The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

Geochemistry and petrogenesis of the Proterozoic Pater metavolcanic suite, Spragge, Ontario /

The Pater metavolcanic suite (PVS) was extruded as part O'f the basal Pater Formation of the Huronian Supergroup ca. 2.4 Ga. They Ars classified as wi thin-plate tholeiites associated with an immature ri-fting episode, and are inter layered with associated vol cani clastic and metasedimentary units. Post-solidif ication alteration caused redistribution o-f the alkalies, Sr, Rb, Ba, Cu, and SiO^. Ce, Y, Zr, CFezOs (as total Fe), Al^Os, TiOa, and, PaOa are considered to have remained essentially immobile in least altered samples. Petrogenetic modelling indicates the PVS was derived from the partial melting of two geochemical ly similar sources in the sub-continental lithosphere. Fractionation was characterized by an oli vine-plagioclase assemblage and a sub-volcanic plagioclase-clinopyroxene assemblage. A comparative study indicates that enrichment of the postulated Huronian source cannot be reconciled by Archean contamination. Enrichment is thought to have been caused by hydrous veined metasomatic heterogeneities in the sub-continental lithosphere, generated by an Archean subduct ion event before 2.68 Ga.

Mode of action of FMRFamide-like peptide on drosophila body wall muscle conractions

Neuropeptides are the largest group of signalling chemicals that can convey the information from the brain to the cells of all tissues. DPKQDFMRFamide, a member of one of the largest families of neuropeptides, FMRFamide-like peptides, has modulatory effects on nerve-evoked contractions of Drosophila body wall muscles (Hewes et aI.,1998) which are at least in part mediated by the ability of the peptide to enhance neurotransmitter release from the presynaptic terminal (Hewes et aI., 1998, Dunn & Mercier., 2005). However, DPKQDFMRFamide is also able to act directly on Drosophila body wall muscles by inducing contractions which require the influx of extracellular Ca 2+ (Clark et aI., 2008). The present study was aimed at identifying which proteins, including the membrane-bound receptor and second messenger molecules, are involved in mechanisms mediating this myotropic effect of the peptide. DPKQDFMRFamide induced contractions were reduced by 70% and 90%, respectively, in larvae in which FMRFamide G-protein coupled receptor gene (CG2114) was silenced either ubiquitously or specifically in muscle tissue, when compared to the response of the control larvae in which the expression of the same gene was not manipulated. Using an enzyme immunoassay (EIA) method, it was determined that at concentrations of 1 ~M- 0.01 ~M, the peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. In addition, the physiological effect of DPKQDFMRFamide at a threshold dose was not potentiated by 3-lsobutyl-1-methylxanthine, a phosphodiesterase inhibitor, nor was the response to 1 ~M peptide blocked or reduced by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. The response to DPKQDFMRFamide was not affected in the mutants of the phosholipase C-~ (PLC~) gene (norpA larvae) or IP3 receptor mutants, which suggested that the PLC-IP3 pathway is not involved in mediat ing the peptide's effects. Alatransgenic flies lacking activity of calcium/calmodul in-dependent protein kinase (CamKII showed an increase in muscle tonus following the application of 1 JlM DPKQDFMRFamide similar to the control larvae. Heat shock treatment potentiated the response to DPKQDFMRFamide in both ala1 and control flies by approximately 150 and 100 % from a non heat-shocked larvae, respectively. Furthermore, a CaMKII inhibitor, KN-93, did not affect the ability of peptide to increase muscle tonus. Thus, al though DPKQDFMRFamide acts through a G-protein coupled FMRFamide receptor, it does not appear to act via cAMP, cGMP, IP3, PLC or CaMKl1. The mechanism through which the FMRFamide receptor acts remains to be determined.

DINOFLAGELLATE CYSTS FROM THE LATEST MIOCENE THROUGH MIDDLE PLEISTOCENE OF THE CARIBBEAN SEA, ODP SITE 1000: BIOSTRATIGRAPHY, PALEOCEANOGRAPHY, AND SHOALING OF THE CENTRAL AMERICAN SEAWAY

This is the first detailed study of organic-walled dinoflagellate cysts (dinocysts) and acritarchs for the latest Miocene–Middle Pleistocene of Ocean Drilling Program Site 1000 in the Caribbean Sea. Well-preserved and moderately diverse dinocysts and other palynomorphs reflect the interplay between neritic (carbonate-platform sourced) and oceanic species. The dinocyst biostratigraphy is tied to an existing marine isotope stratigraphy for the interval 5.5–2.2 Ma. For the interval 5.5–3.8 Ma, palynological samples are coupled to published sea-surface temperature estimates based on planktonic foraminiferal Mg/Ca. Changes in dinocyst assemblage composition are noted at ca. 4.6 Ma when shoaling of the Central American Seaway caused a temperature rise in the Caribbean, ca. 3.8–3.6 Ma, during the cold Marine Isotope Stage M2 when pronounced warming occurred, at ca. 2.7 Ma where possible weak cooling may reflect the onset of Northern Hemisphere glaciation, and in the Middle Pleistocene presumably reflecting global cooling and sea-level fall.

Mode of action of a Drosophila FMRFamide in inducing muscle contraction

Drosophila melanogaster is a model system for examining the mechanisms of action of neuropeptides. DPKQDFMRFamide was previously shown to induce contractions in Drosophila body wall muscle fibres in a Ca(2+)-dependent manner. The present study examined the possible involvement of a G-protein-coupled receptor and second messengers in mediating this myotropic effect after removal of the central nervous system. DPKQDFMRFamide-induced contractions were reduced by 70% and 90%, respectively, in larvae with reduced expression of the Drosophila Fmrf receptor (FR) either ubiquitously or specifically in muscle tissue, compared with the response in control larvae in which expression was not manipulated. No such effect occurred in larvae with reduced expression of this gene only in neurons. The myogenic effects of DPKQDFMRFamide do not appear to be mediated through either of the two Drosophila myosuppressin receptors (DmsR-1 and DmsR-2). DPKQDFMRFamide-induced contractions were not reduced in Ala1 transgenic flies lacking activity of calcium/calmodulin-dependent protein kinase (CamKII), and were not affected by the CaMKII inhibitor KN-93. Peptide-induced contractions in the mutants of the phospholipase C-β (PLCβ) gene (norpA larvae) and in IP3 receptor mutants were similar to contractions elicited in control larvae. The peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. Peptide-induced contractions were not potentiated by 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and were not antagonized by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. Additionally, exogenous application of arachidonic acid failed to induce myogenic contractions. Thus, DPKQDFMRFamide induces contractions via a G-protein coupled FMRFamide receptor in muscle cells but does not appear to act via cAMP, cGMP, IP3, PLC, CaMKII or arachidonic acid.