Calcium is essential for the structural integrity of the cysteine-rich, ligand-binding repeat of the low-density lipoprotein receptor

Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. in the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional H-1 NMR spectral studies: only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.

Inhibition of phenotype modulation, growth, and migration of vascular smooth muscle cells by a guanosine-rich 30-mer phosphorothioate oligodeoxynucleotide

The testing of a 30-mer dG-rich phosphorothioate oligodeoxynucleotide (LG4PS) for effects on the behaviour of vascular smooth muscle cells (VSMC) in vitro and in vivo is described. LG4PS at 0.3 mu M inhibited significantly the phenotype modulation of freshly isolated rabbit VSMC, and cell outgrowth from pig aortic explants was inhibited similar to 80% by 5 mu M LG4PS. The growth of proliferating rabbit and pig VSMC was inhibited similar to 70% by 0.3 mu M and 5 mu M LG4PS, respectively. Though less marked, the antiproliferative effects of LG4PS on human VSMC were comparable to those obtained with heparin. The cytotoxic effects of LG4PS on VSMC in vitro were low. Despite these promising results, adventitial application of 2-200 nmol LG4PS in pluronic gel failed to reduce vascular hyperplasia in balloon-injured rabbit carotid arteries, and the highest dose caused extensive mortality. (C) 1997 Academic Press Limited.

Measurement of grain boundary composition for X52 pipeline steel

Analytical electron microscopy was used to measure the composition of grain boundaries (GBs) and interconstituent boundaries (IBs) of X52 pipeline steel using specimens about 40-60 nm in thickness. All elements of interest were examined with the exception of carbon. With this caveat; there was no segregation at proeutectoid ferrite GBs. This indicated that the commonly expected species S and P are not responsible for preferential corrosion of GBs during intergranular stress corrosion cracking of pipeline steels. Manganese was the only species measured to segregate at the IBs. Manganese segregated to the IBs between proeutectoid ferrite and pearlitic cementite, and desegregated from IBs between proeutectoid ferrite and pearlitic ferrite. The pearlitic cementite was Mn rich. There was no Mn segregation at the IBs between pearlitic ferrite and pearlitic cementite. The pattern of Mn segregation could be explained in terms of diffusion in the process zone ahead of the pearlite during the austenite to pearlite transformation and diffusion in the IBs between the proeutectoid ferrite and pearlite. (C) 1998 Acta Metallurgica Inc. Published by Elsevier Science Ltd. All rights reserved.

Factors controlling the origin of gas in Australian Bowen Basin coals

Open system pyrolysis (heating rate 10 degrees C/min) of coal maturity (vitrinite reflectance, VR) sequence (0.5%, 0.8% and 1.4% VR) demonstrates that there are two stages of thermogenic methane generation from Bowen Basin coals. The first and major stage shows a steady increase in methane generation maximising at 570 degrees C, corresponding to a VR of 2-2.5%. This is followed by a less intense methane generation which has not as yet maximised by 800 degrees C (equivalent to VR of 5%). Heavier (C2+) hydrocarbons are generated up to 570 degrees C after which only the C-1 (CH4, CO and CO2) gases are produced. The main phase of heavy hydrocarbon generation occurs between 420 and 510 degrees C. Over this temperature range,methane generation accounts for only a minor component, whereas the wet gases (C-2-C-5) are either in equal abundance or are more abundant by a factor of two than the liquid hydrocarbons. The yields of non-hydrocarbon gases CO2 and CO are greater then methane during the early stages of gas generation from an immature coal, subordinate to methane during the main phase of methane generation after which they are again dominant. Compositional data for desorbed and produced coal seam gases from the Bowen show that CO2 and wet gases are a minor component. This discrepancy between the proportion of wet gas components produced during open system pyrolysis and that observed in naturally matured coals may be the result of preferential migration of wet gas components, by dilution of methane generated during secondary cracking of bitumen, or kinetic effects associated with different activations for production of individual hydrocarbon gases. Extrapolation of results of artificial pyrolysis of the main organic components in coal to geological significant heating rates suggests that isotopically light methane to delta(13)C of -50 parts per thousand can be generated. Carbon isotope depletions in C-13 are further enhanced, however, as a result of trapping of gases over selected rank levels (instantaneous generation) which is a probable explanation for the range of delta(13)C values we have recorded in methane desorbed from Bowen Basin coals (-51 +/- 9 parts per thousand). Pervasive carbonate-rich veins in Bowen Basin coals are the product of magmatism-related hydrothermal activity. Furthermore, the pyrolysis results suggest an additional organic carbon source front CO2 released at any stage during the maturation history could mix in varying proportions with CO2 from the other sources. This interpretation is supported by C and O isotopic ratios, of carbonates that indicate mixing between magmatic and meteoric fluids. Also, the steep slope of the C and O isotope correlation trend suggests that the carbonates were deposited over a very narrow temperature interval basin-wide, or at relatively high temperatures (i.e., greater than 150 degrees C) where mineral-fluid oxygen isotope fractionations are small. These temperatures are high enough for catagenic production of methane and higher hydrocarbons from the coal and coal-derived bitumen. The results suggests that a combination of thermogenic generation of methane and thermodynamic processes associated with CH4/CO2 equilibria are the two most important factors that control the primary isotope and molecular composition of coal seam gases in the Bowen Basin. Biological process are regionally subordinate but may be locally significant. (C) 1998 Published by Elsevier Science Ltd. All rights reserved.

