beta 2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor

The structures of acetylcholine-binding protein ( AChBP) and nicotinic acetylcholine receptor ( nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 alpha-conotoxins. Here, we used an alpha 3 beta 2 model to identify beta 2 subunit residues in this cleft and investigated their influence on the binding of alpha-conotoxins MII, PnIA, and GID to the alpha 3 beta 2 nAChR by two-electrode voltage clamp analysis. Although a beta 2-L119Q mutation strongly reduced the affinity of all three alpha-conotoxins, beta 2-F117A, beta 2-V109A, and beta 2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of alpha-conotoxins GID and MII was also observed when the beta 2-F117A mutant was combined with the alpha 4 instead of the alpha 3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to beta 2-F117A, beta 2-V109A, and beta 2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 alpha-conotoxin binding to the alpha 3 beta 2 model supported a direct interaction between mutated nAChR residues and alpha-conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the beta subunit contributes to alpha-conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by alpha-conotoxins to block the nAChR.

Tyrosine 394 is phosphorylated in Alzheimer's paired helical filament tau and in fetal tau with c-Abl as the candidate tyrosine kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimer's disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl) pyrazolo[3,4-d] pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimer's disease.

A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated p114a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. p114a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an R-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of p114a revealed a novel signal sequence, indicating that p114a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of p114a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, p114a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50) 1.59 mu M) and neuronal (IC50 = 8.7 mu M for alpha 3 beta 4) and neuromuscular (IC50 = 0.54 mu M for alpha 1 beta 1 is an element of delta) subtypes of the nicotinic acetylcholine receptor ( nAChR). Similarities in sequence and structure are apparent between the middle loop of p114a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

Secondary metabolites from the wood bark of Durio zibethinus and Durio kutejensis

Phytochemical exploration of a wood bark extract from Durio zibethinus afforded two new triterpenoids, namely, methyl 27-O-trans-caffeoylcylicodiscate (1) and methyl 27-O-cis-caffeoylcylicodiscate (2), a new phenolic, 1,2-diarylpropane-3- ol (3), and seven known compounds, fraxidin, eucryphin, boehmenan, threo-carolignan E, (-)-(3R, 4S)-4-hydroxymellein, methyl protocatechuate, and (+)-(R)-de-O-methyllasiodiplodin (4). In addition, chemical analysis of a wood bark extract from Durio kutejensis yielded the new triterpenes 3 beta-O-trans-caffeoyl-2R-hydroxyolean-12-en-28-oic acid (5) and 3 beta-trans-caffeoyl-2R-hydroxytaraxest-12-en-28-oic acid (6) together with four known compounds, maslinic acid, arjunolic acid, 2,6-dimethoxy-p-benzoquinone, and fraxidin. The structures of all compounds were determined on the basis of spectroscopic data.

Spongian Diterpenes from Australian nudibranchs: An anatomically guided chemical study of Glossodoris atromarginata

An Australian population of the nudibranch mollusk Glossodoris atromarginata has been found to contain furanoditerpenes of the spongian series. Spongia-13(16),14-dien-3-one (1) and 3 beta-acetoxy-19-hydroxyspongia-13(16),14-dien-2-one (2) were isolated for the first time from a natural source, along with a series of known diterpenes (3-7). Anatomical dissection of the animals revealed the relative distribution and chemical variation of secondary metabolites. Structural studies have provided a basis for chemical comparisons between populations from different geographic locations.

Structural revision of shatavarins I and IV, the major components from the roots of Asparagus racemosus

The two major steroidal saponins from the roots of Asparagus racemosus were isolated by RP-HPLC and their structure determined by extensive NMR studies. Their structures did not match those reported previously for shatavarins. I and IV and were found to be 3-O-{[beta-D-glueopyranosy](1 -> 2)][alpha-L-rhamnopyranosyl(1 -> 4)]-beta-D-glucopyranosyl}-26-O-(P-D-glu(opyranosyl)-(25S)5 beta-furostan-3p,22 alpha,26-triol and 3-O-{[beta-D-glueopyranosyl(1 -> 2)][alpha-L-rhamnopyranosyl(1 -> 4)]-beta-D-glucopyrariosyl}-(25S)-5 beta-spirostan-3 beta-ol. (c) 2006 Elsevier Ltd. All rights reserved.

