Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed.     

PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity.

Phosphatidylinositol 3-kinase (PI 3-kinase) has a regulatory 85 kDa adaptor subunit whose SH2 domains bind phosphotyrosine in specific recognition motifs, and a catalytic 110 kDa subunit. Mutagenesis of the p110 subunit, within a sequence motif common to both protein and lipid kinases, demonstrates a novel intrinsic protein kinase activity which phosphorylates the p85 subunit on serine at a stoichiometry of approximately 1 mol of phosphate per mol of p85. This protein-serine kinase activity is detectable only upon high affinity binding of the p110 subunit with its unique substrate, the p85 subunit. Tryptic phosphopeptide mapping revealed that the same major peptide was phosphorylated in p85 alpha both in vivo in cultured cells and in the purified recombinant enzyme. N-terminal sequence and mass analyses were used to identify Ser608 as the major phosphorylation site on p85 alpha. Phosphorylation of the p85 subunit at this serine causes an 80% decrease in PI 3-kinase activity, which can subsequently be reversed upon treatment with protein phosphatase 2A. These results have implications for the role of inter-subunit serine phosphorylation in the regulation of the PI 3-kinase in vivo.     

Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.

The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.     

Lipid composition of the peripheral nerves in malnutrition: an experimental study in young rhesus monkeys

An experimental model of protein calorie malnutrition (PCM) was created in young rhesus monkeys. Ulnar and tibial nerves from six monkeys with PCM, six rehabilitated and 12 control monkeys were studied for lipid composition and activity of myelin marker enzyme. Total lipids, myelin marker lipids and activity of myelin marker enzyme, 2', 3'-cyclic nucleotide 3' - phosphohydrolase were decreased, while esterified cholesterol and free fatty acids were increased in PCM as compared to controls. There was partial recovery in myelin marker lipids and complete restoration of other lipids on nutritional rehabilitation.     

Q Fever Knowledge,Attitudes and Vaccination Status of Australia’s Veterinary Workforce in 2014

Q fever, caused by Coxiella burnetii, is a serious zoonotic disease in humans with a worldwide distribution. Many species of animals are capable of transmitting C. burnetii, and consequently all veterinary workers are at risk for this disease. An effective Q fever vaccine has been readily available and used in Australia for many years in at-risk groups, and the European Centre for Disease Prevention and Control has recently also called for the use of this vaccine among at-risk groups in Europe. Little is known about attitudes towards this vaccine and vaccine uptake in veterinary workers. This study aimed to determine the Q fever vaccination status of veterinarians and veterinary nurses in Australia and to assess and compare the knowledge and attitudes towards Q fever disease and vaccination of each cohort. An online cross-sectional survey performed in 2014 targeted all veterinarians and veterinary nurses in Australia. Responses from 890 veterinarians and 852 veterinary nurses were obtained. Binary, ordinal and multinomial logistic regression were used to make comparisons between the two cohorts. The results showed that 74% of veterinarians had sought vaccination compared to only 29% of veterinary nurses. Barriers to vaccination among those not vaccinated did not differ between cohorts, and included a lack of perceived risk, financial expense, time constraints, and difficulty in finding a vaccine provider. Poor knowledge and awareness of Q fever disease and vaccination were additional and notable barriers for the veterinary nursing cohort, suggesting veterinary clinics and veterinarians may not be meeting their legal responsibility to educate staff about risks and risk prevention. Further evaluation is needed to identify the drivers behind seeking and recommending vaccination so that recommendations can be made to improve vaccine uptake.     

Is dipalmitoylphosphatidylcholine a substrate for convertase?

