Binding of arrestin to cytoplasmic loop mutants of bovine rhodopsin

The binding of arrestin to rhodopsin is a multistep process that begins when arrestin interacts with the phosphorylated C terminus of rhodopsin. This interaction appears to induce a conformational change in arrestin that exposes a high-affinity binding site for rhodopsin. Several studies in which synthetic peptides were used have suggested that sites on the rhodopsin cytoplasmic loops are involved in this interaction. However, the precise amino acids on rhodopsin that participate in this interaction are unknown. This study addresses the role of specific amino acids in the cytoplasmic loops of rhodopsin in binding arrestin through the use of site-directed mutagenesis and direct binding assays. A series of alanine mutants within the three cytoplasmic loops of rhodopsin were expressed in HEK-293 cells, reconstituted with 11-cis-retinal, prephosphorylated with rhodopsin kinase, and examined for their ability to bind in vitro-translated, 35S-labeled arrestin. Mutations at Asn-73 in loop I as well as at Pro-142 and Met-143 in loop II resulted in dramatic decreases in the level of arrestin binding, whereas the level of phosphorylation by rhodopsin kinase was similar to that of wild-type rhodopsin. The results indicate that these amino acids play a significant role in arrestin binding.     

Hydrophobic surfaces in oligosaccharides: linear dextrins are amphiphilic chains.

Polysaccharide chains are usually considered to be highly hydrophilic, since they have no obvious nonpolar moieties in them. Yet, it is possible to realise conformations in these chains wherein all the hydroxy groups are disposed in one side or face of the chain and the hydrogens disposed in the other. We experimentally demonstrate that such an amphiphilic surface is present in linear oligomeric dextrins, i.e., alpha-1,4-linked D-glucosides, but not in alpha-1,6-D-glucosides (dextrans) or in beta-1,4-D-glucosides (cellulose). This amphiphilicity is generated as a consequence of the stereochemical constraints, which vary with the structure of the sugar and with the type of linkage. Oligosaccharide chains that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in heteromolecular recognition process than as homomolecular self-aggregation.     

Synthesis and profiling of benzylmorpholine 1,2,4,5-tetraoxane analogue N205: Towards tetraoxane scaffolds with potential for single dose cure of malaria

A series of aryl carboxamide and benzylamino dispiro 1,2,4,5-tetraoxane analogues have been designed and synthesized in a short synthetic sequence from readily available starting materials. From this series of endoperoxides, molecules with in vitro IC50s versus Plasmodium falciparum (3D7) as low as 0.84?nM were identified. Based on an assessment of blood stability and in vitro microsomal stability, N205 (10a) was selected for rodent pharmacokinetic and in vivo antimalarial efficacy studies in the mouse Plasmodium berghei and Plasmodium falciparum Pf3D70087/N9 severe combined immunodeficiency (SCID) mouse models. The results indicate that the 4-benzylamino derivatives have excellent profiles with a representative of this series, N205, an excellent starting point for further lead optimization studies.     

Antimicrobial effects of tea-tree oil and its major components on Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes

Major components of two tea-tree oil samples were identified using thin layer and gas-liquid chromatography (TLC and GLC). Using a TLC-bioautographic technique, the tea-tree oils, terpinen-4-ol, oc-terpineol and α-pinene were found to be active against Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes whereas cineole was inactive against these organisms. The MIC values of the three active compounds increased in the order α-terpineol < terpinen-4-ol < α-pinene for all three micro-organisms. MIC values of the tea-tree oils and terpinen-4-ol were lower for P. acnes than for the two staphylococci. This study supports the use of tea-tree oil in the treatment of acne, and demonstrates that terpinen-4-ol is not the sole active constituent of the oil.     

Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris alpha-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker

Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and α-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris α-amylase inhibitor-1 (αAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris α-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T₁ lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T₁ plants. Transgenic plants could be recovered after 8–10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved.     

