. This is because the level of SMN protein produced by patients dictates the severity of the disease. As all patients retain at least one copy of the SMN2 gene, therapeutic strategies are geared towards increasing full-length SMN protein expression from SMN2. One of the identified therapeutic compounds is valproic acid, or valproate (VPA), a histone deacetylase inhibitor (HDACI) that has been used since the 1970s as an anti-convulsant. Here, we discuss VPA's modes of action and potential side effects in the treatment of SMA.

Constructs of the 5-HT tag for either haemagglutinin (HA) or protein-C (PrC) on the Nterminus of the receptor, or Cyan-Fluorescent Protein (CFP) on the C-terminus. C O S 7 cells were transiently transfected using Fugene 6 and expression of the tagged receptors was assessed by homologous displacement of ['Hlketanserin binding. The 5-HT,,-CFP receptor was expressed most highly then HA-and lastly the PrC-5-HTZA receptor (630,240, and 170 fmol/mg protein respectively; wild type receptor was 440 fmol/mg protein). When the tagged receptors were immunoprecipitated using specific anti-tag antibodies, and visualised on Western blot, the HA-5-HT2, receptor was barely detectable. The PrC-5-HT2, receptor ran as 2 broad bands of 55-65 kDa and 75-90 kDa, whereas the 5-HT2,-CFP receptor ran as a major band of 110-140 with lesser bands at 85,70,62 and 55 kDa. Production of 5-HT-induced [3H]inositol phosphates was measured to assess the function of the tagged receptors. We conclude that the PrC-and the -CFP tagged 5-HTz, receptor constructs will be useful tools to examine receptor-protein interactions.receptor were made with an epitope 2 A