Screening transthyretin amyloid fibril inhibitors: characterization of novel multiprotein,multiligand complexes by mass spectrometry

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.     

Water-use efficiency of a mallee eucalypt growing naturally and in short-rotation coppice cultivation

This study compared mature Eucalyptus kochii subsp. plenissima trees in inner regions or edges of natural bushland to young trees belt-planted through cleared agricultural land as uncut saplings or regenerating coppice over 2.7 years at Kalannie, Western Australia (320 mm annual rainfall). We assessed the ability of the species to alter its gas exchange characteristics, leaf physical attributes, and water-use efficiency of foliar carbon assimilation (WUE _i) or of total dry matter production (WUE _DM). Stomatal conductance (g _s) varied five-fold between treatment means, with coppices exhibiting greatest values and mature bush least. Photosynthetic rates followed this trend. Leaf photosynthetic capacity estimated by chlorophyll content varied 1.3-fold parallel with variations in leaf thickness, with coppices rating lowest and mature edge trees most highly. WUE _i varied 1.5-fold between treatments and was greatest in mature inner-bush and edge trees. Leaf photosynthetic capacity and g _s were both correlated with WUE _i. Carbon isotope composition (δ¹³C values) of new shoot dry matter produced early in a seasonal flush were similar to those of root starch but when averaged over the whole season correlated well with WUE _i and gas exchange characteristics of trees of each treatment. Coppices showed poorest WUE _i and most negative shoot tip δ¹³C but their WUE _DM was high. This discrepancy was suggested to relate to carbon allocation strategies in coppices favouring fast growth of replacement shoots but not of roots. Physiology of coppice growth of E. kochii is usefully geared towards both rapid and water-use efficient production of woody biomass in water limited environments.     

Selection of cell lines resistant to anti-transferrin receptor antibody: evidence for a mutation in transferrin receptor.

Some anti-murine transferrin receptor monoclonal antibodies block iron uptake in mouse cell lines and inhibit cell growth. We report here the selection and characterization of mutant murine lymphoma cell lines which escape this growth inhibition by anti-transferrin receptor antibody. Growth assays and immunoprecipitation of transferrin receptor in hybrids between independently derived mutants or between mutants and antibody-susceptible parental cell lines indicate that all of the selected lines have a similar genetic alteration that is codominantly expressed in hybrids. Anti-transferrin receptor antibodies and transferrin itself still bind to the mutant lines with saturating levels and Kd values very similar to those of the parental lines. However, reciprocal clearing experiments by immunoprecipitation and reciprocal blocking of binding to the cell surface with two anti-transferrin receptor antibodies indicate that the mutant lines have altered a fraction of their transferrin receptors such that the growth-inhibiting antibody no longer binds, whereas another portion of their transferrin receptors is similar to those of the parental lines and binds both antibodies. These results argue that the antibody-selected mutant cell lines are heterozygous in transferrin receptor expression, probably with a mutation in one of the transferrin receptor structural genes.     

Herbivore damage along a latitudinal gradient: relative impacts of different feeding guilds

We present the first broad-scale test for a latitudinal gradient in herbivory made with consistent methods, in similar habitat type, over the entire lifespan of leaves (phyllodes). We assessed the degree of chewing, sap-sucking and mining herbivory on Acacia falcata along its entire coastal latitudinal range (1150 km) in Australia. We found no significant differences in the rate of herbivory among latitudes. Mature phyllodes had a higher rate of herbivory compared to young phyllodes, and mining was higher on mature phyllodes from the most tropical latitude. We found significant differences in phyllode toughness and specific leaf (phyllode) area among latitudes, but no significant differences among latitudes in carbon: nitrogen. This study provides a useful model for further testing of the generalisation that herbivory is more intense in tropical versus temperate regions.     

