Torpedo Californica Research Articles

Mutation in acetylcholinesterase1 associated with triazophos resistance in rice stem borer, Chilo suppressalis (Lepidoptera: Pyralidae)

Two full-length genes encoding different acetylcholinesterases (AChEs), designated as Ch-ace1 and Ch-ace2, were cloned from strains of the rice stem borer ( Chilo suppressalis) susceptible and resistant to the organophosphate insecticide triazophos. Sequence analysis found an amino acid mutation A314S in Ch-ace1 (corresponding to A201 in Torpedo californica AChE) that was consistently associated with the occurrence of resistance. This mutation removed an MspA1 I restriction site from the wild type allele. An assay based on restriction fragment length polymorphism (RFLP) analysis was developed to diagnose A314S genotypes in field populations. Results showed a strong correlation between frequencies of the mutation and phenotypic levels of resistance to triazophos. The assay offers a prospect for rapid monitoring of resistance and assisting with the appropriate choice of insecticide for combating damage caused by C. suppressalis.

Flexibility of Aromatic Residues in the Active-Site Gorge of Acetylcholinesterase: X-ray versus Molecular Dynamics

The high aromatic content of the deep and narrow active-site gorge of acetylcholinesterase (AChE) is a remarkable feature of this enzyme. Here, we analyze conformational flexibility of the side chains of the 14 conserved aromatic residues in the active-site gorge of Torpedo californica AChE based on the 47 three-dimensional crystal structures available for the native enzyme, and for its complexes and conjugates, and on a 20-ns molecular dynamics (MD) trajectory of the native enzyme. The degree of flexibility of these 14 aromatic side chains is diverse. Although the side-chain conformations of F330 and W279 are both very flexible, the side-chain conformations of F120, W233, W432, Y70, Y121, F288, F290 and F331 appear to be fixed. Residues located on, or adjacent to, the Ω-loop (C67–C94), namely W84, Y130, Y442, and Y334, display different flexibilities in the MD simulations and in the crystal structures. An important outcome of our study is that the majority of the side-chain conformations observed in the 47 Torpedo californica AChE crystal structures are faithfully reproduced by the MD simulation on the native enzyme. Thus, the protein can assume these conformations even in the absence of the ligand that permitted their experimental detection. These observations are pertinent to structure-based drug design.

Organophosphates' resistance in the B-biotype of Bemisia tabaci (Hemiptera: Aleyrodidae) is associated with a point mutation in an ace1-type acetylcholinesterase and overexpression of carboxylesterase

Organophosphate (OP) insecticides are inhibitors of the enzyme acetylcholinesterase (AChE), which terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine. Previous biochemical studies in Bemisia tabaci (Hemiptera: Aleyrodidae) proposed the existence of two molecular mechanisms for OPs' resistance: carboxylesterase- (COE) mediated hydrolysis or sequestration and decreased sensitivity of AChE. Here, two acetylcholinesterase genes, ace1 and ace2, have been fully cloned and sequenced from an OP-resistant strain and an OP-susceptible strain of B. tabaci. Comparison of nucleic acid and deduced amino acid sequences revealed only silent nucleotide polymorphisms in ace2, and one mutation, Phe392Trp (Phe331 in Torpedo californica), in ace1 of the resistant strain. The Phe392Trp mutation is located in the acyl pocket of the active site gorge and was recently shown to confer OP insensitivity in Culex tritaeniorhynchus. In addition, we also report on the isolation of two carboxylesterase genes ( coe1 and coe2) from B. tabaci, the first carboxylesterases to be reported from this species. We show that one of the genes, coe1, is overexpressed (∼4-fold) in the OP-resistant strain, and determine, by quantitative PCR, that the elevated expression is not related to gene amplification but probably to modified transcriptional control. Lastly, we bring new biochemical evidence that support the involvement of both AChE insensitivity and COE metabolism in resistance to OP insecticides in the resistant strain.

