A ferrocene functionalised macrocyclic receptor for cations and anions

The isolation and characterisation of a new macrocyclic hexaamine trans-6,13-bis(ferrocenylmethylamino)-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane (L-2) bearing two ferrocenyl groups appended to its exocyclic amines is reported. The crystal structures of L-2 and its dihydrochloride salt L-2. 2HCl . 2H(2)O have been determined. In the latter case cation-anion hydrogen bonding is observed in the solid state. Substrate binding by the electroactive L-2 in MeCN-CH2Cl2 solution has been examined by cyclic voltammetry and reveals the receptor electrochemically to recognise benzoate and chloride anions. The macrocyclic N-donors may also bind transition metal cations such as Cu-II and Zn-II.

The cystine knot motif in toxins and implications for drug design

The cystine knot structural motif is present in peptides and proteins from a variety of species, including fungi, plants, marine molluscs. insects and spiders. It comprises an embedded ring formed by two disulfide bonds and their connecting backbone segments which is threaded by a third disulfide bond. It is invariably associated with nearby beta-sheet structure and appears to be a highly efficient motif for structure stabilization. Because of this stability it makes an ideal framework for molecular engineering applications. In this review we summarize the main structural features of the cystine knot motif, focussing on toxin molecules containing either the inhibitor cystine knot or the cyclic cystine knot. Peptides containing these motifs are 26-48 residues long and include ion channel blockers, haemolytic agents, as well as molecules having antiviral and antibacterial activities. The stability of peptide toxins containing the cystine knot motif, their range of bioactivities and their unique structural scaffold can be harnessed for molecular engineering applications and in drug design. Applications of cystine knot molecules for the treatment of pain. and their potential use in antiviral and antibacterial applications are described. (C) 2000 Elsevier Science Ltd. All rights reserved.

Toxicity and uptake mechanism of cylindrospermopsin and lophyrotomin in primary rat hepatocytes

The toxicities and uptake mechanisms of two hepatotoxins, namely cylindrospermopsin and lophyrotomin, were investigated on primary rat hepatocytes by using microcystin-LIZ (a well-known hepatotoxin produced by cyanobacteria) as a comparison. Isolated rat hepatocytes were incubated with different concentrations of hepatotoxins for 0, 24, 48 and 72 h. The cell viability was assayed by the tetrazolium-based (MTT) assay. Microcystin-LR, cylindrospermopsin and lophyrotomin all exhibited toxic effects on the primary rat hepatocytes with 72-h LC50 of 8, 40 and 560 ng/ml, respectively. The involvement of the bile acid transport system in the hepatotoxin-induced toxicities was tested in the presence of two bile acids, cholate and taurocholate. Results showed that the bile acid transport system was responsible for the uptake, and facilitated the subsequent toxicities of lophyrotomin on hepatocytes. This occurred to a much lesser extent with cylindrospermopsin. With its smaller molecular weight, passive diffusion might be one of the possible mechanisms for cylindrospermopsin uptake into hepatocytes. This was supported by incubating a permanent cell line, KB (devoid of bile acid transport system), with cylindrospermopsin which showed cytotoxic effects. No inhibition of protein phosphatase 2A by cylindrospermopsin or lophyrotomin was found. This indicated that other toxic mechanisms besides protein phosphatase inhibition were producing the toxicities of cylindrospermopsin and lophyrotomin, and that they were unlikely to be potential tumor promoters. (C) 2001 Elsevier Science Ltd. All rights reserved.

Human Fatalities from Cyanobacteria: Chemical and Biological Evidence for Cyanotoxins