Synthesis and characterization of Pd(II) and Pt(II) complexes with triazolopyrimidine derivatives: The crystal structure of [Pd2L2Cl4] where L=5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine

The Pd(II) and Pt(II) complexes with triazolopyrimidine C-nucleosides L-1 (5,7-dimethyl-3-(2',3',5'-tri-O-benzoyl-beta-D-ribofuranosyl-s-triazolo)[4,3-a]pyrimidine), L-2 (5,7-dimethyl-3-beta-D-ribofuranosyl-s-triazolo [4,3-a]pyrimidine) and L-3 (5,7-dimethyl[1,5-a]-s-triazolopyrimidine), [Pd(en)(L-1)](NO3)(2), (Pd(bpy)(L-1)](NO3)(2), cis-Pd(L-3)(2)Cl-2, [Pd-2(L-3)(2)Cl-4]center dot H2O, cis-Pd(L-2)(2)Cl-2 and [Pt-3(L-1)(2)Cl-6] were synthesized and characterized by elemental analysis and NMR spectroscopy. The structure of the [Pd-2(L-3)(2)Cl-4]center dot H2O complex was established by Xray crystallography. The two L-3 ligands are found in a head to tail orientation, with a (PdPd)-Pd-... distance of 3.1254(17) angstrom.L-1 coordinates to Pd(II) through N8 and N1 forming polymeric structures. L-2 coordinates to Pd(II) through N8 in acidic solutions (0.1 M HCl) forming complexes of cis-geometry. The Pd(II) coordination to L-2 does not affect the sugar conformation probably due to the high stability of the C-C glycoside bond. (c) 2006 Elsevier B.V. All rights reserved.

Asparinins, asparosides, curillins, curillosides and shavatarins: structural clarification with the isolation of shatavarin V, a new steroidal saponin from the root of Asparagus racemosus

A new steroidal saponin, shatavarin V, (3-O-{[alpha-L-rhamnopyranosy](1-2)][beta-D-glucopyranosyl(1 -> 4)]-beta-D-glucopyranosyl}-(25S)-5 beta-spirostan-3 beta-ol), was isolated from the roots of Asparagus racemosus by RP-HPLC, and its structure determined by 1D and 2D NMR studies. This data permits clarification of the structures reported for several known saponins: asparinins A and B; asparosides A and B; curillin H; curillosides G and H and shavatarins I and IV. (c) 2006 Elsevier Ltd. All rights reserved.

The origin of the difference in the superconducting critical temperatures of the beta(H) and beta(L) phases of (BEDT-TTF)(2)I-3

Incommensurate lattice fluctuations are present in the beta(L) phase (T-c similar to 1.5 K) of ET2I3 (where ET is BEDT-TTF - bis(ethylenedithio)tetrathiafulvalene) but are absent in the beta(H) phase (T-c similar to 7 K). We propose that the disorder in the conformational degrees of freedom of the terminal ethylene groups of the ET molecules, which is required to stabilise the lattice fluctuations, increases the quasiparticle scattering rate and that this leads to the observed difference in the Superconducting critical temperatures, T-c, of the two phases. We calculate the dependence of T-c on the interlayer residual resistivity. Our theory has no free parameters. Our predictions are shown to be consistent with experiment. We describe experiments to conclusively test our hypothesis.

Overexpressed Ca-v beta 3 inhibits N-type (Ca(v)2.2) calcium channel currents through a hyperpolarizing shift of "ultra-slow" and "closed-state" inactivation

It has been shown that P auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a hovel inhibitory effect of beta3 Subunit on macroscopic Ba2+ currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/ cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at physiological holding potentials (HPs) of -60 and -80 mV At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca,.2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced Current suppression was reversed at a HP of - 120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba-2divided by (40 mM) as a charge carrier also largely diminished the effect of P3 at -80 mV Therefore, experimental conditions (HP, divalent cation concentration, and P3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel P3 effect. Steady-state inactivation curves revealed that N-type channels exhibited closed-state inactivation without P3, and that P3 caused an similar to40 mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of P3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV Similar ultra-slow inactivation was observed for N-type channels Without P3. Thus, P3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by Causing a hyperpolarizing shift of the inactivation without affecting ultra-slow and closed-state inactivation properties.