Small molecular probes for G-protein-coupled C5a receptors: Conformationally constrained antagonists derived from the C terminus of the human plasma protein C5a

Activation of the human complement system of plasma proteins in response to infection or injury produces a 4-helix bundle glycoprotein (74 amino acids) known as C5a. C5a binds to G-protein-coupled receptors on cell surfaces triggering receptor-ligand internalization, signal transduction, and powerful inflammatory responses. Since excessive levels of C5a are associated with autoimmune and chronic inflammatory disorders, inhibitors of receptor activation may have therapeutic potential. We now report solution structures and receptor-binding and antagonist activities for some of the first small molecule antagonists of C5a derived from its hexapeptide C terminus. The antagonist NMe-Phe-Lys-Pro-D-Cha-Trp-D-Arg-CO2H (1) surprisingly shows an unusually well-defined solution structure as determined by H-1 NMR spectroscopy. This is one of the smallest acyclic peptides found to possess a defined solution conformation, which can be explained by the constraining role of intramolecular hydrogen bonding. NOE and coupling constant data, slow deuterium exchange, and a low dependence on temperature for the chemical shift of the D-Cha-NH strongly indicate an inverse gamma turn stabilized by a D-Cha-NH ... OC-Lys hydrogen bond. Smaller conformational populations are associated with a hydrogen bond between Trp-NH ... OC-Lys, defining a type II beta turn distorted by the inverse gamma turn incorporated within it. An excellent correlation between receptor-affinity and antagonist activity is indicated for a limited set of synthetic peptides. Conversion of the C-terminal carboxylate of 1 to an amide decreases antagonist potency 5-fold, but potency is increased up to 10-fold over 1 if the amide bond is made between the C-terminal carboxylate and a Lys/Orn side chain to form a cyclic analogue. The solution structure of cycle 6 also shows gamma and beta turns; however, the latter occurs in a different position, and there are clear conformational changes in 6 vs 1 that result in enhanced activity. These results indicate that potent C5a antagonists can be developed by targeting site 2 alone of the C5a receptor and define a novel pharmacophore for developing powerful receptor probes or drug candidates.

An investigation of localised soil heterogeneities on solute transport using a multisegment percolation system

A multisegment percolation system (MSPS) consisting of 25 individual collection wells was constructed to study the effects of localised soil heterogeneities on the transport of solutes in the vadose zone. In particular, this paper discusses the transport of water and nutrients (NO3-, Cl-, PO43-) through structurally stable, free-draining agricultural soil from Victoria, Australia. A solution of nutrients was irrigated onto the surface of a large undisturbed soil core over a 12-h period. This was followed by a continuous irrigation of distilled water at a fate which did not cause pending for a further 18 days. During this time, the volume of leachate and the concentration of nutrients in the leachate of each well were measured. Very significant variation in drainage patterns across a small spatial scale was observed. Leaching of nitrate-nitrogen and chloride from the core occurred two days after initial application. However, less than 1% of the total applied phosphate-phosphorus leached from the soil during the 18-day experiment, indicating strong adsorption. Our experiments indicate considerable heterogeneity in water flow patterns and solute leaching on a small spatial scale. These results have significant ramifications for modelling solute transport and predicting nutrient loadings on a larger scale.

Quantum open-systems approach to current noise in resonant tunneling junctions

A quantum Markovian master equation is derived to describe the current noise in resonant tunneling devices. This equation includes both incoherent and coherent quantum tunneling processes. We show how to obtain the population master equation by adiabatic elimination of quantum coherences in the presence of elastic scattering. We calculate the noise spectrum for a double well device and predict subshot noise statistics for strong tunneling between the wells. The method is an alternative to Green's function methods and population master equations for very small coherently coupled quantum dots.

Isolation of a cDNA for an octopamine-like, G-protein coupled receptor from the cattle tick, Boophilus microplus

Octopamine is a biogenic amine neurotransmitter of invertebrates that binds to a G-protein coupled receptor that has seven transmembrane domains. Formamidine pesticides like amitraz are highly specific agonists of the octopamine receptor. Amitraz is used extensively to control the cattle tick, Boophilus microplus, and many other ticks but now there are strains of ticks that are resistant to amitraz. We have isolated a cDNA from the cattle tick, B. miciroplus, that belongs to the biogenic amine family of receptors. The predicted amino acid sequence from this cDNA is most similar to octopamine receptors from insects. The nucleotide sequence of this gene from amitraz-resistant and amitraz-susceptible cattle ticks was identical. Thus, a point mutation/s did not confer resistance to amitraz in the strains we studied. Alternative explanations for resistance to amitraz in B. microplus are discussed. (C) 1999 Elsevier Science Ltd. All rights reserved.

