Evidence for multiple calcium response mechanisms in mammalian olfactory receptor neurons

Olfactory receptor neurons employ a diversity of signaling mechanisms for transducing and encoding odorant information. The simultaneous activation of subsets of receptor neurons provides a complex pattern of activation in the olfactory bulb that allows for the rapid discrimination of odorant mixtures. While some transduction elements are conserved among many species, some species-specificity occurs in certain features that may relate to their particular physiology and ecological niche. However, studies of olfactory transduction have been limited to a relatively small number of vertebrate and invertebrate species. To better understand the diversity and evolution of olfactory transduction mechanisms, we studied stimulus-elicited calcium fluxes in olfactory neurons from a previously unstudied mammalian species, the domestic cat. Isolated cells from cat olfactory epithelium were stimulated with odorant mixtures and biochemical agents, and cell responses were measured with calcium imaging techniques. Odorants elicited either increases or decreases in intracellular calcium; odorant-induced calcium increases were mediated either by calcium fluxes through the cell membrane or by mobilization of intracellular stores. Individual cells could employ multiple signaling mechanisms to mediate responses to different odorants. The physiological features of these olfactory neurons suggest greater complexity than previously recognized in the role of peripheral neurons in encoding complex odor stimuli. The investigation of novel and unstudied species is important for understanding the mechanisms of odorant signaling that apply to the olfactory system in general and suggests both broadly conserved and species-specific evolutionary adaptations.     

Part 2: Design, synthesis and evaluation of hydroxyproline-derived α2δ ligands

Conformational constraint has been used to design a potent series of α₂δ ligands derived from the readily available starting material (2S,4R)-hydroxy-l-proline. The ligands have improved physicochemistry and potency compared to their linear counterparts (described in our earlier publication) and the lead compound has been progressed to clinical development.     

Changes in Parkinsonian gait kinematics with self-generated and externally-generated cues: a comparison of responders and non-responders

Abstract

Purpose: Rhythmic auditory stimulation such as listening to music can alleviate gait bradykinesia in people with Parkinson disease (PD) by increasing spatiotemporal gait features. However, evidence about what specific kinematic alterations lead to these improvements is limited, and differences in responsiveness to cueing likely affect individual motor strategies. Self-generated cueing techniques, such as singing or mental singing, provide similar benefits but no evidence exists about how these techniques affect lower limb joint movement. In this study, we assessed immediate effects of external and self-generated cueing on lower limb movement trajectories during gait.

Methods: Using 3D motion capture, we assessed sagittal plane joint angles at the hip, knee, and ankle across 35 participants with PD, divided into responders (n?=?23) and non-responders (n?=?12) based on a clinically meaningful change in gait speed. Joint motion was assessed as overall range of motion as well as at two key time points during the gait cycle: initial contact and toe-off.

Results: Responders used both cue types to increase gait speed and induce increases in overall joint ROM at the hip while only self-generated cues also increased ROM at the ankle. Increased joint excursions for responders were also evident at initial contact and toe-off.

Conclusions: Our results indicate that self-generated rhythmic cues can induce similar increases in joint excursions as externally-generated cues and that some people may respond more positively than others. These results provide important insight into how self-generated cueing techniques may be tailored to meet the varied individual needs of people with PD.     

Novel species-specific glycoprotein on the surface of Mytilus edulis and M. trossulus eggs

Protein–protein interactions play a central role in the gamete attraction, binding, and fusion stages of gamete interactions and fertilization for broadcast spawning species, such as marine mussels in the Mytilus edulis species complex. Although assortative gamete interaction has been implicated in the level of reproductive isolation among the three species in this complex, the molecular basis of these interactions has not been elucidated. Using mass spectrometry peptide sequencing, cDNA sequencing, and bioinformatics approaches, we have investigated species-level variation in the proteins expressed on the surface of mussel eggs. We herein describe an extracellular protein, MESP-1, from the surface of the eggs of M. edulis and M. trossulus that has a unique domain structure when compared to protein structures that have heretofore been identified. Given variation in the size of MESP-1 predicted from cDNA sequences versus those estimated from SDS-PAGE gels, we conclude this protein is subject to significant species-specific post-translation modifications. Further, bioinformatic analysis of the novel structure of MESP-1 suggests that this protein may be an integral membrane protein involved in sperm–egg fusion, and/or released to the vitelline envelope.     

Molecular Evolution at the Cytochrome Oxidase Subunit 2 Gene Among Divergent Populations of the Intertidal Copepod, Tigriopus californicus

The cytochrome c oxidase subunit 2 gene (COII) encodes a highly conserved protein that is directly responsible for the initial transfer of electrons from cytochrome c to cytochrome c oxidase (COX) crucial to the production of ATP during cellular respiration. Despite its integral role in electron transport, we have observed extensive intraspecific nucleotide and amino acid variation among 26 full-length COII sequences sampled from seven populations of the marine copepod, Tigriopus californicus. Although intrapopulation divergence was virtually nonexistent, interpopulation divergence at the COII locus was nearly 20% at the nucleotide level, including 38 nonsynonymous substitutions. Given the high degree of interaction between the cytochrome c oxidase subunit 2 protein (COX2) and the nuclear-encoded subunits of COX and cytochrome c (CYC), we hypothesized that some codons in the COII gene are likely to be under positive selection in order to compensate for amino acid substitutions in other subunits. Estimates of the ratio of nonsynonymous to synonymous substitution (ω), obtained using a series of maximum likelihood models of codon substitution, indicated that the majority of codons in T. californicus COII are under strong purifying selection (ω << 1), while approximately 4% of the sites in this gene appear to evolve under relaxed selective constraint (ω = 1). A branch-site maximum likelihood model identified three sites that may have experienced positive selection within the central California sequence clade in our COII phylogeny; these results are consistent with previous studies showing functional and fitness consequences among interpopulation hybrids between central and northern California populations. [Reviewing Editor: Dr. Willie Swanson]     

Isolation and characterization of cytochrome c from the marine copepod Tigriopus californicus

Mitochondrial energy production requires complex interactions among proteins encoded in both the nuclear and mitochondrial genomes. The intergenomic coevolution of interacting gene products has been previously suggested based on interspecific comparisons of cytochrome c (encoded by the nuclear CYC gene) and cytochrome c oxidase (partly encoded in the mitochondrial DNA by the COX1, COX2 and COX3 genes). In the intertidal copepod, Tigriopus californicus, non-synonymous substitutions in the COX1 gene have previously been found in interpopulation comparisons. In order to determine if CYC also shows interpopulation variation, this gene was isolated from a cDNA library using a degenerate primer/polymerase chain reaction approach. Characterization of a cDNA sequence and 25 genomic DNA sequences derived from four T. californicus populations yielded the following results: (1) the T. californicus CYC gene is interrupted by an intron that occurs at the same position as the intron found in vertebrate CYC genes; (2) there is extensive sequence variation within both the coding region and intron of this gene and the vast majority of this variation occurs between sequences drawn from geographically distinct populations; (3) the coding sequence variation includes a minimum of five amino acid replacement substitutions; (4) segregation of length variants among offspring in an interpopulation cross revealed genotypic ratios consistent with the proposed allelic nature of the CYC variants. These results demonstrate that the requisite genetic variation required for intergenomic coevolution exists in the CYC-COX system in T. californicus.     

Chloroplast DNA variation in pearl millet and related species

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16S and 5S ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sa/I fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp Eco RI fragment. The third region was a 6-kbp EcoRI fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.—A survey of 12 geographically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.