Low-molecular-weight peptidic and cyclic antagonists of the receptor for the complement factor C5a

Activation of the human complement system of plasma proteins during immunological host defense can result in overproduction of potent proinflammatory peptides such as the anaphylatoxin C5a. Excessive levels of C5a are associated with numerous immunoinflammatory diseases, but there is as yet no clinically available antagonist to regulate the effects of C5a. We now describe a series of small molecules derived from the C-terminus of C5a, some of which are the most potent low-molecular-weight C5a receptor antagonists reported to date for the human polymorphonuclear leukocyte (PMN) C5a receptor. H-1 NMR spectroscopy was used to determine solution structures for two cyclic antagonists and to indicate that antagonism is related to a turn conformation, which can be stabilized in cyclic molecules that are preorganized for receptor binding. While several cyclic derivatives were of similar antagonistic potency, the most potent antagonist was a hexapeptide-derived macrocycle AcF[OPdChaWR] with an IC50 = 20 nM against a maximal concentration of C5a (100 nM) on intact human PMNs. Such potent C5a antagonists may be useful probes to investigate the role of C5a in host defenses and to develop therapeutic agents for the treatment of many currently intractable inflammatory conditions.

Chemical synthesis and folding pathways of large cyclic polypeptide: Studies of the cystine knot polypeptide kalata B1

Kalata B1 is a member of a new family of polypeptides, isolated from. plants, which have a cystine knot structure embedded within an amide-cyclized backbone. This family of molecules are the largest known cyclic peptides, and thus, the mechanism of synthesis and folding is of great interest. To provide information about both these phenomena, we have synthesized kalata B1 using two distinct strategies. In the first, oxidation of the cysteine residues of a linear precursor peptide to form the correct disulfide bonds results in folding of the three-dimensional structure and preorganization of the termini in close proximity for subsequent cyclization. The second approach involved cyclization prior to oxidation. In the first method, the correctly folded peptide was produced only in the presence of partially hydrophobic solvent conditions. These conditions are presumably required to stabilize the surface-exposed hydrophobic residues. However,; in the synthesis,involving cyclization prior to oxidation, the cyclic reduced peptide folded to a significant degree in the absence of hydrophobic solvents and even more efficiently in the presence of hydrophobic solvents. Cyclization clearly has a major effect on the folding pathway and facilitates formation of the correctly disulfide-bonded form in aqueous solution; In addition to facilitating folding to a compact stable structure cyclization has an important effect on biological activity as assessed by hemolytic activity.

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using H-1-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.

Ontological changes in the crystallin composition of the eye lenses of the territorial damselfish Parma microlepis and their possible effects on trace-metal accumulation

The eye lenses of Parma microlepis from the rocky barrens of Sydney (New South Wales, Australia) were found to contain Ba, Hg, Rb, and Sr at concentrations above the quantitative detection limits of solution-based inductively-coupled plasma-mass spectrometry (ICP-MS). Lenses were separated into the hard central nucleus and the softer surrounding cortex. Nuclei contained lower (equal for Ba) concentrations of these metals. Biochemical analysis of the protein composition of these lenses revealed differences in the ratio of gamma-crystallin to beta-crystallin in the lens nucleus and cortex. These changes were shown to be attributable both to protein degradation and changes in protein synthesis as fish age. Such changes may lead to the loss of sequestered metals from older cell layers, or change the affinity of new layers for particular trace metals. Differential binding affinities of these crystallins may, therefore, partially account for trace-metal differences observed in the lens nucleus and cortex.

Increasing the efficiency of solute leaching: impacts of flow interruption with drainage of the "preferential flow paths"

Most soils contain preferential flow paths that can impact on solute mobility. Solutes can move rapidly down the preferential flow paths with high pore-water velocities, but can be held in the less permeable region of the soil matrix with low pore-water velocities, thereby reducing the efficiency of leaching. In this study, we conducted leaching experiments with interruption of the flow and drainage of the main flow paths to assess the efficiency of this type of leaching. We compared our experimental results to a simple analytical model, which predicts the influence of the variations in concentration gradients within a single spherical aggregate (SSA) surrounded by preferential flow paths on leaching. We used large (length: 300 mm, diameter: 216 mm) undisturbed field soil cores from two contrasting soil types. To carry out intermittent leaching experiments, the field soil cores were first saturated with tracer solution (CaBr2), and background solution (CaCl2) was applied to mimic a leaching event. The cores were then drained at 25- to 30-cm suction to empty the main flow paths to mimic a dry period during which solutes could redistribute within the undrained region. We also conducted continuous leaching experiments to assess the impact of the dry periods on the efficiency of leaching. The flow interruptions with drainage enhanced leaching by 10-20% for our soils, which was consistent with the model's prediction, given an optimised equivalent aggregate radius for each soil. This parameter quantifies the time scales that characterise diffusion within the undrained region of the soil, and allows us to calculate the duration of the leaching events and interruption periods that would lead to more efficient leaching. Application of these methodologies will aid development of strategies for improving management of chemicals in soils, needed in managing salts in soils, in improving fertiliser efficiency, and in reclaiming contaminated soils. (C) 2000 Elsevier Science B.V. All rights reserved.