Both beta(2)- and beta(1)-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of fallot infants, consistent with selective coupling of beta(2)-adrenergic receptors to G(s)-protein

Background-In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. Methods and Results-Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 2-1/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC50 [mol/L] NE 5.5+/-0.1, n=12; -EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC50 [mol/L] NE 5.8+/--0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC50 [mol/L] NE 5.7+/-0.2: EPI 5.8+/-0.1), Ventricular membranes from Fallot infants, labeled with (-)-[I-125]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. Conclusions-Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.

Mirabilin G: A new alkaloid from a southern Australian marine sponge, Clathria species

A Clathria sp. collected during scientific trawling operations in the Great Australian Bight, Australia, has yielded the new alkaloid mirabilin G (1). A structure was secured for 1 by detailed spectroscopic analysis and comparison to known marine alkaloids.

Modelling solute transport in structured soils: Performance evaluation of the ADR and TRM models

The movement of chemicals through the soil to the groundwater or discharged to surface waters represents a degradation of these resources. In many cases, serious human and stock health implications are associated with this form of pollution. The chemicals of interest include nutrients, pesticides, salts, and industrial wastes. Recent studies have shown that current models and methods do not adequately describe the leaching of nutrients through soil, often underestimating the risk of groundwater contamination by surface-applied chemicals, and overestimating the concentration of resident solutes. This inaccuracy results primarily from ignoring soil structure and nonequilibrium between soil constituents, water, and solutes. A multiple sample percolation system (MSPS), consisting of 25 individual collection wells, was constructed to study the effects of localized soil heterogeneities on the transport of nutrients (NO3-, Cl-, PO43-) in the vadose zone of an agricultural soil predominantly dominated by clay. Very significant variations in drainage patterns across a small spatial scale were observed tone-way ANOVA, p < 0.001) indicating considerable heterogeneity in water flow patterns and nutrient leaching. Using data collected from the multiple sample percolation experiments, this paper compares the performance of two mathematical models for predicting solute transport, the advective-dispersion model with a reaction term (ADR), and a two-region preferential flow model (TRM) suitable for modelling nonequilibrium transport. These results have implications for modelling solute transport and predicting nutrient loading on a larger scale. (C) 2001 Elsevier Science Ltd. All rights reserved.

Scorpidotrema longistipes n. g., n. sp. (Digenea: Opecoelidae) from Scorpis georgiana (Teleostei: Scorpididae) from southern Western Australia

Scorpidotrema longistipes n. g., n. sp. is described from the intestine of Scorpis georgiana Valenciennes (Scorpididae) from off Point Peron, Western Australia. The new genus is distinguished by the combination of a remarkably long and retractable ventral sucker peduncle, a possible uroproct, well-developed cirrus-sac and a uterine seminal receptacle. The subfamilial relationships of the new genus are troublesome. It incorporates features of the Opecoelinae, Stenakrinae and Plagioporinae. The absence of a canalicular seminal receptacle suggests a relationship with the Opecoelinae and Stenakrinae, whereas the well-developed cirrus-sac suggests a relationship with the Plagioporinae and Stenakrinae. The overall arrangement of the gonads is not similar to that of existing genera of Stenakrinae. It is concluded that the genus is best placed in the Stenakrinae although that subfamily may now be an artificial assemblage. This new genus forms part of a distinctive fauna of trematodes restricted to Australian southern temperate fishes.

