Block by amiloride derivatives of odor-evoked discharge in lobster olfactory receptor neurons through action on a presumptive TRP channel

Amiloride and its derivatives inhibit a number of sensory transduction processes, including some types of chemosensory transduction. Here, we report that pyrazine derivatives of amiloride reversibly inhibit odorant-evoked activity in lobster olfactory receptor neurons. The potency sequence is as follows-(IC50, mM): 5-(N,N-hexamethylene)amiloride (0.015) approximately 5-(N-methyl-N-isobutyl)amiloride (0.02) approximately 5-(N-ethyl-N-isopropyl)amiloride (0.03) > 5-(N,N-dimethyl)amiloride (0.48); 3',4'-dichlorobenzamil (0.4), phenamil (0.5), and amiloride itself (2) are ineffective. The same derivatives with the similar potency sequence also block a presumptive transient receptor potential (TRP) channel that is the likely downstream target of phosphoinositide signaling in these cells. Our results suggest that pyrazine derivatives of amiloride are useful probes to study more detailed mechanisms of chemosensory transduction in this system and possibly in other chemosensory systems in which TRP channels are the known or suspected downstream effector.     

NMR studies on fully hydrated membrane proteins, with emphasis on bacteriorhodopsin as a typical and prototype membrane protein

The 3D structures or dynamic feature of fully hydrated membrane proteins are very important at ambient temperature, in relation to understanding their biological activities, although their data, especially from the flexible portions such as surface regions, are unavailable from X-ray diffraction or cryoelectron microscope at low temperature. In contrast, high-resolution solid-state NMR spectroscopy has proved to be a very convenient alternative means to be able to reveal their dynamic structures. To clarify this problem, we describe here how we are able to reveal such structures and dynamic features, based on intrinsic probes from high-resolution solid-state NMR studies on bacteriorhodopsin (bR) as a typical membrane protein in 2D crystal, regenerated preparation in lipid bilayer and detergents. It turned out that their dynamic features are substantially altered upon their environments where bR is present. We further review NMR applications to study structure and dynamics of a variety of membrane proteins, including sensory rhodopsin, rhodopsin, photoreaction centers, diacylglycerol kinases, etc.     

Temporal order and the evolution of complex acoustic signals

The evolution of complex signals may be favoured by hidden preferences or pre-existing sensory biases. Females of two species of grey treefrogs (Hyla chrysoscelis and Hyla versicolor) were tested with combinations of a conspecific advertisement call and acoustic appendages. Appendages consisted of aggressive calls and segments of advertisement calls from conspecific males and males of three other species and bursts of filtered noise. When a wide variety of these acoustic appendages followed the advertisement call, the resulting compound signal was often more attractive than the same advertisement call alone. When the same appendages led advertisement calls, however, the compound signal was never more attractive and sometimes less attractive. The order effect was especially strong in tests of H. versicolor in which advertisement-call duration was decreased. These results cannot be explained by a general pre-existing bias for extra stimulation per se. Rather, order and other effects may constrain the evolution and subsequent modification of complex and extravagant signals, examples of which have been reported for a wide range of taxa.     

Is a short concentrate-finishing period in lambs raised on alfalfa pasture effective at reducing fat indoles contents and lightening meat colour?

Pasture-feeding in lambs has been associated with dark-coloured meat and high meat contents of volatile indoles (skatole and indole), which may be responsible for sensory defects. The risk of off-flavours is even higher when lambs graze alfalfa, because it is particularly rich in rapidly degradable proteins. Here, we investigated whether a short concentrate stall-finishing period in lambs that previously grazed alfalfa influences meat sensory quality traits. We compared three feeding treatments, using three groups of 10 male Romane lambs: grazing alfalfa (A), stall-feeding with concentrate and straw (S), and stall-finishing with concentrate and straw for 21 days after previously grazing alfalfa (AS). During stall-feeding, the concentrate was given at a level adjusted to achieve a similar growth pattern in all treatment groups, and the length of the trough was sufficient for all lambs to have access to the feeds at the same time. Lamb live weight and age at slaughter, and carcass conformation and fatness were similar for all treatment groups. Perirenal fat skatole content was lower in AS and S lambs than in A lambs (P < 0.001 and P < 0.05, respectively), and not significantly different between AS and S lambs. Perirenal fat indole and dorsal fat skatole concentrations were lower in AS lambs than in A lambs (P < 0.05 and P < 0.005, respectively), the other pairwise comparisons being not significant. There was no treatment effect on dorsal fat indole content. Longissimus et lumborum muscle colour coordinates differed between pasture-fed and stall-fed lambs, with a lower lightness (P < 0.005) and greater redness and hue angle (P < 0.001 and P < 0.01, respectively) in pasture-fed lambs, but muscle colour coordinates did not differ between A and AS lambs. A 21-d concentrate-based stall-finishing period in lambs previously raised on alfalfa pasture therefore markedly reduced fat indoles concentrations compared to lambs grazed on alfalfa until slaughter but did not change meat colour.     

Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin

Mastigocladopsis repens halorhodopsin (MrHR) is a Cl^?-pumping rhodopsin that belongs to a distinct cluster far from other Cl^? pumps. We investigated its pumping function by analyzing its photocycle and the effect of amino acid replacements. MrHR can bind I^? similar to Cl^? but cannot transport it. I^?-bound MrHR undergoes a photocycle but lacks the intermediates after L, suggesting that, in the Cl^?-pumping photocycle, Cl^? moves to the cytoplasmic (CP) channel during L decay. A photocycle similar to that of the I^?-bound form was also observed for a mutant of the Asp200 residue, which is superconserved and assumed to be deprotonated in most microbial rhodopsins. This residue is probably close to the Cl^?-binding site and the protonated Schiff base, in which a chromophore retinal binds to a specific Lys residue. However, the D200N mutation affected neither the Cl^?-binding affinity nor the absorption spectrum, but completely eliminated the Cl^?-pumping function. Thus, the Asp200 residue probably protonates in the dark state but deprotonates during the photocycle. Indeed, a H⁺ release was detected for photolyzed MrHR by using an indium?tin oxide electrode, which acts as a good time-resolved pH sensor. This H⁺ release disappeared in the I^?-bound form of the wild-type and Cl^?-bound form of the D200N mutant. Thus, Asp200 residue probably deprotonates during L decay and then drives the Cl^? movement to the CP channel.     

Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK

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Evolutionary diversification of the auditory organ sensilla in Neoconocephalus katydids (Orthoptera: Tettigoniidae) correlates with acoustic signal diversification over phylogenetic relatedness and life history

Neoconocephalus Tettigoniidae are a model for the evolution of acoustic signals as male calls have diversified in temporal structure during the radiation of the genus. The call divergence and phylogeny in Neoconocephalus are established, but in tettigoniids in general, accompanying evolutionary changes in hearing organs are not studied. We investigated anatomical changes of the tympanal hearing organs during the evolutionary radiation and divergence of intraspecific acoustic signals. We compared the neuroanatomy of auditory sensilla (crista acustica) from nine Neoconocephalus species for the number of auditory sensilla and the crista acustica length. These parameters were correlated with differences in temporal call features, body size, life histories and different phylogenetic positions. By this, adaptive responses to shifting frequencies of male calls and changes in their temporal patterns can be evaluated against phylogenetic constraints and allometry. All species showed well‐developed auditory sensilla, on average 32–35 between species. Crista acustica length and sensillum numbers correlated with body size, but not with phylogenetic position or life history. Statistically significant correlations existed also with specific call patterns: a higher number of auditory sensilla occurred in species with continuous calls or slow pulse rates, and a longer crista acustica occurred in species with double pulses or slow pulse rates. The auditory sensilla show significant differences between species despite their recent radiation, and morphological and ecological similarities. This indicates the responses to natural and sexual selection, including divergence of temporal and spectral signal properties. Phylogenetic constraints are unlikely to limit these changes of the auditory systems.     

Newly raised anti‐VAChT and anti‐ChAT antibodies detect cholinergic cells in chicken embryos

The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O‐acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia.     

Proximate factors and potential benefits influencing selection of Psychotria suterella for shelter by the harvestman Jussara spec.

Harvestmen (Arachnida, Opiliones) are arachnids that rely on chemicals for communication and are particularly dependent on high humidity. The harvestman Jussara spec. (Sclerosomatidae) clearly prefers to rest on the stem and leaves of Psychotria suterella Muell. Arg. (Rubiaceae), a plant having a complex architecture of overlapping branches. So far, few studies have focused on understanding how harvestmen find their host plant and the benefits associated with the selected plant. Here, we investigated cues harvestmen may exploit to find P. suterella and the potential benefits for harvestmen of this interaction. To address how harvestmen find the plant, we tested two non‐exclusive hypotheses: harvestmen use chemicals from conspecifics, and/or harvestmen use chemicals from plants. For the first hypothesis, we assessed the number of harvestmen choosing P. suterella with or without chemicals of Jussara spec. on the main stem. We did not find evidence that Jussara spec. uses chemicals from conspecifics to select P. suterella, at least in isolation, without any mechanical stimuli such as stem texture or size. For the second hypothesis, we tested harvestman behavior exposed to volatiles from P. suterella, a non‐preferred host plant – Impatiens walleriana Hook.f. (Balsaminaceae) – and a blank treatment, both in a triangular arena and in a Y‐tube olfactometer. We also found no evidence that the harvestmen use plant volatiles to find it. We also tested two non‐exclusive hypotheses regarding the benefits of selecting P. suterella for harvestmen. The first hypothesis is that P. suterella offers a higher humidity than other plants in the micro‐environment surrounding the leaves because of a high leaf transpiration rate. Harvestmen could benefit from this because they rest with the body in contact with the leaf or at less than 5 mm from it. We did not find evidence that leaf transpiration rate is important for Jussara spec., as P. suterella did not present high rates compared to other local species. The second hypothesis is that the leaves of P. suterella provide a better shadow than other plants, acting as a sun shade due to its complex architecture with overlapping leaves. We measured light passage through the leaves of P. suterella and three other local species and found that less light passes through the leaves of P. suterella, which therefore provides darker shadow. This potentially provides a favorable micro‐climate for harvestmen, which might help to explain the micro‐habitat preference of Jussara spec.