Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells [1]. ARNO is a member of the family of guanine nucleotide exchange factors for ARFs which includes cytohesin-1 and GRP-1 [2] [3-5]. Members of this family contain a carboxy-terminal pleckstrin homology (PH) domain which, in the case of GRP-1, has been shown to bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) in preference to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) in vitro [3,4]. Here, we show that recombinant ARNO has the binding characteristics of a PIP3 receptor and that this activity is restricted to the PH domain. When expressed in murine 3T3 L1 adipocytes, ARNO tagged using green fluorescent protein (GFP) is localised exclusively in the cytoplasm. Stimulation with insulin, however, causes a rapid (< 50 second) PH-domain-dependent translocation of GFP-ARNO to the plasma membrane. This translocation is blocked by the PI(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin and LY294002, and by co-expression with a dominant-negative p85 mutant, suggesting that the translocation is a consequence of insulin stimulation of PI 3-kinase. Our data strongly suggest that ARNO binds PIP3 in vivo and that this interaction causes a translocation of ARNO to the plasma membrane where it might activate ARF6 and regulate subsequent plasma membrane cycling events.

Clustering of inhibitory γ-aminobutyric acidA (GABAA) and glycine receptors at synapses is thought to involve key interactions between the receptors, a “scaffolding” protein known as gephyrin and the RhoGEF collybistin. We report the identification of a balanced chromosomal translocation in a female patient presenting with a disturbed sleep-wake cycle, late-onset epileptic seizures, increased anxiety, aggressive behavior, and mental retardation, but not hyperekplexia. Fine mapping of the breakpoint indicates disruption of the collybistin gene (ARHGEF9) on chromosome Xq11, while the other breakpoint lies in a region of 18q11 that lacks any known or predicted genes. We show that defective collybistin transcripts are synthesized and exons 7–10 are replaced by cryptic exons from chromosomes X and 18. These mRNAs no longer encode the pleckstrin homology (PH) domain of collybistin, which we now show binds phosphatidylinositol-3-phosphate (PI3P/ PtdIns-3-P), a phosphoinositide with an emerging role in membrane trafficking and signal transduction, rather than phosphatidylinositol 3,4,5-trisphosphate (PIP3/PtdIns-3,4,5-P) as previously suggested in the “membrane activation model” of gephyrin clustering. Consistent with this finding, expression of truncated collybistin proteins in cultured neurons interferes with synaptic localization of endogenous gephyrin and GABAA receptors. These results suggest that collybistin has a key role in membrane trafficking of gephyrin and selected GABAA receptor subtypes involved in epilepsy, anxiety, aggression, insomnia, and learning and memory.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that are regulators of vesicle trafficking in eukaryotic cells. GRP1 is a member of a family of ARF guanine-nucleotide-exchange factors that binds in vitro the lipid second messenger phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P3]. In order to study the effects of PtdIns(3,4,5)P3 on the function of GRP1, we have cloned the human homologue of GRP1, encoding for a protein which is 98.8% identical to mouse brain GRP1. Human GRP1 binds, via its pleckstrin homology (PH) domain, the inositol head group of PtdIns(3,4,5)P3, inositol 1, 3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], with high affinity (Kd 32. 2+/-5.2 nM) and inositol phosphate specificity [Kd values for Ins(1, 3,4,5,6)P5, InsP6, Ins(1,3,4)P3 and Ins(1,4,5)P3: 283+/-32, >10000, >10000 and >10000 nM, respectively). Furthermore, GRP1 can accommodate addition of glycerol or diacetylglycerol to the 1-phosphate of Ins(1,3,4,5)P4, data that are consistent with its proposed role as a putative PtdIns(3,4,5)P3 receptor. To address whether GRP1 binds PtdIns(3,4,5)P3 in vivo, we have expressed a chimaera of green fluorescent protein (GFP) fused to the N-terminus of GRP1 in PC12 cells and, using confocal microscopy, examined its resultant localization in live cells. Stimulation with either nerve growth factor or epidermal growth factor (both at 100 ng/ml) results in a rapid, PH-domain dependent, translocation of GFP-GRP1 from the cytosol to the plasma membrane, which occurs with a time course that parallels the production of PtdIns(3,4,5)P3. This translocation is dependent on the activation of phosphatidylinositol 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by the co-expression with dominant negative p85. Taken together these data strongly suggest that GRP1 interacts in vivo with plasma membrane-located PtdIns(3,4,5)P3 and hence constitutes a true PtdIns(3,4,5)P3 receptor.

Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neurons in vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking.

Type 2 diabetes (T2D), which has currently become a global pandemic, is a metabolic disease largely characterised by impaired insulin secretion and action. Significant progress has been made in understanding T2D aetiology and pathogenesis, which is discussed in this review. Extrapancreatic pathology is also summarised, which demonstrates the highly multifactorial nature of T2D. Glucagon-like peptide (GLP)-1 is an incretin hormone responsible for augmenting insulin secretion from pancreatic beta-cells during the postprandial period. Given that native GLP-1 has a very short half-life, GLP-1 mimetics with a much longer half-life have been developed, which are currently an effective treatment option for T2D by enhancing insulin secretion in patients. Interestingly, there is continual emerging evidence that these therapies alleviate some of the post-diagnosis complications of T2D. Additionally, these therapies have been shown to induce weight loss in patients, suggesting they could be an alternative to bariatric surgery, a procedure associated with numerous complications. Current GLP-1-based therapies all act as orthosteric agonists for the GLP-1 receptor (GLP-1R). Interestingly, it has emerged that GLP-1R also has allosteric binding sites and agonists have been developed for these sites to test their therapeutic potential. Recent studies have also demonstrated the potential of bi- and tri-agonists, which target multiple hormonal receptors including GLP-1R, to more effectively treat T2D. Improved understanding of T2D aetiology/pathogenesis, coupled with the further elucidation of both GLP-1 activity/targets and GLP-1R mechanisms of activation via different agonists, will likely provide better insight into the therapeutic potential of GLP-1-based therapies to treat T2D.

Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of ␣9 integrin promotes axon regeneration, so we have investigated ␣9␤1 trafficking and transport in axons and at the growth cone. We have previously found that ␣9 and ␤1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied ␣9 and ␤1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of ␤1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

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...