Modulation of Nicotinic Acetylcholine Receptor Conformational State by Free Fatty Acids and Steroids

Steroids and free fatty acids (FFA) are noncompetitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is purportedly located at the lipid-AChR interface, but their exact mechanism of action is still unknown. Here we studied the effect of structurally different FFA and steroids on the conformational equilibrium of the AChR in Torpedo californica receptor-rich membranes. We took advantage of the higher affinity of the fluorescent AChR open channel blocker, crystal violet, for the desensitized state than for the resting state. Increasing concentrations of steroids and FFA decreased the K(D) of crystal violet in the absence of agonist; however, only cis-unsaturated FFA caused an increase in K(D) in the presence of agonist. This latter effect was also observed with treatments that caused the opposite effects on membrane polarity, such as phospholipase A(2) treatment or temperature increase (decreasing or increasing membrane polarity, respectively). Quenching by spin-labeled fatty acids of pyrene-labeled AChR reconstituted into model membranes, with the label located at the gammaM4 transmembrane segment, disclosed the occurrence of conformational changes induced by steroids and cis-unsaturated FFA. The present work is a step forward in understanding the mechanism of action of this type of molecules, suggesting that the direct contact between exogenous lipids and the AChR transmembrane segments removes the AChR from its resting state and that membrane polarity modulates the AChR activation equilibrium by an independent mechanism.

Crystal Structure of Thioflavin T Bound to the Peripheral Site of Torpedo californica Acetylcholinesterase Reveals How Thioflavin T Acts as a Sensitive Fluorescent Reporter of Ligand Binding to the Acylation Site

Acetylcholinesterase plays a key role in cholinergic synaptic transmission by hydrolyzing the neurotransmitter acetylcholine with one of the highest known catalytic rate constants. Hydrolysis occurs in a narrow and deep gorge that contains two sites of ligand binding: A peripheral site, or P-site, near the gorge entrance that contributes to catalytic efficiency both by transiently trapping substrate molecules as they enter the gorge and by allosterically accelerating the transfer of the substrate acyl group to a serine hydroxyl in an acylation site or A-site at the base of the gorge. Thioflavin T is a useful reporter of ligand interactions with the A-site. It binds specifically to the P-site with fluorescence that is enhanced approximately 1000-fold over that of unbound thioflavin T, and the enhanced fluorescence is quenched 1.5- to 4-fold when another ligand binds to the A-site in a ternary complex. To clarify the structural basis of this advantageous signal change, we here report the X-ray structure of the complex of thioflavin T with Torpedo californica acetylcholinesterase. The two aromatic rings in thioflavin T are coplanar and are packed snugly parallel to the aromatic side chains of Trp279, Tyr334, and Phe330. Overlays of this structure with the crystal structures of Torpedo californica acetylcholinesterase complexes with either edrophonium or m-( N, N, N-trimethylammonio)-2,2,2-trifluoroacetophenone, two small aromatic ligands that bind specifically to the A-site, indicate that the phenyl side chain of Phe330 must rotate to sterically accommodate both thioflavin T and the A-site ligand in the ternary complex. This rotation may allow some relaxation of the strict coplanarity of the aromatic rings in the bound thioflavin T and result in partial quenching of its fluorescence.

Potential role of caveolin-1-positive domains in the regulation of the acetylcholine receptor's activable pool: Implications in the pathogenesis of a novel congenital myasthenic syndrome

Cholesterol modulates the plasmalemma's biophysical properties and influences the function and trafficking of membrane proteins. A fundamental phenomenon that remains obscure is how the plasmalemma's lipid composition regulates the activatable pool of membrane receptors. An outstanding model to study this phenomenon is the nicotinic acetylcholine receptor (nAChR), since the nAChR activatable pool has been estimated to be but a small fraction of the receptors present in the plasmalemma. Studies on the effect of cholesterol depletion in the function of the Torpedo californica nAChR, using the lipid-exposed nAChR mutation (αC418W) that produces a congenital myasthenic syndrome (CMS), demonstrated that cholesterol depletion causes a remarkable increase in the αC418W nAChR's macroscopic current whereas not in the wild type (WT). A variety of approaches were used to define the mechanism responsible for the cholesterol depletion mediated-increase in the αC418W nAChR's macroscopic current. The present study suggests that a substantial fraction of the αC418W nAChRs is located in caveolin-1-positive domains, "trapped" in a non-activatable state, and that membrane cholesterol depletion results in the relocation of these receptors to the activatable pool. Co-fractionation and co-immunoprecipitation of the αC418W nAChR and the membrane raft protein caveolin-1 (cav1) support the notion that interactions at lipid-exposed domains regulate the partition of the receptor into membrane raft microdomains. These results have potential implications as a novel mechanism to fine-tune cholinergic transmission in the nervous system and in the pathogenesis associated to the αC418W nAChR.