Role of the crystallite phase of TiO2 in heterogeneous photocatalysis for phenol oxidation in water

A series of TiO2 samples with different anatase-to-rutile ratios was prepared by calcination, and the roles of the two crystallite phases of titanium(IV) oxide (TiO2) on the photocatalytic activity in oxidation of phenol in aqueous solution were studied. High dispersion of nanometer-sized anatase in the silica matrix and the possible bonding of Si-O-Ti in SiO2/TiO2 interface were found to stabilize the crystallite transformation from anatase to rutile. The temperature for this transformation was 1200 degrees C for the silica-titania (ST) sample, much higher than 700 degrees C for Degussa P25, a benchmarking photocatalyst. It is shown that samples with higher anatase-to-rutile ratios have higher activities for phenol degradation. However, the activity did not totally disappear after a complete crystallite transformation for P25 samples, indicating some activity of the rutile phase. Furthermore, the activity for the ST samples after calcination decreased significantly, even though the amount of anatase did not change much. The activity of the same samples with different anatase-to-rutile ratios is more related to the amount of the surface-adsorbed water and hydroxyl groups and surface area. The formation of rutile by calcination would reduce the surface-adsorbed water and hydroxyl groups and surface area, leading to the decrease in activity.

Synthesis and complexes of the sexidentate macrocycle 15-methyl-1,4,7,10,13-pentaazacyclohexadecan-15-amine

The potentially sexidentate polyamine macrocycle 15-methyl-1,4,7,10,13-pentaazacyclohexadecan-15-amine (1) was prepared via a copper(II)-templated route from 3,6,9-triazaundecan-1,ll-diamine, formaldehyde and nitroethane which first formed the copper(II) complex of the macrocycle 15-methyl-15-nitro-1,4,7,10,13-pentaazacyclohexadecane (2), reduced subsequently with zinc and aqueous acid to yield 1. The hexaamine 1, with five secondary amine groups in the macrocyclic ring and one pendant primary amine group, forms inert sexidentate octahedral complexes with cobalt(III), chromium(III) and iron(III). An X-ray structure of [Co(1)](ClO4)(3) defines the distorted octahedron of the complex cation and shows it is a symmetrical isomer with all nitrogens bound and the central aza group trans to the pendant primary amine group. The [M(1)](3+) ions are all stable indefinitely in aqueous solution and exhibit spectra consistent with MN6 d(3) (Cr), low-spin d(5) (Fe) and low-spin d(6) (Co) electronic ground states. For each complex, a reversible M(III/II) redox couple is observed. (C) 2000 Elsevier Science S.A. All rights reserved.

A two-dimensional analytical solution of groundwater responses to tidal loading in an estuary and ocean

Previous studies on tidal dynamics of coastal aquifers have focussed on the inland propagation of oceanic tides in the cross-shore direction, a configuration that is essentially one-dimensional. Aquifers at natural coasts can also be influenced by tidal waves in nearby estuaries, resulting in a more complex behaviour of head fluctuations in the aquifers. We present an analytical solution to the two-dimensional depth-averaged groundwater flow equation for a semi-infinite aquifer subject to oscillating head conditions at the boundaries. The solution describes the tidal dynamics of a coastal aquifer that is adjacent to a cross-shore estuary. Both the effects of oceanic and estuarine tides on the aquifer are included in the solution. The analytical prediction of the head fluctuations is verified by comparison with numerical solutions computed using a standard finite-difference method. An essential feature of the present analytical solution is the interaction between the cross- and along-shore tidal waves in the aquifer area near the estuary's entry. As the distance from the estuary or coastline increases, the wave interaction is weakened and the aquifer response is reduced, respectively, to the one-dimensional solution for oceanic tides or the solution of Sun (Sun H. A two-dimensional analytical solution of groundwater response to tidal loading in an estuary, Water Resour Res 1997;33:1429-35) for two-dimensional non-interacting tidal waves. (C) 2000 Elsevier Science Ltd. All rights reserved.

Solution of a two-leg spin ladder system

A model for a spin-1/2 ladder system with two legs is introduced. It is demonstrated that this model is solvable via the Bethe ansatz method for arbitrary values of the rung coupling J. This is achieved by a suitable mapping from the Hubbard model with appropriate twisted boundary conditions. We determine that a phase transition between gapped and gapless spin excitations occurs at the critical value J(c) = 1/2 of the rung coupling.