Convertase has homology with carboxylesterases, but its substrate(s) is not known. Accordingly, we determined whether dipalmitoylphosphatidylcholine (DPPC), the major phospholipid in surfactant, was a substrate for convertase. We measured [(3)H]choline release during cycling of the heavy subtype containing [(3)H]choline-labeled DPPC with convertase, phospholipases A(2), B, C, and D, liver esterase, and elastase. Cycling with liver esterase or peanut or cabbage phospholipase D produced the characteristic profile of heavy and light peaks observed on cycling with convertase. In contrast, phospholipases A(2), B, and C and yeast phospholipase D produced a broad band of radioactivity across the gradient without distinct peaks. [(3)H]choline was released when natural surfactant containing [(3)H]choline-labeled DPPC was cycled with yeast phospholipase D but not with convertase or peanut and cabbage phospholipases D. Similarly, yeast phospholipase D hydrolyzed [(3)H]choline from [(3)H]choline-labeled DPPC after incubation in vitro, whereas convertase, liver esterase, or peanut and cabbage phospholipases D did not. Thus convertase, liver esterase, and plant phospholipases D did not hydrolyze choline from DPPC either on cycling or during incubation with enzyme in vitro. In conclusion, conversion of heavy to light subtype of surfactant by convertase may require a phospholipase D type hydrolysis of phospholipids, but the substrate in this reaction is not DPPC.     

Oxidative stress in zinc-induced dopaminergic neurodegeneration: implications of superoxide dismutase and heme oxygenase-1

The study was undertaken to investigate the effect of zinc (Zn) on glutathione S-transferase (GST) and superoxide dismutases (SOD) activities and on the expressions of cytosolic Cu, Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2), γ-glutamyl cysteine synthetase (γ-GCS) and heme oxygenase-1 (HO-1) in the nigrostriatal tissue of rats. Additionally, Zn-induced alterations in the neurobehavioral parameters, lipid peroxidation (LPO), striatal dopamine and its metabolites and tyrosine hydroxylase (TH) protein expression were measured to assess their correlations with the oxidative stress. Zn exposure reduced the locomotor activity, rotarod performance, striatal dopamine and its metabolites and TH protein expression. LPO, total SOD, SOD1 and SOD2 activities were increased while GST and catalase were reduced in a dose and time dependent manner. Expressions of SOD1 and HO-1 were increased while no change was observed in SOD2 and γ-GCS expressions. The results obtained suggest that Zn-induced augmentation of total SOD, SOD1, SOD2 and HO-1 was associated with increased oxidative stress and neurodegenerative indexes indicating the involvement of both cytosolic and mitochondrial machinery in Zn-induced oxidative stress leading to dopaminergic neurodegeneration.     

Comparative Pathology and Ecological Implications of Two Myxosporean Parasites in Native Australian Frogs and the Invasive Cane Toad

Myxosporean parasites Cystodiscus axonis and C. australis are pathogens of native and exotic Australian frog species. The pathology and ecological outcomes of infection with these parasites were investigated in this study. Gliosis was correlated to Cystodiscus axonis plasmodia in the brains of (9/60) tadpoles and (3/9) adult endangered Green and golden bell frogs using ordinal regression. Severe host reactions to C. axonis (haemorrhage, necrosis, and vasulitis) were observed in the brains of threatened Southern bell frogs (8/8), critically endangered Booroolong frogs (15/44) and Yellow spotted bell frogs (3/3). Severe brain lesions were associated with behavioural changes, neurological dysfunction, and spontaneous death. Both C. axonis and C. australis develop in the bile ducts of tadpoles, the plasmodia were significantly associated with biliary hyperplasia, inflammation and the loss of hepatocytes in (34/72) Green and golden bell frog tadpoles using ordinal regression. These lesions were so severe that in some cases 70% of the total liver was diseased. Normal liver function in tadpoles is necessary for metamorphosis, metabolism, and immune function. We postulate that this extensive liver damage would have significant host health impacts. Severe hepatic myxosporidiosis was more prevalent in tadpoles examined in autumn and winter (overwintered), suggestive of delayed metamorphosis in infected tadpoles, which would have serious flow-on effects in small populations. We compared the sensitivity of histopathology and species-specific PCR in the detection of C. australis and C. axonis. PCR was determined to be the most sensitive method (detection limit 1 myxospore equivalent of ribosomal DNA). Histology, however, had the advantage of assessing the impact of the parasite on the host. It was concluded that these parasites have the potential for significant ecological impacts, because of their high prevalence of infection and their ability to cause disease in some frogs.