Petrosamine, a potent anticholinesterase pyridoacridine alkaloid from a Thai marine sponge Petrosia n. sp

Two pyridoacridine alkaloids, including a known petrosamine and a new 2-bromoamphimedine were isolated from a Thai marine sponge Petrosia n. sp. The alkaloids were characterized on the basis of 1D and 2D NMR, MS, and IR spectroscopy. Only petrosamine showed strong acetylcholinesterase inhibitory activity approximately six times higher than that of the reference galanthamine. A computational docking study of petrosamine with the enzyme from the electric eel Torpedo californica ( TcAChE) showed the major contribution to the petrosamine- TcAChE interaction to be arising from the quaternary ammonium group of petrosamine.

Biophysical and Ion Channel Functional Characterization of the Torpedo californica Nicotinic Acetylcholine Receptor in Varying Detergent–Lipid Environments

The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, high-resolution structural studies have been hampered by the lack of mechanistic molecular models that describe how detergents influence membrane protein stability and function. Furthermore, elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examines the effects of nine detergents on: (1) nAChR-lipid composition (gas chromatography with flame ionization; GC-FID and/or mass selective detectors; GC-MSD), (2) stability and aggregation state (analytical size exclusion chromatography; A-SEC and electron microscopy; EM) and (3) ion channel function (planar lipid bilayers). Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analogue detergents CHAPS {(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate}, FC-12 (n-dodecylphosphocholine) and sodium cholate (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid) maintaining stability and supporting ion channel function, and non-lipid-analogue detergents Cymal-6 (6-cyclohexyl-1-hexyl-beta-D-maltoside), DDM (n-dodecyl-beta-D-maltopyranoside), LDAO (lauryldimethylamine-N-oxide) and OG (n-octyl-beta-d-glucopyranoside) decreasing stability and significantly reducing or completely suppressing ion channel function. Anapoe-C(12)E(9 )(polyoxyethylene-[9]-dodecyl ether) and BigCHAP (N,N'-bis-[3-d-gluconamidopropyl] cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function compared to lipid-analogue detergents. Therefore, the nAChR can be stable and functional in lipid-analogue detergents or in detergents that retain moderate amounts of residual native lipids, but not in non-lipid-analogue detergents.

Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

Fluorescent Agonists for the Torpedo Nicotinic Acetylcholine Receptor

We have synthesized a series of fluorescent acylcholine derivatives carrying different linkers that vary in length and structure and connect the acylcholine unit to the environment-sensitive fluorophores 7-(diethylamino)coumarin-3-carbonyl (DEAC) or N-(7-nitrobenz-2-oxa-1,3-diazol-yl) (NBD). The pharmacological properties of the fluorescent analogues were investigated on heterologously expressed nicotinic acetylcholine receptor (nAChR) from Torpedo californica and on oocytes transplanted with nAChR-rich Torpedo marmorata membranes. Agonist action strongly depends on the length and the structure of the linker. One particular analogue, DEAC-Gly-C6-choline, showed partial agonist behavior with about half of the maximum response of acetylcholine, which is at least 20 times higher than those observed with previously described fluorescent dansyl- and NBD-acylcholine analogues. Binding of DEAC-Gly-C6-choline to Torpedo nAChR induces a strong enhancement of fluorescence intensity. Association and displacement kinetic experiments revealed dissociation constants of 0.5 nM for the alphadelta-binding site and 15.0 nM for the alphagamma-binding site. Both the pharmacological and the spectroscopic properties of this agonist show great promise for characterizing the allosteric mechanism behind the function of the Torpedo nAChR, as well as for drug-screening studies.

