Optimization of phenylacetic acid derivatives for balanced CRTH2 and DP dual antagonists

Our first generation CRTH2 and DP dual antagonists, represented by AMG 009, are more potent toward the CRTH2 receptor than to the DP receptor. Here we report our efforts in the discovery of CRTH2 and DP dual antagonists with more balanced potencies to both receptors, such as compound 15.     

Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (G(i) or G(s)), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-x(L) BH4(4-23) and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-x(L) BH4(4-23) attenuated ATP release in response to incubation of erythrocytes with the β-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.     

Deep sequencing reveals predominant expression of miR-21 amongst the small non-coding RNAs in retinal microvascular endothelial cells

The retinal vascular endothelium is essential for angiogenesis and is involved in maintaining barrier selectivity and vascular tone. The aim of this study was to identify and quantify microRNAs and other small regulatory non-coding RNAs (ncRNAs) which may regulate these crucial functions. Primary bovine retinal microvascular endothelial cells (RMECs) provide a well-characterized in vitro system for studying angiogenesis. RNA extracted from RMECs was used to prepare a small RNA library for deep sequencing (Illumina Genome Analyzer). A total of 6.8 million reads were mapped to 250 known microRNAs in miRBase (release 16). In many cases, the most frequent isomiR differed from the sequence reported in miRBase. In addition, five novel microRNAs, 13 novel bovine orthologs of known human microRNAs and multiple new members of the miR-2284/2285 family were detected. Several ～30 nucleotide sno-miRNAs were identified, with the most highly expressed being derived from snoRNA U78. Highly expressed microRNAs previously associated with endothelial cells included miR-126 and miR-378, but the most highly expressed was miR-21, comprising more than one-third of all mapped reads. Inhibition of miR-21 with an LNA inhibitor significantly reduced proliferation, migration, and tube-forming capacity of RMECs. The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs and other small RNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in RMECs. Knockdown of miR-21 suggests a role for this microRNA in regulation of angiogenesis in the retinal microvasculature.     

Cerebellar function: coordination, learning or timing?

Theories of cerebellar function have largely involved three ideas: movement coordination, motor learning or timing. New evidence indicates these distinctions are not particularly meaningful, as the cerebellum influences movement execution by feedforward use of sensory information via temporally specific learning.     

Nicotine Uses Neuron-Glia Communication to Enhance Hippocampal Synaptic Transmission and Long-term Memory

Nicotine enhances synaptic transmission and facilitates long-term memory. Now it is known that bi-directional glia-neuron interactions play important roles in the physiology of the brain. However, the involvement of glial cells in the effects of nicotine has not been considered until now. In particular, the gliotransmitter D-serine, an endogenous co-agonist of NMDA receptors, enables different types of synaptic plasticity and memory in the hippocampus. Here, we report that hippocampal long-term synaptic plasticity induced by nicotine was annulled by an enzyme that degrades endogenous D-serine, or by an NMDA receptor antagonist that acts at the D-serine binding site. Accordingly, both effects of nicotine: the enhancement of synaptic transmission and facilitation of long-term memory were eliminated by impairing glial cells with fluoroacetate, and were restored with exogenous D-serine. Together, these results show that glial D-serine is essential for the long-term effects of nicotine on synaptic plasticity and memory, and they highlight the roles of glial cells as key participants in brain functions.     

Armament imbalances: match and mismatch in plant-pollinator traits of highly specialized long-spurred orchids

Background

Some species of long-spurred orchids achieve pollination by a close association with long-tongued hawkmoths. Among them, several Habenaria species present specialized mechanisms, where pollination success depends on the attachment of pollinaria onto the heads of hawkmoths with very long proboscises. However, in the Neotropical region such moths are less abundant than their shorter-tongued relatives and are also prone to population fluctuations. Both factors may give rise to differences in pollinator-mediated selection on floral traits through time and space.

Methodology/Principal Findings

We characterized hawkmoth assemblages and estimated phenotypic selection gradients on orchid spur lengths in populations of three South American Habenaria species. We examined the match between hawkmoth proboscis and flower spur lengths to determine whether pollinators may act as selective agents on flower morphology. We found significant directional selection on spur length only in Habenaria gourlieana, where most pollinators had proboscises longer than the mean of orchid spur length.

Conclusions/Significance

Phenotypic selection is dependent on the mutual match between pollinator and flower morphologies. However, our findings indicate that pollinator-mediated selection may vary through time and space according to local variations in pollinator assemblages.