Limitations of feeding experiments in studying alkaloid biosynthesis in Peganum harmala callus cultures

Feeding and trapping experiments to Peganum harmala callus cultures were limited by compartmentation; exogenous substrates were detoxified by precipitation, presumably as polymers or conjugates, or by conversion to water-soluble products, such as phenols and glucosides, easily stored in vacuoles. Alkaloid-producing and non-alkaloid-producing callus cultures were readily able to convert tryptamine to 5-hydroxytryptamine and harmaline to dihydroruine (8-hydroxyglucosylharmaline). Phenolic substrates, including 5- and 6-hydroxy-tryptophan, 5- and 6-hydroxytryptamine and harmalol, were not metabolized. In alkaloid-producing callus cultures, radioactivity from [methylene-¹⁴C]-l-tryptophan and [methyl-¹⁴C-]-harmaline was incorporated into harmine. The dilution of radioactivity was 30000- and 2-fold respectively.     

PERIZONIUM AND INITIAL VALVE FORMATION IN THE DIATOM NAVICULA CUSPIDATA (BACILLARIOPHYCEAE)1

This paper describes the perizonium and initial valve formation in Navicula cuspidata Kütz., based on light microscope (LM) and scanning electron microscope (SEM) observations. The perizonium consists of concentric over-lapping bands, laid down sequentially at the tips of the expanding biconical auxospore during its elongation. The central perizonial band has fimbriate edges and is considerably more rigid than the more distal bands. During auxospore elongation and the band secretion, the chloroplasts continuously oscillate between the two ends of the cell; this oscillation ceases once the elongation is complete. The initial valves, formed within the perizonium, are molded into the basically biconical shape of the perizonium except for a central flattening of each valve face. In contrast to the raphes in gametangial and vegetative valves which are surrounded by a smooth axial area, the raphes in initial valves lie within a raised ridge running along the apical axis of the valve. The regular pattern of apically oriented ridges on the outer surface of vegetative valves is also lacking on initial valves. Comparison of pore–pore spacing within striae of gametangial valves, initial values and post-initial valves (first division and vegetative cells) reveals that the pore–pore distance within striae is conserved at all sexual stages. However, the distance between striae is considerably larger in initial valves than in gametangial and post-initial valves. Vegetative interstriae spacing as well as the planar morphology of the valve face is regained at the first division of the initial cell. This suggests that the spacing between striae is dependent on the sexual stage of the cell during valve formation (i.e. not directly dependent on the cell size) and can be altered independently of the pore–pore spacing.     

Binding of bovine seminal plasma protein BSP-A1/-A2 to model membranes: lipid specificity and effect of the temperature

Bovine seminal plasma (BSP) contains a family of phospholipid-binding proteins. The affinity of the protein BSP-A1/-A2 for lipid membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and POPC containing 30% (mol/mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) or cholesterol, has been investigated by the isothermal titration calorimetry (ITC). This study confirms the association of these proteins to lipid bilayers, and provides a direct characterization of this exothermic process, at 37 degrees C. The measurements indicate that the protein affinity for lipid bilayers is modulated by the lipid composition, the lipid/protein ratio, and the temperature. The saturation lipid/protein ratio was increased in the presence of cholesterol and, to a lesser extent, of phosphatidylethanolamine, suggesting that it is modulated by the lipid acyl chain order. For all the investigated systems, the binding of BSP-A1/-A2 could not be modeled using a simple partitioning of the proteins between the aqueous and lipid phases. The existence of "binding sites", and lipid phase separations is discussed. The decrease of temperature, from 37 to 10 degrees C, converts the exothermic association of the proteins to the POPC bilayers to an endothermic process. A complementary 1-D and 2-D infrared spectroscopy study excludes the thermal denaturation of BSP-A1/-A2 as a contributor in the temperature dependence of the protein affinity for lipid bilayers. The reported findings suggest that changes in the affinity of BSP-A1/-A2 for lipid bilayers could be involved in modulating the association of these proteins to sperm membranes as a function of space and time; this would consequently modulate the extent of lipid extraction, including cholesterol, at a given place and given time.     

Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.