Assaying Small-Molecule−Receptor Interactions by Continuous Flow Competitive Displacement Chromatography/Mass Spectrometry

Small-molecule screening techniques that employ mass spectrometry detection have been highly successful. However, the inability of conventional techniques, such as frontal affinity chromatography-mass spectrometry (FAC-MS), to easily identify weak binding molecules (i.e., Kd >or= 1 microM) using small amounts of target protein (subpicomole levels) represents a significant impediment to the widespread use of the method in the routine screening of low-abundance membrane receptors. This limitation is particularly notable in the early stages of the drug discovery process, as weak binding molecules can serve as useful leads for targets with no known ligand or when existing tight binding ligands have little therapeutic value. Competitive assay methods involving the displacement of an indicator ligand offer a more sensitive alternative, as the ability to generate an appreciable signal through various methods, including transient overconcentrations of indicator compounds, provides an unambiguous means for identifying weak affinity ligands. In this work we describe a continuous flow competitive assay based on the principles of FAC-MS that can be widely used to identify and characterize weak affinity ligands using low levels of the nicotinic acetylcholine receptor from Torpedo californica (nAChR). The validity of the assay is shown through the ability to identify nicotine (Kd approximately 1 microM) with columns containing <2 pmol of binding sites. Multiple injections of nicotine on a single column produce reproducible peaks in the signal of the indicator compound, epibatidine (Kd approximately 2 nM) showing minimal degradation in signal intensity between trials. The intensity of the peaks is dependent on the concentration of nicotine being injected, and binding curves can be generated through multiple injections on the same column. We investigate and optimize various parameters, including assay speed and concentrations, and demonstrate an automated assay format with the potential for use as a high-throughput screening tool. The ability to screen for weak binders of more pharmacologically relevant membrane receptors in a high-throughput screening format is discussed.

Modulation of the Nicotinic Acetylcholine Receptor Function by Cholesterol

The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. The nAChR is also found in the electric organs of electric rays (e.g. Torpedo californica). Cholesterol is a key lipid for maintaining the correct functionality of membrane proteins and has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs when expressed in cell membranes with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P of Xenopus laevis oocytes expressing the neuronal α‐7, muscle‐type or Torpedo californica nAChRs in the functioning of the nAChR. Using the two‐electrode voltage clamp technique it was found that the neuronal ‐7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle‐type nAChR. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration renders a significant fraction of the neuronal ‐7 and Torpedo nAChRs “inactivated” whereas the muscle‐type nAChR tends to resist this functional inhibition. This work was supported by NIH grants RO1GM56371‐11, NCRR 1S0RR13705 and S06‐GM08102‐27 to JAL‐D. NDH‐R and CAB‐P were supported by the NIH‐MBRS RISE Grant R25GM61151.

Molecular properties of local anesthetics as predictors of affinity for nicotinic acetylcholine receptors

In spinal anesthesia, the effects of local anesthetics (LAs) are not completely explained by sodium channel inhibition. Other targets include neuronal nicotinic acetylcholine receptors (nAChRs). LA affinities for the Torpedo californica nAChR were measured by inhibition of [(3)H]TCP binding and correlated with molecular volume, surface area, molecular weight, and log of the octanol-water partition coefficients (P and D). To understand the molecular determinants important for interaction with the nAChR, ester and amide LAs were compared separately. Also, correlations with the aromatic/linker half and the hydrophilic half of the LA molecules were considered individually. The IC(50)s of the ester LAs correlated better with the molecular volume, surface area, molecular weight, and log P of the aromatic/linker half of the molecules; whereas the IC(50)s for amide LAs correlated better with the four parameters based on the hydrophilic half. These correlations were used to predict the IC(50) of various LAs (including several not studied here) and to compare these values with the published values. The predicted values of IC(50) correlated well with the published results both for neuronal and for electroplaque-desensitized nAChR, suggesting that the results can be generalized to include neuronal nAChRs.

