Protein Kinase C-Dependent Signaling Controls the Midgut Epithelial Barrier to Malaria Parasite Infection in Anopheline Mosquitoes

Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC) gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members – PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC − in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC-dependent signaling is a negative regulator of epithelial barrier function and a potential new target for transmission-blocking strategies.     

Discovery of GSK143, a highly potent, selective and orally efficacious spleen tyrosine kinase inhibitor

The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.     

BRD4 Short Isoform Interacts with RRP1B,SIPA1 and Components of the LINC Complex at the Inner Face of the Nuclear Membrane

Recent studies suggest that BET inhibitors are effective anti-cancer therapeutics. Here we show that BET inhibitors are effective against murine primary mammary tumors, but not pulmonary metastases. BRD4, a target of BET inhibitors, encodes two isoforms with opposite effects on tumor progression. To gain insights into why BET inhibition was ineffective against metastases the pro-metastatic short isoform of BRD4 was characterized using mass spectrometry and cellular fractionation. Our data show that the pro-metastatic short isoform interacts with the LINC complex and the metastasis-associated proteins RRP1B and SIPA1 at the inner face of the nuclear membrane. Furthermore, histone binding arrays revealed that the short isoform has a broader acetylated histone binding pattern relative to the long isoform. These differential biochemical and nuclear localization properties revealed in our study provide novel insights into the opposing roles of BRD4 isoforms in metastatic breast cancer progression.     

Phosphorothioate analogues of phosphoribosyldiphosphate are substrates of quinolinate phosphoribosyltransferase

Phosphorothioate analogues of phosphoribosyldiphosphate (P-Rib-PP) can act as substrates of quinolinate phosphoribosyltransferase. The order of effectiveness as substrates, with Mg2+ as the activating metal ion, was P-Rib-PP greater than P-Rib-PPbeta S greater than P-Rib-PP alpha S(Sp) greater than P-Rib-PP alsph S(Rp). Cd2+ was more effective than Mg2+ with the Rp diastereommer.     

Multifaceted shared care intervention for late life depression in residential care: randomised controlled trial

ObjectiveTo evaluate the effectiveness of a population based, multifaceted shared care intervention for late life depression in residential care.DesignRandomised controlled trial, with control and intervention groups studied one after the other and blind follow up after 9.5 months.SettingPopulation of residential facility in Sydney living in self care units and hostels.Participants220 depressed residents aged ⩾65 without severe cognitive impairment.InterventionThe shared care intervention included: (a) multidisciplinary consultation and collaboration, (b) training of general practitioners and carers in detection and management of depression, and (c) depression related health education and activity programmes for residents. The control group received routine care.ResultsIntention to treat analysis was used. There was significantly more movement to “less depressed” levels of depression at follow up in the intervention than control group (Mantel-Haenszel stratification test, P=0.0125). Multiple linear regression analysis found a significant intervention effect after controlling for possible confounders, with the intervention group showing an average improvement of 1.87 points on the geriatric depression scale compared with the control group (95% confidence interval 0.76 to 2.97, P=0.0011).ConclusionsThe outcome of depression among elderly people in residential care can be improved by multidisciplinary collaboration, by enhancing the clinical skills of general practitioners and care staff, and by providing depression related health education and activity programmes for residents.

Key messages

• Large numbers of depressed elderly people live in residential care but few receive appropriate management
• A population based, multifaceted shared care intervention for late life depression was more effective than routine care in improving depression outcome
• The outcome of late life depression can be improved by enhancing the clinical skills of general practitioners and care staff and by providing depression related health education and activity programmes for residents
• The intervention needs further refining and evaluation to improve its effectiveness and to determine how best to implement it in other residential care settings

     

Rapid vertical accretion on a ‘young’ shore-detached turbid zone reef: Offshore Paluma Shoals, central Great Barrier Reef, Australia

We report on the age structure and net accretion rates determined for an open water turbid zone reef, known as Offshore Paluma Shoals, located on the inner central Great Barrier Reef. Twenty-eight radiocarbon dates from 5 cores through the reef structure indicate that this reef began growing ~1,700 years ago and that net vertical accretion through the main phase of reef development was rapid (averaging 7.8 mm yr^?1), this despite the reef growing in highly turbid waters. The most rapid growth phases coincided with the accumulation of mud-rich terrigenoclastic sediments within the reef fabric. The study emphasises the capacity of turbid zone reefs to vertically accrete at rates matching or exceeding many clear water reefs despite seemingly detrimental water quality conditions.     

Splice junctions are constrained by protein disorder

We have discovered that positions of splice junctions in genes are constrained by the tolerance for disorder-promoting amino acids in the translated protein region. It is known that efficient splicing requires nucleotide bias at the splice junction; the preferred usage produces a distribution of amino acids that is disorder-promoting. We observe that efficiency of splicing, as seen in the amino-acid distribution, is not compromised to accommodate globular structure. Thus we infer that it is the positions of splice junctions in the gene that must be under constraint by the local protein environment. Examining exonic splicing enhancers found near the splice junction in the gene, reveals that these (short DNA motifs) are more prevalent in exons that encode disordered protein regions than exons encoding structured regions. Thus we also conclude that local protein features constrain efficient splicing more in structure than in disorder.     

Electron paramagnetic resonance measurements of the hydration of Mn(II) in ternary complexes with GDP and ras p21 proteins

Electron paramagnetic resonance (EPR) spectroscopy has been used to determine the hydration numbers of Mn(II) in complexes with GDP and three forms of ras p21. EPR signals of Mn(II) in the GDP complex with viral-Harvey p21pRAS1 (Arg 12, Thr 59), p21EC (Gly 12, Thr 59), and p21EJ (Val 12, Thr 59) have narrow line-widths that permit ready observation of inhomogeneous broadening from unresolved superhyperfine coupling with the nuclear spin of 17O of directly coordinated oxygen ligands. Quantitative analysis of the lineshapes for the samples in H2 17O-enriched water indicates that four water ligands coordinate to the metal ion in the GDP complexes with all three proteins. The four solvent ligands, together with an oxygen from the beta-phosphate group of GDP, leave space for only one ligand from the protein. An X-ray diffraction-derived model for the MgII beta-gamma-imidoguanosine-5'-triphosphate complex with p21 shows coordination of Mg(II) to the beta- and gamma-phosphate groups of the nucleotide as well as to the hydroxyl groups of Thr 35 and Ser 17 (Pai, E.F., Kabusch, W., Krengel, U., Holmes, K. H., John, J., and Wittinghofer, A., 1989, Nature (London) 341, 209-214). Thus, upon conversion of the nucleotide from a triphosphate to a diphosphate, solvent replaces both the gamma-phosphate of the nucleotide and one of the protein ligands. The EPR results are consistent with a recent X-ray crystallographic model for the p21-MgIIGDP complex (Milburn, M. V., Tong, L., DeVos, A. M., Brunger, A., Yamaizumi, Z., Nishimura, S., and Kim, S.-H., 1990, Science 247, 939-945). EPR spectra of complexes with the three forms of ras p21 differ with respect to the intrinsic linewidths of the EPR signals. These subtle differences in linewidth appear to originate from slight differences in local disorder near the metal-nucleotide binding site.