Oxygen transport capacity in the air-breathing fish, Megalops cyprinoides: compensations for strenuous exercise

Tarpon have high resting or routine hematocrits (Hct) (37.6+/-3.4%) and hemoglobin concentrations (120.6+/-7.3 g 1(-1)) that increased significantly following bouts of angling-induced exercise (51.9+/-3.7% and 142.8+/-13.5 g 1(-1), respectively). Strenuous exercise was accompanied by an approximately tenfold increase in blood lactate and a muscle metabolite profile indicative of a high energy demand teleost. Routine blood values were quickly restored only when this facultative air-breathing fish was given access to atmospheric air. In vitro studies of oxygen transport capacity, a function of carrying capacity and viscosity, revealed that the optimal Hct range corresponded to that observed in fish under routine behaviour. During strenuous exercise however, further increase in viscosity was largely offset by a pronounced reduction in the shear-dependence of blood which conformed closely to an ideal Newtonian fluid. The mechanism for this behaviour of the erythrocytes appears to involve the activation of surface adrenergic receptors because pre-treatment with propranolol abolished the response. High levels of activity in tarpon living in hypoxic habitats are therefore supported by an elevated Hct with adrenergically mediated viscosity reduction, and air-breathing behaviour that enables rapid metabolic recovery. (C) 2002 Elsevier Science Inc. All rights reserved.

Ion and urea regulation in elasmobranch fish

In recent years our understanding of the control of ion and urea metabolism in elasmobranch fish has increased with many more species being investigated. This has demonstrated that many species regarded as stenohaline marine are at least, partially euryhaline and may survive in environments less concentrated than full seawater. This presentation will review these recent findings and then compare the osmoregulatory strategies of a partially euryhaline species, Scyliorhinus canicula, with a fully euryhaline migratory species Carcharinus leucas. This will include new data for both species and will generate new models for the control of ion and urea metabolism in elasmobranchs on which to base future research.

Improved VDC casting of aluminium alloys through an understanding of the surface properties of the molten metal

Vertical direct chill (VDC) casting of aluminium alloys is a mature process that has evolved over many decades through gradual change to both equipment design and casting practice. Today, air-pressurised, continuous lubrication, hot top mould systems with advanced station automation are selected as the process of choice for producing extrusion billet. Specific sets of operating parameters are employed on these stations for each alloy and size combination to produce optimal billet quality. The designs and parameters are largely derived from past experience and accumulated know-how. Recent experimental work at the University of Queensland has concentrated on understanding the way in which the surface properties of liquid aluminium alloys, e.g., surface tension, wetting angle and oxide skin strength, influence the size and shape of the naturally-stab le meniscus for a given alloy, temperature and atmosphere. The wide range of alloy-and condition-dependent values measured has led to the consideration of how these properties impact the stability of the enforced molten metal meniscus within the hot top mould cavity. The actual shape and position of the enforced meniscus is controlled by parameters such as the upstream conduction distance (UCD) from sub-mould cooling and the molten metal head. The degree of deviation of this actual meniscus from the predicted stable meniscus is considered to be a key driver in surface defect formation. This paper reports on liquid alloy property results and proposes how this knowledge might be used to better design VDC mould systems and casting practices.

Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

Determination of the solution structures of conantokin-G and conantokin-T by CD and NMR spectroscopy

Conantokin-G and conantokin-T are two paralytic polypeptide toxins originally isolated from the venom of the fish-hunting cone snails of the genus Conus. Conantokin-G and conantokin-T are the only naturally occurring peptidic compounds which possess N-methyl-D-aspartate receptor antagonist activity, produced by a selective non-competitive antagonism of polyamine responses, They are also structurally unusual in that they contain a disproportionately large number of acid labile post-translational gamma-carboxyglutamic acid (Gla) residues, Although no precise structural information has previously been published for these peptides, early spectroscopic measurements have indicated that both conantokin-G and conantokin-T form alpha-helical structures, although there is some debate whether the presence of calcium ions is required for these peptides to adopt this fold, We now report a detailed structural study of synthetic conantokin-G and conantokin-T in a range of solution conditions using CD and H-1 NMR spec troscopy. The three-dimensional structures of conantokin-T and conantokin-G were calculated from H-1 NMR-derived distance and dihedral restraints. Both conantokins were found to contain a mixture of alpha- and 3(10) helix, that give rise to curved and straight helical conformers. Conantokin-G requires the presence of divalent cations (Zn2+, Ca2+, Cu2+, Or Mg2+) to form a stable iv-helix, while conantokin-T adopts a stable alpha-helical structure in aqueous conditions, in the presence or absence of divalent cations (Zn2+, Ca2+, Cu2+, Or Mg2+).