Synaptic arrangement of the neuroligin/beta-neurexin complex revealed by X-ray and neutron scattering.

Neuroligins are postsynaptic cell-adhesion proteins that associate with their presynaptic partners, the neurexins. Using small-angle X-ray scattering, we determined the shapes of the extracellular region of several neuroligin isoforms in solution. We conclude that the neuroligins dimerize via the characteristic four-helix bundle observed in cholinesterases, and that the connecting sequence between the globular lobes of the dimer and the cell membrane is elongated, projecting away from the dimer interface. X-ray scattering and neutron contrast variation data show that two neurexin monomers, separated by 107 A, bind at symmetric locations on opposite sides of the long axis of the neuroligin dimer. Using these data, we developed structural models that delineate the spatial arrangements of different neuroligin domains and their partnering molecules. As mutations of neurexin and neuroligin genes appear to be linked to autism, these models provide a structural framework for understanding altered recognition by these proteins in neurodevelopmental disorders.

Conformation-Sensitive Steroid and Fatty Acid Sites in the Transmembrane Domain of the Nicotinic Acetylcholine Receptor

The mechanism by which some hydrophobic molecules such as steroids and free fatty acids (FFA) act as noncompetitive inhibitors of the nicotinic acetylcholine receptor (AChR) is still not known. In the present work, we employ Förster resonance energy transfer (FRET) between the intrinsic fluorescence of membrane-bound Torpedo californica AChR and the fluorescent probe Laurdan using the decrease in FRET efficiency (E) caused by steroids and FFA to identify potential sites of these hydrophobic molecules. Structurally different steroids produced similar changes (DeltaE) in FRET, and competition studies between them demonstrate that they occupy the same site(s). They also share their binding site(s) with FFA. Furthermore, the FRET conditions define the location of the sites at the lipid-protein interface. Endogenous production of FFA by controlled phospholipase A2 enzymatic digestion of membrane phospholipids yielded DeltaE values similar to those obtained by addition of exogenous ligand. This finding, together with the preservation of the sites in membranes subjected to controlled proteolysis of the extracellular AChR moiety with membrane-impermeable proteinase K, further refines the topology of the sites at the AChR transmembrane domain. Agonist-induced desensitization resulted in the masking of the sites observed in the absence of agonist, thus demonstrating the conformational sensitivity of FFA and steroid sites in the AChR.

Interactions between α-conotoxin MI and the Torpedo marmorata receptor α–δ interface

The muscle-type nicotinic receptor has two distinguishable acetylcholine binding sites at the α–γ and α–δ subunit interfaces; α-conotoxins can bind them selectively. Moreover, we previously reported that α-conotoxin MI can interact with Torpedo californica and Torpedo marmorata receptors showing that conotoxins can also detect receptors from different species of the same genus [L. Cortez, S.G. del Canto, F. Testai, M.B. de Jiménez Bonino, Conotoxin MI inhibits the acetylcholine binding site of the Torpedo marmorata receptor, Biochem. Biophys. Res. Commun. 295 (2002) 791–795]. Herein, to identify T. marmorata receptor regions involved in α-conotoxin MI binding, a photoactivatable reagent was used and labeled sites were mapped by enzymatic proteolysis, MALDI-TOF-MS and Edman degradation. α-Conotoxin MI binding determinants were found and studies revealed a second binding motif at the α/δ interface. A proposal for receptor–toxin interaction is discussed based on experimental results and docking studies.

