Clay mineralogy of ODP Site 131-808 sediments

Drill core recovered at Ocean Drilling Program Site 808 (Leg 131) proves that the wedge of trench sediment within the central region of the Nankai Trough comprises approximately 600 m of hemipelagic mud, sandy turbidites, and silty turbidites. The stratigraphic succession thickens and coarsens upward, with hemipelagic muds and volcanic-ash layers of the Shikoku Basin overlain by silty and sandy trench-wedge deposits. Past investigations of clay mineralogy and sand petrography within this region have led to the hypothesis that most of the detritus in the Nankai Trough was derived from the Izu-Honshu collision zone and transported southwestward via axial turbidity currents. Shipboard analyses of paleocurrent indicators, on the other hand, show that most of the ripple cross-laminae within silty turbidites of the outer marginal trench-wedge facies are inclined to the north and northwest; thus, many of the turbidity currents reflected off the seaward slope of the trench rather than moving straight down the trench axis. Shore-based analyses of detrital clay minerals demonstrate that the hemipelagic muds and matrix materials within sandy and silty turbidites are all enriched in illite; chlorite is the second-most abundant clay mineral, followed by smectite. In general, the relative mineral percentages change relatively little as a function of depth, and the hemipelagic clay-mineral population is virtually identical to the turbidite-matrix population. Comparisons between different size fractions (<2 µm and 2-6 µm) show modest amounts of mineral partitioning, with chlorite content increasing in the coarser fraction and smectite increasing in the finer fraction. Values of illite crystallinity index are consistent with conditions of advanced anchimetamorphism and epimetamorphism within the source region. Of the three mica polytypes detected, the 2M1 variety dominates over the 1M and 1Md polytypes; these data are consistent with values of illite crystallinity. Measurements of mica bo lattice spacing show that the detrital illite particles were eroded from a zone of intermediate-pressure metamorphism. Collectively, these data provide an excellent match with the lithologic and metamorphic character of the Izu-Honshu collision zone. Data from Leg 131, therefore, confirm the earlier interpretations of detrital provenance. The regional pattern of sediment dispersal is dominated by a combination of southwest-directed axial turbidity currents, radial expansion of the axial flows, oblique movement of suspended clouds onto and beyond the seaward slope of the Nankai Trough, and flow reflection back toward the trench axis.

Differential effects on the hepatitis C virus (HCV) internal ribosome entry site by vitamin B-12 and the HCV core protein

To investigate the role of the hepatitis C virus internal ribosome entry site (HCV IRES) domain IV in translation initiation and regulation, two chimeric IRES elements were constructed to contain the reciprocal domain IV in the otherwise HCV and classical swine fever virus IRES elements. This permitted an examination of the role of domain IV in the control of HCV translation. A specific inhibitor of the HCV IRES, vitamin B-12 was shown to inhibit translation directed by all IRES elements which contained domain IV from the HCV and the GB virus B IRES elements, whereas the HCV core protein could only suppress translation from the wild-type HCV IRES. Thus, the mechanisms of translation inhibition by vitamin B-12 and the core protein differ, and they target different regions of the IRES.

Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by H-1 NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 Angstrom, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H-alpha protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.

Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site

The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites ( Ile(116), Arg(175), Arg(206), Glu(199), Asp(282), and Val(286)). Agonist peptide 1 (Phe-Lys-Pro-D-cyclohexylalanine-cyclohexylalanine-D-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 ( peptide 2). Conversely, mutation of C5aR transmembrane residue Ile(116) to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394 - 3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] ( peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile(116), the latter is not essential for either activation or antagonism. Arg(206) and Arg(175) both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp(282) has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu(199) may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.

Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site

Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering have been used to determine the identity of the metals incorporated into the active site in vivo. We have recombinantly expressed the phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in medium supplemented with 1 mM CoCl2 and in unsupplemented medium. Anomalous scattering data, collected from a single crystal at the Fe-K, Co-K and Zn-K edges, indicate that iron and cobalt are the primary constituents of the two metal-binding sites in the catalytic centre (alpha and P) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the beta-position of the active site, which results in an increase in the catalytic efficiency of the enzyme. These results suggest an essential role for iron in the catalytic mechanism of bacterial phosphotriesterases, and that these phosphotriesterases are natively heterobinuclear iron-zinc enzymes.

Structure-function analysis of RAMP1 by Alanine Mutagenesis

Receptor activity modifying protein 1 (RAMP1) is an integral component of several receptors including the calcitonin gene-related peptide (CGRP) receptor. It forms a complex with the calcitonin receptor-like receptor (CLR) and is required for receptor trafficking and ligand binding. The N-terminus of RAMP1 comprises three helices. The current study investigated regions of RAMP1 important for CGRP or CLR interactions by alanine mutagenesis. Modeling suggested the second and third helices were important in protein-protein interactions. Most of the conserved residues in the N-terminus (M48, W56, Y66, P85, N66, H97, F101, D113, P114, P115), together with a further 13 residues spread throughout three helices of RAMP1, were mutated to alanine and coexpressed with CLR in Cos 7 cells. None of the mutations significantly reduced RAMP expression. Of the nine mutants from helix 1, only M48A had any effect, producing a modest reduction in trafficking of CLR to the cell surface. In helix 2 Y66A almost completely abolished CLR trafficking; L69A and T73A reduced the potency of CGRP to produce cAMP. In helix 3, H97A abolished CLR trafficking; P85A, N86A, and F101A had caused modest reductions in CLR trafficking and also reduced the potency of CGRP on cAMP production. F93A caused a modest reduction in CLR trafficking alone and L94A increased cAMP production. The data are consistent with a CLR recognition site particularly involving Y66 and H97, with lesser roles for adjacent residues in helix 3. L69 and T73 may contribute to a CGRP recognition site in helix 2 also involving nearby residues.

