Arf GAPs: multifunctional proteins that regulate membrane traffic and actin remodelling

The ADP-ribosylation factor (Arf) Arf GTPase-activating proteins (GAPs) are a family of proteins that induce hydrolysis of GTP bound to Arf. A conserved domain containing a zinc finger motif mediates catalysis. The substrate, Arf.GTP, affects membrane trafficking and actin remodelling. Consistent with activity as an Arf regulator, the Arf GAPs affect both of these pathways. However, the Arf GAPs are likely to have Arf-independent activities that contribute to their cellular functions. Structures of the Arf GAPs are diverse containing catalytic, protein-protein interaction and lipid interaction domains in addition to the Arf GAP domain. Some Arf GAPs have been identified and characterized on the basis of activities other than Arf GAP. Here, we describe the Arf GAP family, enzymology of some members of the Arf GAP family and known functions of the proteins. The results discussed illustrate roles for both Arf-dependent and -independent activities in the regulation of cellular architecture.     

CIN85 associates with multiple effectors controlling intracellular trafficking of epidermal growth factor receptors

CIN85 is a multidomain adaptor protein involved in Cbl-mediated down-regulation of epidermal growth factor (EGF) receptors. CIN85 src homology 3 domains specifically bind to a proline-arginine (PxxxPR) motif in Cbl, and this association seems to be important for EGF receptor endocytosis. Here, we report identification of novel CIN85 effectors, all containing one or more PxxxPR motifs, that are indispensable for their mutual interactions. These effectors include phosphatidyl-inositol phosphatases SHIP-1 and synaptojanin 2B1, Arf GTPase-activating proteins ASAP1 and ARAP3, adaptor proteins Hip1R and STAP1, and a Rho exchange factor, p115Rho GEF. Acting as a molecular scaffold, CIN85 clusters its effectors and recruits them to high-molecular-weight complexes in cytosolic extracts of cells. Further characterization of CIN85 binding to ASAP1 revealed that formation of the complex is independent on cell stimulation. Overexpression of ASAP1 increased EGF receptor recycling, whereas ASAP1 containing mutated PxxxPR motif failed to promote this event. We propose that CIN85 functions as a scaffold molecule that binds to numerous endocytic accessory proteins, thus controlling distinct steps in trafficking of EGF receptors along the endocytic and recycling pathways.     

High tumour necrosis factor-alpha levels are associated with exercise intolerance and neurohormonal activation in chronic heart failure patients

Immune activation plays an important role in the progression of chronic heart failure (CHF). We sought to investigate whether different degrees of tumor necrosis factor-alpha (TNF-alpha) activation are associated with exercise intolerance, neurohormonal activation and alterations in muscle mass and function in patients with CHF without cardiac cachexia. Patients were divided into quartiles according to their TNF levels (first quartile: 0.98-4.90 pg/ml, second quartile: 5.00-6.60 pg/ml; third quartile 6.80-9.00 pg/ml; fourth quartile 9.80-32.00 pg/ml). Patients underwent cardiopulmonary exercise testing, quadriceps muscle strength test, quadriceps fatigue test, and assessment of thigh muscle and fat cross-sectional area (CSA) by computerized tomography scanning. Patients in the highest TNF quartile had the lowest peak oxygen consumption [13.1 (+/-4.1) ml/kg/min vs 18.1 (+/-5.3), 18.8 (+/-4.8) and 18.7 (+/-5.6) ml/kg/min, P<0.01] the greatest relation of ventilation and dioxide production (VE/VCO(2)) slope (P<0.05) and the most elevated catecholamine levels (P<0.05) compared to patients in the first three quartiles. Patients with the lowest TNF levels had preserved thigh muscle size and quadriceps strength. Strength/muscle CSA was similar in the four groups. Muscle strength during fatigue testing was significantly lower in the fourth quartile (P=0.01) compared with the other three groups. In CHF patients only the highest levels of TNF are associated with poor functional status and neurohormonal activation. This group of patients may represent the appropriate target population for TNF antagonism.     

Differential expression of calcineurin and SR Ca2+ handling proteins in equine muscle fibers during early postnatal growth.

During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load,resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isoforms and PLB were expressed in almost all fiber types at birth. From 4 weeks of age onward, SERCA1 was exclusively expressed in fast MyHC fibers and SERCA2a and PLB in slow MyHC fibers. At all time points, CnA and CnB proteins were expressed at a basal level in all fibers, but with a higher expression level in MyHC type 1 fibers. From 4 weeks onward, expression of only CnA was also higher in MyHC type 2a and 2ad fibers. We propose a double function of calcineurin in calcium homeostasis and maintenance of slow MyHC fiber type identity. Although equine muscle is already functional at birth, expression patterns of the monitored proteins still show adaptation, depending on the MyHC fiber type.     