Characterization of the acyl carrier protein gene and the fab gene locus in Xanthomonas albilineans

A genomic region containing the fatty acid biosynthetic (fab) genes was isolated from the sugarcane leaf-scald pathogen Xanthomonasalbilineans. The order and predicted products of fabG (beta -ketoacyl reductase), acpP (acyl carrier protein), fabF(ketoacyl synthase II) and downstream genes in X. albilineans are very similar to those in Escherichia coli, with one exception. Sequence analysis, confirmed by insertional knockout and specific substrate feeding experiments, shows that the position occupied by pabC (encoding aminodeoxychorismate lyase) in other bacteria is occupied instead by pabB (encoding aminodeoxychorismate synthase component I) in X. albilineans. Downstream of pabB, X. albilineans resumes the arrangement common to characterized Gram-negative bacteria, with three transcriptionally coupled genes, encoding an ORF340 protein of undefined function, thymidylate kinase and delta' subunit of DNA polymerase III holoenzyme (HolB). Different species may obtain a common advantage from coordinated regulation of the same biosynthetic pathways using different genes in this region. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Functional analysis, using in vitro mutagenesis, of amino acids located in the phenylalanine hydroxylase active site

The 3-dimensionaI structure determination of rat phenylalanine hydroxylase (PAH) has identified potentially important amino acids lining the active site cleft with the majority of these having hydrophobic side-chains including several with aromatic side chains. Here we have analyzed the effect on rat PAH enzyme kinetics of in vitro mutagenesis of a number of these amino acids lining the PAH active site. Mutation of F299, Y324, F331, and Y343 caused a significant decrease in enzyme activity but no change in the K-m for substrate or cofactor. me conclude that these aromatic residues are essential for activity but are not significantly involved in binding of the substrate or cofactor. in contrast the PAH mutant, S349T, showed an 18-fold increase in K-m for phenylalanine, showing the first functional evidence that this residue was binding at or near the phenylalanine binding site. This confirms the recently published model for the binding of phenylalanine to the PAH active site that postulated S349 interacts with the amino group on the main chain of the phenylalanine molecule. This result differs with that found for the equivalent mutation (S395T), in the closely related tyrosine hydroxylase, which had no effect on substrate K-m, showing that while the architecture of the two active sites are very similar the amino acids that bind to the respective substrates are different. (C) 2000 Academic Press.

Transfer matrix eigenvalues of the anisotropic multiparametric U model

A multiparametric extension of the anisotropic U model is discussed which maintains integrability. The R-matrix solving the Yang-Baxter equation is obtained through a twisting construction applied to the underlying U-q(sl (2/1)) superalgebraic structure which introduces the additional free parameters that arise in the model. Three forms of Bethe ansatz solution for the transfer matrix eigenvalues are given which we show to be equivalent.

Analytical solution to the zero-inertia problem for surge flow phenomena in nonprismatic channels

Surge flow phenomena. e.g.. as a consequence of a dam failure or a flash flood, represent free boundary problems. ne extending computational domain together with the discontinuities involved renders their numerical solution a cumbersome procedure. This contribution proposes an analytical solution to the problem, It is based on the slightly modified zero-inertia (ZI) differential equations for nonprismatic channels and uses exclusively physical parameters. Employing the concept of a momentum-representative cross section of the moving water body together with a specific relationship for describing the cross sectional geometry leads, after considerable mathematical calculus. to the analytical solution. The hydrodynamic analytical model is free of numerical troubles, easy to run, computationally efficient. and fully satisfies the law of volume conservation. In a first test series, the hydrodynamic analytical ZI model compares very favorably with a full hydrodynamic numerical model in respect to published results of surge flow simulations in different types of prismatic channels. In order to extend these considerations to natural rivers, the accuracy of the analytical model in describing an irregular cross section is investigated and tested successfully. A sensitivity and error analysis reveals the important impact of the hydraulic radius on the velocity of the surge, and this underlines the importance of an adequate description of the topography, The new approach is finally applied to simulate a surge propagating down the irregularly shaped Isar Valley in the Bavarian Alps after a hypothetical dam failure. The straightforward and fully stable computation of the flood hydrograph along the Isar Valley clearly reflects the impact of the strongly varying topographic characteristics on the How phenomenon. Apart from treating surge flow phenomena as a whole, the analytical solution also offers a rigorous alternative to both (a) the approximate Whitham solution, for generating initial values, and (b) the rough volume balance techniques used to model the wave tip in numerical surge flow computations.

Flexibility revealed by the 1.85 angstrom crystal structure of the beta sliding-clamp subunit of Escherichia coli DNA polymerase III

The beta subunit of the Escherichia coli replicative DNA polymerase III holoenzyme is the sliding clamp that interacts with the alpha (polymerase) subunit to maintain the high processivity of the enzyme. The beta protein is a ring-shaped dimer of 40.6 kDa subunits whose structure has previously been determined at a resolution of 2.5 Angstrom [Kong et al. (1992), Cell, 69, 425-437]. Here, the construction of a new plasmid that directs overproduction of beta to very high levels and a simple procedure for large-scale purification of the protein are described. Crystals grown under slightly modified conditions diffracted to beyond 1.9 Angstrom at 100 K at a synchrotron source. The structure of the beta dimer solved at 1.85 Angstrom resolution shows some differences from that reported previously. In particular, it was possible at this resolution to identify residues that differed in position between the two subunits in the unit cell; side chains of these and some other residues were found to occupy alternate conformations. This suggests that these residues are likely to be relatively mobile in solution. Some implications of this flexibility for the function of beta are discussed.