An outbreak of acute liver failure occurred at a dialysis center in Caruaru, Brazil (8 degrees 17 'S, 35 degrees 58 'W), 134 km from Recife, the state capital of Pernambuco. At the clinic, 116 (89%) of 131 patients experienced visual disturbances, nausea, and vomiting after routine hemodialysis treatment on 13-20 February 1996. Subsequently, 100 patients developed acute liver failure, and of these 76 died. As of December 1996, 52 of the deaths could be attributed to a common syndrome now called Caruaru syndrome. Examination of phytoplankton from the dialysis clinic's water source, analyses of the clinic's water treatment system, plus serum and liver tissue of clinic patients led to the identification of two groups of cyanobacterial toxins, the hepatotoxic cyclic peptide microcystins and the hepatotoxic alkaloid cylindrospermopsin. Comparison of victims' symptoms and pathology using animal studies of these two cyanotoxins leads us to conclude that the major contributing factor to death of the dialyses patients was intravenous exposure to microcystins, specifically microcystin-YR, -LR, and -AR. From liver concentrations and exposure volumes, it was estimated that 19.5 mug/L microcystin was in the water used for dialysis treatments. This is 19.5 times the level set as a guideline for safe drinking water supplies by the World. Health Organization.

Modulation of ligand selectivity by mutation of the first extracellular loop of the human C5a receptor

The cyclic C5a receptor antagonist, phenylalanine [L-ornithine-proline-D-cyclohexylalanine-tryptophan-arginine] (F-[OPchaWR]), has similar to 1000-fold less affinity for the C5a receptor (C5aR) on murine polymorphonuclear leukocytes than on human. Analysis of C5aR from different species shows that a possible cause of this difference is the variation in the sequence of the first extracellular loop of the receptor. The mouse receptor contains Y at a position analogous to P-103 in the human receptor, and D at G(105). To test this hypothesis, we expressed human C5aR mutants ((PY)-Y-103, G(105)D and the double mutant, (PY)-Y-103/G(105)D) in RBL-2H3 cells and investigated the effects of these mutations on binding affinity and receptor activation. All three mutant receptors had a higher affinity for human C5a than the wild-type receptor, but showed no significant difference in the ability of F-[OPchaWR] to inhibit human C5a binding. However, all of the mutant receptors had substantially lower affinities for the weak agonist, C5a des Arg(74) (C5adR(74)), and two altered receptors (G(105)D and (PY)-Y-103/G(105)D) had much lower affinities for the C-terminal C5a agonist peptide analogue, L-tyrosine-serine-phenylalanine-lysine-proline-methionine-proline-leucine-D-alanine-arginine (YSFKPMPLaR). Although it is unlikely that differences at these residues are responsible for variations in the potency of F-[OPchaWR] across species, residues in the first extracellular loop are clearly involved in the recognition of both C5a and C5a agonists. The complex effects of mutating these residues on the affinity and response to C5a, C5adR(74), and the peptide analogues provide evidence of different binding modes for these ligands on the C5aR. (C) 2001 Elsevier Science Inc. All rights reserved.

Species dependence for binding of small molecule agonist and antagonists to the C5a receptor on polymorphonuclear leukocytes

This study investigated the receptor binding affinities of a C5a agonist and cyclic antagonists for polymorphonuclear leukocytes (PMNs) isolated from human, sheep, pig, dog, rabbit, guinea pig, rat and mouse. The affinities of the two small molecule antagonists, F-[OPdChaWR] and AcF-[OPdChaWR], and the agonist, YSFKPMPLaR, revealed large differences in C5a receptor (C5aR) affinities between species. The antagonists bound to human, rat and dog PMNs with similar high affinities, but with lower affinities to PMNs from all other species. The C5a agonist also bound with varying affinities between species, but showed a different affinity profile to the antagonists. In contrast, recombinant human C5a had similar affinity for PMNs of all species investigated. The low correlation between the affinities of the antagonists and the agonist between species either suggests that different receptor residues are important for distinguishing between agonist/antagonist binding, or that the agonist and antagonist peptides bind to two distinct sites within the C5aR.

Synthesis and structural properties of patellamide a derivatives and their copper(II) compounds