Transplacental Transmission of Bluetongue Virus Serotype 1 and Serotype 8 in Sheep: Virological and Pathological Findings

The Bluetongue virus serotype 8 (BTV-8) strain, which emerged in Europe in 2006, had an unusually high ability to cause foetal infection in pregnant ruminants. Other serotypes of BTV had already been present in Europe for more than a decade, but transplacental transmission of these strains had never been demonstrated. To determine whether transplacental transmission is a unique feature of BTV-8 we compared the incidence and pathological consequences of transplacental transmission of BTV-8 to that of BTV-1. Nine pregnant ewes were infected with either BTV-8 or BTV-1. The BTV strains used for the infection were field strains isolated on embryonated chicken eggs and passaged twice on mammalian cells. Blood samples were taken to monitor the viraemia in the ewes. Four weeks after the infection, the foetuses were examined for pathological changes and for the presence of BTV. BTV-8 could be demonstrated in 12 foetuses (43%) from 5 ewes (56%). %). BTV-1 was detected in 14 foetuses (82%) from 6 ewes (67%). Pathological changes were mainly found in the central nervous system. In the BTV-8 group, lympho-histiocytic infiltrates, gliosis and slight vacuolation of the neuropil were found. BTV-1infection induced a severe necrotizing encephalopathy and severe meningitis, with macroscopic hydranencephaly or porencephaly in 8 foetuses. In our experimental setting, using low passaged virus strains, BTV-1 was able to induce transplacental transmission to a higher incidence compared to BTV-8, causing more severe pathology.     

Influence of DNMT Genotype on Global and Site Specific DNA Methylation Patterns in Neonates and Pregnant Women

This study examines the relationship between common genetic variation within DNA methyltransferase genes and inter-individual variation in DNA methylation. Eleven polymorphisms spanning DNMT1 and DNMT3B were genotyped. Global and gene specific (IGF2, IGFBP3, ZNT5) DNA methylation was quantified by LUMA and bisulfite Pyrosequencing assays, respectively, in neonatal cord blood and in maternal peripheral blood. Associations between maternal genotype and maternal methylation (n ^≈ 333), neonatal genotype and neonatal methylation (n ^≈ 454), and maternal genotype and neonatal methylation (n ^≈ 137) were assessed. The findings of this study provide some support to the hypothesis that genetic variation in DNA methylating enzymes influence DNA methylation at global and gene-specific levels; however observations were not robust to correction for multiple testing. More comprehensive analysis of the influence of genetic variation on global and site specific DNA methylation is warranted.     

Coastal Fish Assemblages Reflect Geological and Oceanographic Gradients Within An Australian Zootone

Distributions of mobile animals have been shown to be heavily influenced by habitat and climate. We address the historical and contemporary context of fish habitats within a major zootone: the Recherche Archipelago, southern western Australia. Baited remote underwater video systems were set in nine habitat types within three regions to determine the species diversity and relative abundance of bony fishes, sharks and rays. Constrained ordinations and multivariate prediction and regression trees were used to examine the effects of gradients in longitude, depth, distance from islands and coast, and epibenthic habitat on fish assemblage composition. A total of 90 species from 43 families were recorded from a wide range of functional groups. Ordination accounted for 19% of the variation in the assemblage composition when constrained by spatial and epibenthic covariates, and identified redundancy in the use of distance from the nearest emergent island as a predictor. A spatial hierarchy of fourteen fish assemblages was identified using multivariate prediction and regression trees, with the primary split between assemblages on macroalgal reefs, and those on bare or sandy habitats supporting seagrass beds. The characterisation of indicator species for assemblages within the hierarchy revealed important faunal break in fish assemblages at 122.30 East at Cape Le Grand and subtle niche partitioning amongst species within the labrids and monacanthids. For example, some species of monacanthids were habitat specialists and predominantly found on seagrass (Acanthaluteres vittiger, Scobinichthys granulatus), reef (Meuschenia galii, Meuschenia hippocrepis) or sand habitats (Nelusetta ayraudi). Predatory fish that consume molluscs, crustaceans and cephalopods were dominant with evidence of habitat generalisation in reef species to cope with local disturbances by wave action. Niche separation within major genera, and a sub-regional faunal break, indicate future zootone mapping should recognise both cross-shelf and longshore environmental gradients.     