Characterization of heparin-binding site of tissue transglutaminase:its importance in cell surface targeting, matrix deposition, and cell signaling

Tissue transglutaminase (TG2) is a multifunctional Ca2+ activated protein crosslinking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a non-transamidating mechanism via its association with fibronectin (FN), heparan sulphates (HS) and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modelling and mutagenesis we have identified the HS binding site of TG2 202KFLKNAGRDCSRRSSPVYVGR222. We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate the RGD-induced loss of cell adhesion on FN via binding to syndecan-4, leading to activation of PKCa, pFAK-397 and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.

Investigations on sediment coring site PS1823 in the Riiser-Larsen Sea, Southern Ocean

Physical and sedimentological investigations were carried out on a 14 m long gravity core and a 0.5 m long box core from 4440 m water depth off Queen Maud Land, East-Antarctica. Strongly bioturbated hemipelagic muds of predominantly terrigenous origin and a very small biogenic part build up the 'Normal-Facies'. Several sandy to silty layers are inserted in the 'Normal-Facies'. These layers are seperated by lithology, structure and the investigated parameters of this study and are interpreted as turbidites. The source area for the turbidity currents is supposed to be at the uppermost continental margin, close to the shelf break and there is evidenee for this gravity transport within the erosive Ritscher-Canyon, which extends close to the core position. The distribution of biogenic components indicates an age of 1.3 million years or more, with an average sedimentation rate of about 1 cm/1000 years. Early diagenetic proeesses caused water loss by compaction, errosion and dissolution of biogenic components and precipitation and recrystallization of manganese micronodules. Cyclic fluctuations of the sediment-parameters within the 'Normal-Facies' enable the distinction of a 'Glazial'- and an 'Interglazial'-Facies. The 'Glazial'-Facies reflects glacial sedimentary conditions and shows a dark olive gray colour, high susceptibility, low silt/clay-ratios, only a few biogenic components and the regular occurence of interrelated turbidite layers. In contrast, the 'Interglazial'-Facies is dominated by a light olive or olive-brown colour, low susceptibility, high silt/clay-ratios and an increased number of biogenic components. This facies corresponds to interglacial conditions. Three main processes are supposed to have been responsible for the observed facies changes: (1) the bottom water mass circulation, (2) the gravity transport by turbidity currents and (3) the biogenic surface production. These processes are related to the quaternary climatic changes. The extension of the ice shelves directed the gravity transport to the deep sea and the formation of Antarctic Bottom Water, which in turn influenced the silt/clay-ratios in the sediment record. Fluctuations in sea ice coverage controlled the biogenic surface production.

RECQ5 promotes recombination and mutagenesis at targeted nicks through disruption of RAD51 filaments

Thesis (Ph.D.)--University of Washington, 2016-08

Final report : offsite manufacture in Australia Final report off-site manufacture in Australia

Off-site Manufacture (OSM) has long been recognised, both in Australia and internationally, as offering numerous benefits to all parties in the construction process. More importantly, it is recognised as a key vehicle for driving improvement within the construction industry. The uptake of OSM in construction is however limited, despite well documented benefits. The research aims to determine the ‘state-of-the-art’ of OSM in Australia. It confirms the benefits and identifies the real and perceived barriers to the widespread adoption of OSM. Further the project identifies opportunities for future investment and research. Although numerous reports have been produced in the UK on the state of OSM adoption within that region, no prominent studies exist for the Australian context. This scoping study is an essential component upon which to build any initiatives that can take advantage of the benefits of OSM in construction. The Construction 2020 report predicted that OSM is set to increase in use over the next 5-15 years, further justifying the need for such a study. The long-term goal of this study is to contribute to the improvement of the Australian construction industry through a realisation of the potential benefits of OSM.

Off-site Manufacture in Australia : Current State and Future Directions

Off-site manufacture (OSM) offers numerous benefits to all parties in the construction process. The uptake of OSM in Australia has, however, been limited. This limited uptake corresponds to similar trends in the UK and US, although the level of OSM there appears to be increasing. This project undertook three workshops — one each in Victoria, Queensland and Western Australia — and 18 interviews with key stakeholders to assist in identifying the general benefits and barriers to OSM uptake in the Australian construction industry. Seven case studies were also undertaken, involving construction projects that used OSM, ranging from civil projects through to residential. Each of these case studies has been analysed to identify what worked and what didn’t, and suggest the lessons to be learned from each project.