Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity

Cyclotides are a recently discovered family of disulfide rich proteins from plants that contain a circular protein backbone. They are exceptionally stable, as exemplified by their use in native medicine of the prototypic cyclotide kalata B1. The peptide retains uterotonic activity after the plant from which it is derived is boiled to make a medicinal tea. The circular backbone is thought to be in part responsible for the stability of the cyclotides, and to investigate its role in determining structure and biological activity, an acyclic derivative, des-(24-28)-kalata B1, was chemically synthesized and purified. This derivative has five residues removed from the 29-amino acid circular backbone of kalata B1 in a loop region corresponding to a processing site in the biosynthetic precursor protein. Two-dimensional NMR spectra of the peptide were recorded, assigned, and used to identify a series of distance, angle, and hydrogen bonding restraints. These were in turn used to determine a representative family of solution structures. Of particular interest was a determination of the structural similarities and differences between des-(2428)-kalata B1 and native kalata B1. Although the overall three-dimensional fold remains very similar to that of the native circular protein, removal of residues 24-28 of kalata B1 causes disruption of some structural features that are important to the overall stability. Furthermore, loss of hemolytic activity is associated with backbone truncation and linearization.

Characterisation of alkaloids from some Australian Stephania (Menispermaceae) species

Chemical investigations of some Stephania species native to Australia and reportedly employed by Aboriginal people as therapeutic agents. are described. The alkaloids from the forest vines Stephania bancroftii F.M. Bailey and S. aculeata F.M. Bailey (Menispermaceae) have been isolated and characterised. The major alkaloids in the tuber of the former species are (-)-tetra-hydropalmatine and (-)-stephanine, whereas these are minor components in the leaves, from which a C-7 hydroxylated aporphine has been identified. The major tuber alkaloids in S. aculcata are (+)-laudanidine, and the morphinoid, (-)-amurine, whose absolute stereochemistry has been established by X-ray structural analysis of the methiodide derivative. No significant levels of alkaloids were detected in S. japonica. Complete and unambiguous H-1 and C-13 NMR data are presented for these alkaloids. (C) 2003 Elsevier Ltd. All rights reserved.

On the induced electric field gradients in the human body for magnetic stimulation by gradient coils in MRI

Prior theoretical studies indicate that the negative spatial derivative of the electric field induced by magnetic stimulation may he one of the main factors contributing to depolarization of the nerve fiber. This paper studies this parameter for peripheral nerve stimulation (PNS) induced by time.-varying gradient fields during MRI scans. The numerical calculations are based on an efficient, quasi-static, finite-difference scheme and an anatomically realistic human, full-body model. Whole-body cylindrical and planar gradient sets in MRI systems and various input signals have been explored. The spatial distributions of the induced electric field and their gradients are calculated and attempts are made to correlate these areas with reported experimental stimulation data. The induced electrical field pattern is similar for both the planar coils and cylindrical coils. This study provides some insight into the spatial characteristics of the induced field gradients for PNS in MRI, which may be used to further evaluate the sites where magnetic stimulation is likely to occur and to optimize gradient coil design.

alpha-conotoxins PnIA and [A10L] PnIA stabilize different states of the alpha 7-L247T nicotinic acetylcholine receptor

The effects of the native alpha-conotoxin PnIA, its synthetic derivative [ A10L] PnIA and alanine scan derivatives of [ A10L] PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L] PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nM, respectively. Rates of onset of inhibition were similar for PnIA and [ A10L] PnIA; however, the rate of recovery was slower for [ A10L] PnIA, indicating that the increased potency of [ A10L] PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [ A10L] PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [ A10L] PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nM. In contrast, [ A10L] PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [ A10L] PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [ A10L] PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.

Cytotoxic iron chelators: Characterization of the structure, solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

Di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and a range of its analogues comprise a series of monobasic acids that are capable of binding iron (Fe) as tridentate (N,N,O) ligands. Recently, we have shown that these chelators are highly cytotoxic, but show selective activity against cancer cells. Particularly interesting was the fact that cytotoxicity of the HPKIH analogues is maintained even after complexation with Fe. To understand the potent anti-tumor activity of these compounds, we have fully characterized their chemical properties. This included examination of the solution chemistry and X-ray crystal structures of both the ligands and Fe complexes from this class and the ability of these complexes to mediate redox reactions. Potentiometric titrations demonstrated that all chelators are present predominantly in their charge-neutral form at physiological pH (7.4), allowing access across biological membranes. Keto-enol tautomerism of the ligands was identified, with the tautomers exhibiting distinctly different protonation constants. Interestingly, the chelators form low-spin (diamagnetic) divalent Fe complexes in solution. The chelators form distorted octahedral complexes with Fe-II, with two tridentate ligands arranged in a meridional fashion. Electrochemistry of the Fe complexes in both aqueous and non-aqueous solutions revealed that the complexes are oxidized to their ferric form at relatively high potentials, but this oxidation is coupled to a rapid reaction with water to form a hydrated (carbinolamine) derivative, leading to irreversible electrochemistry. The Fe complexes of the HPKIH analogues caused marked DNA degradation in the presence of hydrogen peroxide. This observation confirms that Fe complexes from the HPKIH series mediate Fenton chemistry and do not repel DNA. Collectively, studies on the solution chemistry and structure of these HPKIH analogues indicate that they can bind cellular Fe and enhance its redox activity, resulting in oxidative damage to vital biomolecules.

Bridgehead arylation: A direct route to advanced intermediates for the synthesis of C-20 diterpene alkaloids

[GRAPHICS] Rapid access to the ABCE ring system of the C-20 diterpene alkaloids was achieved by silver(I)-promoted intramolecular Friedel-Crafts arylation of a functional group-specific 5-bromo-3-azabicyclo[3.3.1]nonane derivative.