Bio-functionalized graphene-graphene oxide nanocomposite based electrochemical immunosensing

We report a novel in-situ electrochemical synthesis approach for the formation of functionalized graphene-graphene oxide (fG-GO) nanocomposite on screen-printed electrodes (SPE). Electrochemically controlled nanocomposite film formation was studied by transmission electron microscopy (TEM) and Raman spectroscopy. Further insight into the nanocomposite has been accomplished by the Fourier transformed infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) spectroscopy. Configured as a highly responsive screen-printed immunosensor, the fG-GO nanocomposite on SPE exhibits electrical and chemical synergies of the nano-hybrid functional construct by combining good electronic properties of functionalized graphene (fG) and the facile chemical functionality of graphene oxide (GO) for compatible bio-interface development using specific anti-diuron antibody. The enhanced electrical properties of nanocomposite biofilm demonstrated a significant increase in electrochemical signal response in a competitive inhibition immunoassay format for diuron detection, promising its potential applicability for ultra-sensitive detection of range of target analytes.     

Interactions among DIV voltage-sensor movement,fast inactivation,and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the Na_Vβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed Na_V1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of Na_Vβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.     

Ligand-Induced Protein Mobility in Complexes of Carbonic Anhydrase II and Benzenesulfonamides with Oligoglycine Chains

This paper describes a biophysical investigation of residual mobility in complexes of bovine carbonic anhydrase II (BCA) and para-substituted benzenesulfonamide ligands with chains of 1–5 glycine subunits, and explains the previously observed increase in entropy of binding with chain length. The reported results represent the first experimental demonstration that BCA is not the rigid, static globulin that has been typically assumed, but experiences structural fluctuations upon binding ligands. NMR studies with ¹⁵N-labeled ligands demonstrated that the first glycine subunit of the chain binds without stabilization or destabilization by the more distal subunits, and suggested that the other glycine subunits of the chain behave similarly. These data suggest that a model based on ligand mobility in the complex cannot explain the thermodynamic data. Hydrogen/deuterium exchange studies provided a global estimate of protein mobility and revealed that the number of exchanged hydrogens of BCA was higher when the protein was bound to a ligand with five glycine subunits than when bound to a ligand with only one subunit, and suggested a trend of increasing number of exchanged hydrogens with increasing chain length of the BCA-bound ligand, across the series. These data support the idea that the glycine chain destabilizes the structure of BCA in a length-dependent manner, causing an increase in BCA mobility. This study highlights the need to consider ligand-induced mobility of even “static” proteins in studies of protein-ligand binding, including rational ligand design approaches.     

QTL Mapping of Agronomic Waterlogging Tolerance Using Recombinant Inbred Lines Derived from Tropical Maize (Zea mays L) Germplasm

Waterlogging is an important abiotic stress constraint that causes significant yield losses in maize grown throughout south and south-east Asia due to erratic rainfall patterns. The most economic option to offset the damage caused by waterlogging is to genetically incorporate tolerance in cultivars that are grown widely in the target agro-ecologies. We assessed the genetic variation in a population of recombinant inbred lines (RILs) derived from crossing a waterlogging tolerant line (CAWL-46-3-1) to an elite but sensitive line (CML311-2-1-3) and observed significant range of variation for grain yield (GY) under waterlogging stress along with a number of other secondary traits such as brace roots (BR), chlorophyll content (SPAD), % stem and root lodging (S&RL) among the RILs. Significant positive correlation of GY with BR and SPAD and negative correlation with S&RL indicated the potential use of these secondary traits in selection indices under waterlogged conditions. RILs were genotyped with 331 polymorphic single nucleotide polymorphism (SNP) markers using KASP (Kompetitive Allele Specific PCR) Platform. QTL mapping revealed five QTL on chromosomes 1, 3, 5, 7 and 10, which together explained approximately 30% of phenotypic variance for GY based on evaluation of RIL families under waterlogged conditions, with effects ranging from 520 to 640 kg/ha for individual genomic regions. 13 QTL were identified for various secondary traits associated with waterlogging tolerance, each individually explaining from 3 to 14% of phenotypic variance. Of the 22 candidate genes with known functional domains identified within the physical intervals delimited by the flanking markers of the QTL influencing GY and other secondary traits, six have previously been demonstrated to be associated with anaerobic responses in either maize or other model species. A pair of flanking SNP markers has been identified for each of the QTL and high throughput marker assays were developed to facilitate rapid introgression of waterlogging tolerance in tropical maize breeding programs.