Applications of NMR in drug design: Sructure-activity relationships in disulfide-rich peptides

NMR is a powerful technique for determining structures of biologically active molecules in solution. In recent years. our laboratory has focussed on the structure determination of small disulfide-rich proteins from both plants and animals which are valuable targets in drug design applications. This article will review these structural studies and their implications in drug design.

Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods, These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA, To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters, In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [H-3]N-OH-IQ to DNA, ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and similar to 50% of that seen with acetyl coenzyme A (AcCoA), In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites, With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein, The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Indicators used to measure the innovation process: defects and possible remedies

Some diverse indicators used to measure the innovation process are considered, They include those with art aggregate, and often national, focus, and rely on data from scientific publications, patents and R&D expenditures, etc. Others have a firm-level perspective, relying primarily on surveys or case studies. Also included are indicators derived from specialized databases, or consensual agreements reached through foresight exercises. There is an obvious need for greater integration of the various approaches to capture move effectively the richness of available data and better reflect the reality of innovation. The focus for such integration could be in the area of technology strategy, which integrates the diverse scientific, technological, and innovation activities of firms within their operating environments; improved capacity to measure it has implications for policy-makers, managers and researchers.

Heterogeneous distribution of isoactins in cultured vascular smooth muscle cells does not reflect segregation of contractile and cytoskeletal domains

We have previously demonstrated that or-smooth muscle (alpha -SM) actin is predominantly distributed in the central region and beta -non-muscle (beta -NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, alpha -actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of beta -NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and ol-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of alpha -SM actin in the central portion of stress fibers was related to continuous myosin and punctate alpha -actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sar-comere-like units with alpha -actinin-rich dense bodies analogous to Z-lines, are the contractile vimentin structures of cultured SMCs that link to the network of vimentin-containing intermediate alpha -actinin filaments through the dense bodies and dense plaques.

Simultaneous steady state and dynamic design of process systems using structural indicators

The simultaneous design of the steady-state and dynamic performance of a process has the ability to satisfy much more demanding dynamic performance criteria than the design of dynamics only by the connection of a control system. A method for designing process dynamics based on the use of a linearised systems' eigenvalues has been developed. The eigenvalues are associated with system states using the unit perturbation spectral resolution (UPSR), characterising the dynamics of each state. The design method uses a homotopy approach to determine a final design which satisfies both steady-state and dynamic performance criteria. A highly interacting single stage forced circulation evaporator system, including control loops, was designed by this method with the goal of reducing the time taken for the liquid composition to reach steady-state. Initially the system was successfully redesigned to speed up the eigenvalue associated with the liquid composition state, but this did not result in an improved startup performance. Further analysis showed that the integral action of the composition controller was the source of the limiting eigenvalue. Design changes made to speed up this eigenvalue did result in an improved startup performance. The proposed approach provides a structured way to address the design-control interface, giving significant insight into the dynamic behaviour of the system such that a systematic design or redesign of an existing system can be undertaken with confidence.

Charge ordering and antiferromagnetic exchange in layered molecular crystals of the theta type

We consider the electronic properties of layered molecular crystals of the type theta -D(2)A where A is an anion and D is a donor molecule such as bis-(ethylenedithia-tetrathiafulvalene) (BEDT-TTF), which is arranged in the theta -type pattern within the layers. We argue that the simplest strongly correlated electron model that can describe the rich phase diagram of these materials is the extended Hubbard model on the square lattice at one-quarter filling. In the limit where the Coulomb repulsion on a single site is large, the nearest-neighbor Coulomb repulsion V plays a crucial role. When V is much larger than the intermolecular hopping integral t the ground state is an insulator with charge ordering. In this phase antiferromagnetism arises due to a novel fourth-order superexchange process around a plaquette on the square lattice. We argue that the charge ordered phase is destroyed below a critical nonzero value V, of the order of t. Slave-boson theory is used to explicitly demonstrate this for the SU(N) generalization of the model, in the large-N limit. We also discuss the relevance of the model to the all-organic family beta-(BEDT-TTF)(2)SF5YSO3 where Y=CH2CF2, CH2, CHF.

The Las Campanas/AAT rich cluster survey - I. Precision and reliability of the photometric catalogue

The Las Campanas Observatory and Anglo-Australian Telescope Rich Cluster Survey (LARCS) is a panoramic imaging and spectroscopic survey of an X-ray luminosity-selected sample of 21 clusters of galaxies at 0.07 < z < 0.16. Charge-coupled device (CCD) imaging was obtained in B and R of typically 2 degrees wide regions centred on the 21 clusters, and the galaxy sample selected from the imaging is being used for an on-going spectroscopic survey of the clusters with the 2dF spectrograph on the Anglo-Australian Telescope. This paper presents the reduction of the imaging data and the photometric analysis used in the survey. Based on an overlapping area of 12.3 deg(2) we compare the CCD-based LARCS catalogue with the photographic-based galaxy catalogue used for the input to the 2dF Galaxy Redshift Survey (2dFGRS) from the APM, to the completeness of the GRS/APM catalogue, b(J) = 19.45. This comparison confirms the reliability of the photometry across our mosaics and between the clusters in our survey. This comparison also provides useful information concerning the properties of the GRS/APM. The stellar contamination in the GRS/APM galaxy catalogue is confirmed as around 5-10 per cent, as originally estimated. However, using the superior sensitivity and spatial resolution in the LARCS survey evidence is found for four distinct populations of galaxies that are systematically omitted from the GRS/APM catalogue. The characteristics of the 'missing' galaxy populations are described, reasons for their absence examined and the impact they will have on the conclusions drawn from the 2dF Galaxy Redshift Survey are discussed.