Characteristics of cases with unknown stage prostate cancer in a population-based cancer registry

BackgroundThe New South Wales Central Cancer Registry (NSW CCR) is the only population-based cancer registry in Australia that has routinely collected summary stage at diagnosis since its inception in 1972. However, a large proportion of prostate cancer cases have “unknown” stage recorded by the registry. We investigated the characteristics of prostate cancer cases with “unknown” stage recorded by the NSW CCR, and examined survival for this group.MethodsData were obtained from the NSW CCR for all first primary prostate cancer cases diagnosed in 1999–2007. Summary stage was recorded as localised, regional, distant or “unknown”. Associations between disease stage and patient characteristics (age, place of residence at diagnosis, year of diagnosis and country of birth) and prostate cancer specific survival were investigated using multivariable logistic regression and Cox proportional hazards models respectively.ResultsOf 39 852 prostate cancer cases, 41.8% had “unknown” stage recorded by the NSW CCR. This proportion decreased significantly over time, increased with increasing age at diagnosis and was higher for those living in socio-economically disadvantaged areas. The proportion with “unknown” stage varied across area health services. Prostate cancer specific survival for cases with “unknown” stage was significantly poorer than for those with localised stage but better than for those with regional or distant stage.ConclusionsResearchers or others using cancer registry stage data to examine prostate cancer outcomes need to consider the differences between cases with “unknown” stage at diagnosis and those with known stage recorded by the registry, and what impact this may have on their results.     

Bicarbonate-dependent pH(i) regulation by chondrocytes within the superficial zone of bovine articular cartilage

Control of chondrocyte pH (pH(i)) determines articular cartilage matrix metabolism. However, the transporters of chondrocytes in situ throughout cartilage zones are unclear, and we tested the hypothesis that chondocytes within the superficial zone (SZ) utilise a HCO(3) (-)-dependent system absent from other zones. Imaging of single BCECF-labelled cells was used to monitor the pH(i) of in situ chondrocytes within the cartilage zones, and also that of cells isolated from the SZ or full depth (FD) explants. Resting pH(i) and intrinsic buffering power (beta(i)) in HEPES-buffered saline was not different between SZ and DZ cells, however the pH(i) of SZ chondrocytes was lower in HCO(3) (-) saline. Ammonium pre-pulse was used to acid-load cells and pH(i) recovery by in situ or isolated SZ chondrocytes shown to be totally dependent on HCO(3) (-). pH(i) recovery rate was significantly (P < 0.05) greater for in situ cells, suggesting that isolation damaged the HCO(3) (-)-dependent system. Recovery of pH(i) by in situ cells was blocked by the anion transport inhibitor DIDS, and partially inhibited by EIPA probably non-specifically. Recovery of pH(i) by acidified MZ or DZ cells or those isolated from FD explants was not affected by HCO(3) (-) (P > 0.05). Na(+)-dependent HCO(3) (-)-(NBC) transporters were identified in SZ chondrocytes by fluorescence immunohistochemistry suggesting that this system might account for the HCO(3) (-)-dependent recovery of pH(i). Bovine articular cartilage chondrocytes possess a HCO(3) (-)-dependent transporter which plays a key role in pH(i) regulation in cells in the SZ, but not in chondrocytes within deeper cartilage zones.     

Novel structures for alpha-actinin:F-actin interactions and their implications for actin-membrane attachment and tension sensing in the cytoskeleton

We have applied correspondence analysis to electron micrographs of 2-D rafts of F-actin cross-linked with alpha-actinin on a lipid monolayer to investigate alpha-actinin:F-actin binding and cross-linking. More than 8000 actin crossover repeats, each with one to five alpha-actinin molecules bound, were selected, aligned, and grouped to produce class averages of alpha-actinin cross-links with approximately 9-fold improvement in the stochastic signal-to-noise ratio. Measurements and comparative molecular models show variation in the distance separating actin-binding domains and the angle of the alpha-actinin cross-links. Rafts of F-actin and alpha-actinin formed predominantly polar 2-D arrays of actin filaments, with occasional insertion of filaments of opposite polarity. Unique to this study are the numbers of alpha-actinin molecules bound to successive crossovers on the same actin filament. These "monofilament"-bound alpha-actinin molecules may reflect a new mode of interaction for alpha-actinin, particularly in protein-dense actin-membrane attachments in focal adhesions. These results suggest that alpha-actinin is not simply a rigid spacer between actin filaments, but rather a flexible cross-linking, scaffolding, and anchoring protein. We suggest these properties of alpha-actinin may contribute to tension sensing in actin bundles.