The proteome survey of an electricity‐generating organ (Torpedo californica electric organ)

Torpedo californica is a species in class Chondrichthyes. Electric rays have evolved the electric organ, which is similar to the mammalian neuromuscular junction (NMJ). Here, we took a combined cDNA sequencing and proteomic approach to define the molecular constituents of the T. californica electric organ. For soluble proteins, 2-DE was used and 224 protein spots were mapped. Plasma membrane fractions were analyzed using the shotgun approach (LC-MS/MS). A Torpedo cDNA library was constructed and 607 cDNA clones were sequenced. Identification of electric organ proteins was done using cross-species comparisons, and a custom database was constructed from cDNA translations. We unambiguously identified 121 proteins and transcripts, 103 of which were novel additions to the existing databases of Torpedo fish. Fifteen proteins of known function, but not previously associated with either the electroplaque or NMJ, were present at high abundance. These included the heat shock and oxidative stress proteins, annexin V (calelectrin), and plectin 1. Most interesting were the unambiguous matches to 11 human ORFs of unknown function, including four potential RNA splicing proteins, a vacuolar sorting protein, and a tetraspanin containing protein. This analysis identified proteins that may play a role in the higher vertebrate neuromuscular junction or other electrical synapses.

α‐Conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors

Alpha-conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of alpha-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into alpha-conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in alpha-conotoxins GI and SI, or D12K substitution in alpha-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent alpha-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in alpha-conotoxin [A10L]PnIA, a blocker of neuronal alpha7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two alpha7-like anion-selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to alpha-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of alpha-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type alpha-conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.

Morphometric minefields—towards a measurement standard for chondrichthyan fishes

Size measurements are crucial for studies on the growth, maturation, maximum size, and population structure of cartilaginous fishes. However, researchers use a variety of measurement techniques even when working on the same species. Accurate comparison of results among studies is only possible if the measurement technique used is adequately defined and, if different techniques are used, a conversion equation can be derived. These conditions have not always been met, leading to invalid comparisons and incorrect conclusions. This paper reviews methods used for measuring chondrichthyans, and summarises the variety of constraints that influence the choice of a measurement technique. Estimates of the variability present in some measurement techniques are derived for shortfin mako shark, Isurus oxyrinchus, porbeagle shark, Lamna nasus, blue shark, Prionace glauca, Antarctic thorny skate, Amblyraja georgiana, and Pacific electric ray, Torpedo californica. Total length measured with the tail in the natural position (sharks) and disc widths (batoids) have higher variability than other methods, and are not recommended. Instead, the longest longitudinal axis should be measured where possible and practical; i.e., flexed total length for sharks, total length for batoids (excluding suborder Myliobatoidei), pelvic length for batoids of the suborder Myliobatoidei, and chimaera length (snout to posterior end of supracaudal fin) for chimaeroids (except for Callorhinchus, for which fork length should be measured from the anterior edge of the snout protuberance). Straight-line measurements are preferred to measurements over the curve of the body. Importantly, measurement methods must be clearly defined, giving information on the anterior reference point, the posterior reference point, and how the measurement was made between these two. Measurements using at least two different methods are recommended on at least a subsample of the fish in order to develop conversion regression relationships.

Ester derivatives of annulated tetrahydroazocines: A new class of selective acetylcholinesterase inhibitors

A series of ester derivatives of annulated tetrahydroazocines, namely 2,3,6,11-tetrahydro-1 H-azocino[4,5- b]indoles ( 5– 10), 2,3,6,7-tetrahydro-1 H-azocino[5,4- b]indoles ( 11– 14), and 4,7,8,9-tetrahydro-1 H-pyrrolo[2,3- d]azocines ( 15– 18), synthesized through an efficient 6 → 8 membered ring expansion procedure, were investigated for their acetylcholinesterase (AChE) inhibitory activities. Most of the compounds acted as AChE inhibitors in vitro, with IC 50 values ranging from 5 to 40 μM. The most potent compounds 11 and 15, both as racemic mixtures, proved selective toward AChE, exhibiting selectivity ratios versus butyrylcholinesterase (BuChE) of ca. 15 and more than 20, respectively. Structure–activity studies highlighted, among other factors, lipophilicity as a property modulating the AChE inhibition potency, as shown by a reasonable parabolic correlation between pIC 50 and experimental 1-octanol/water partition coefficient (log P), which described the prevailing behavior of the examined compounds ( r 2 = 0.665). Molecular docking simulations using the X-ray crystal structure of AChE from Torpedo californica suggested possible binding modes of the tetrahydroazocine ester derivatives 11 and 15.