The synthesis, characterization and copper(II) coordination chemistry of three new cyclic peptide ligands, PatJ(1) (cyclo-(Ile -Thr- (Gly)Thz-lle-Thr(Gly)Thz)), PatJ(2) (cyclo-(Ile-Thr(Gly)Thz-(D)-Ile-Thr-(Gly)Thz)), and PatL (cyclo-(Ile-Ser-(Gly)Thz-Ile-Ser(Gly)Thz)) are reported. All of these cyclic peptides and PatN (cyclo-(Ile-Ser(Gly)Thz-Ile-Thr-(Gly)Thz)) are derivatives of patellamide A and have a [24]azacrown-8 macrocyclic structure. All four synthetic cyclic peptides have two thiazole rings but, in contrast to patellamide A, no oxazoline rings. The molecular structure of PatJ1, determined by X-ray crystallography, has a saddle conformation with two close-to-co-parallel thiazole rings, very similar to the geometry of patellamide D. The two coordination sites of PatJ1 with thiazole-N and amide-N donors are each well preorganized for transition metal ion binding. The coordination of copper(II) was monitored by UV/Vis spectroscopy, and this reveals various (meta)stable mono- and dinuclear copper(II) complexes whose stoichiometry was confirmed by mass spectra. Two types of dinuclear copper(II) complexes, [Cu-2(H4L)(OH2)(n)](2+) (n = 6, 8) and [Cu-2(H4L)(OH2)(n)] (n=4, 6; L=PatN, PatL, PatJ1, PatJ2) have been identified and analyzed structurally by EPR spectroscopy and a combination of spectra simulations and molecular mechanics calculations (MM-EPR). The four structures are similar to each other and have a saddle conformation, that is, derived from the crystal structure of PatJ(1) by a twist of the two thiozole rings. The small but significant structural differences are characterized by the EPR simulations.

Medium ring ethers by ring expansion-ring contraction: Synthesis of lauthisan

[GRAPHICS] A new general method for the construction of medium ring ethers has been developed. This involves the ring expansion of halo-O,S-acetals followed by a Ramburg-Backlund ring contraction reaction with concomitant extrusion of the sulfur atom. This methodology has been utilized for the synthesis of cis- and trans-lauthisan.

The first non-turnover voltammetric response from a molybdenum enzyme: direct electroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

The first direct voltammetric response from a molybdenum enzyme under non-turnover conditions is reported. Cyclic voltammetry of dimethylsulfoxide reductase from Rhodobacter capsulatus reveals a reversible Mo-VI/V response at + 161 mV followed by a reversible Mo-V/IV response at -102 mV versus NHE at pH 8. The higher potential couple exhibits a pH dependence consistent with protonation upon reduction to the Mo-V state and we have determined the pK(a) for this semi-reduced species to be 9.0. The lower potential couple is pH independent within the range 5 < pH < 10. The optical spectrum of the Mo chromophore has been investigated with spectroelectrochemistry. At high potential, in its resting state, the enzyme exhibits a spectrum characteristic of the Mo-VI form. This changes significantly following bulk electrolysis (-400 mV versus NHE) at an optically transparent, indium-doped tin oxide working electrode, where a single visible electronic maximum at 632 nm is observed, which is comparable with spectra reported previously for the dithionite-reduced enzyme. This two-electron process is chemically reversible by reoxidizing the enzyme at the electrode in the absence of mediators or promoters. The activity of the enzyme has been established by observation of a catalytic current in the presence of DMSO at pH 8, where a sigmoidal (steady state) voltammogram is seen. Electronic supplementary material to this paper (Fig. S 1) can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0374-y.

Myocardial abnormalities in hypertensive patients with normal and abnormal left ventricular filling: a study of ultrasound tissue characterization and strain

Abnormal left ventricular (LV) filling is common, but not universal, in hypertensive LV hypertrophy (LVH). We sought to elucidate the relative contributions of myocardial structural changes, loading and hypertrophy to LV dysfunction in 113 patients: 85 with hypertensive LVH and 28 controls without LVH and with normal filling. Patients with normal dobutamine stress echocardiography and no history of coronary artery disease were selected, in order to exclude a contribution from ischaemia or scar. Abnormal LV filling was identified in 65 LVH patients, based on Doppler measurement of transmitral filling and annular velocities. All patients underwent grey-scale and colour tissue Doppler imaging from three apical views, which were stored and analysed off line. Integrated backscatter (113) and strain rate imaging were used to detect changes in structure and function; average cyclic variation of 113, strain rate and peak systolic strain were calculated by averaging each segment. Calibrated 113 intensity, corrected for pericardial 113 intensity, was measured in the septum and posterior wall from the parasternal long-axis view. Patients with LVH differed significantly from controls with respect to all backscatter and strain parameters, irrespective of the presence or absence of abnormal LV filling. LVH patients with and without abnormal LV filling differed with regard to age, LV mass and incidence of diabetes mellitus, but also showed significant differences in cyclic variation (P < 0.01), calibrated 113 in the posterior wall (P < 0.05) and strain rate (P < 0.01), although blood pressure, heart rate and LV systolic function were similar. Multivariate logistic regression analysis demonstrated that age, LV mass index and calibrated IB in the posterior wall were independent determinants of abnormal LV filling in patients with LVH. Thus structural and functional abnormalities can be detected in hypertensive patients with LVH with and without abnormal LV filling. In addition to age and LVH, structural (not functional) abnormalities are likely to contribute to abnormal LV filling, and may be an early sign of LV damage. 113 is useful for the detection of myocardial abnormalities in patients with hypertensive LVH.

Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa

Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasl::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604,2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket or Vfr. This allele (VfrDeltaEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrDeltaEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrDeltaEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa

It has been reported that mutations in the quorum-sensing genes lasI and rhlI in Pseudomonas aeruginosa result in, among many other things, loss of twitching motility (A. Glessner, R. S. Smith, B. H. Iglewski, and J. B. Robinson, J. Bacteriol. 181:1623-1629, 1999). We constructed knockouts of lasI and rhlI and the corresponding regulatory genes lasR and rhlR and found no effect on twitching motility. However, twitching-defective variants accumulated during culturing of lasI and rhlI mutants. Further analysis showed that the stable twitching-defective variants of lasI and rhlI mutants had arisen as a consequence of secondary mutations in vfr and algR, respectively, both of which encode key regulators affecting a variety of phenotypes, including twitching motility. In addition, when grown in shaking broth culture, lasI and rhlI mutants, but not the wild-type parent, also accumulated unstable variants that lacked both twitching motility and swimming motility and appeared to be identical in phenotype to the S1 and S2 variants that were recently reported to occur at high frequencies in P. aeruginosa strains grown as a biofilm or in static broth culture (E. Deziel, Y. Comeau, and R. Villemur, J. Bacteriol. 183:1195-1204, 2001). These results indicate that mutations in one regulatory system may create distortions that select during subsequent culturing for compensatory mutations in other regulatory genes within the cellular network. This problem may have compromised some past studies of regulatory hierarchies controlled by quorum sensing and of bacterial regulatory systems in general.

Discovery and structures of the cyclotides: novel macrocyclic peptides from plants

Circular disulfide-rich polypeptides were unknown a decade ago but over recent years a large family of such molecules has been discovered, which we now refer to as the cyclotides. They are typically about 30 amino acids in size, contain an N- to C-cyclised backbone and incorporate three disulfide bonds arranged in a cystine knot motif. In this motif, an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments is penetrated by the third disulfide bond. The combination of this knotted and strongly braced structure with a circular backbone renders the cyclotides impervious to enzymatic breakdown and makes them exceptionally stable. This article describes the discovery of the cyclotides in plants from the Rubiaceae and Violaceae families, their chemical synthesis, folding, structural characterisation, and biosynthetic origin. The cyclotides have a diverse range of biological applications, ranging from uterotonic action, to anti-HIV and neurotensin antagonism. Certain plants from which they are derived have a history of uses in native medicine, with activity being observed after oral ingestion of a tea made from the plants. This suggests the possibility that the cyclotides may be orally bioavailable. They therefore have a range of potential applications as a stable peptide framework.

The cyclotides: Novel macrocyclic peptides as scaffolds in drug design

Cyclotides are a novel class of circular, disulfide-rich peptides (similar to 30 amino acids) that display a broad range of bioactivities and have exceptionally high stability. Their physical properties, which include resistance to thermal and enzymatic degradation, can be attributed to their unique cyclic backbone and knotted arrangement of disulfide bonds. The applicability of linear peptides as drugs is potentially limited by their susceptibility to proteolytic cleavage and poor bioavailability. Such limitations may be overcome by using the cyclotide framework as a scaffold onto which new activities may be engineered. The potential use of cyclotides for drug design is evaluated here, with reference to rapidly increasing knowledge of natural cyclotides and the emergence of new techniques in peptide engineering.