Detecting Clusters of Mutations

Positive selection for protein function can lead to multiple mutations within a small stretch of DNA, i.e., to a cluster of mutations. Recently, Wagner proposed a method to detect such mutation clusters. His method, however, did not take into account that residues with high solvent accessibility are inherently more variable than residues with low solvent accessibility. Here, we propose a new algorithm to detect clustered evolution. Our algorithm controls for different substitution probabilities at buried and exposed sites in the tertiary protein structure, and uses random permutations to calculate accurate P values for inferred clusters. We apply the algorithm to genomes of bacteria, fly, and mammals, and find several clusters of mutations in functionally important regions of proteins. Surprisingly, clustered evolution is a relatively rare phenomenon. Only between 2% and 10% of the genes we analyze contain a statistically significant mutation cluster. We also find that not controlling for solvent accessibility leads to an excess of clusters in terminal and solvent-exposed regions of proteins. Our algorithm provides a novel method to identify functionally relevant divergence between groups of species. Moreover, it could also be useful to detect artifacts in automatically assembled genomes

Activation of TREK currents by riluzole in three subgroups of cultured mouse nodose ganglion neurons

Two-pore domain potassium channels (K2P) constitute major candidates for the regulation of background potassium currents in mammalian cells. Channels of the TREK subfamily are also well positioned to play an important role in sensory transduction due to their sensitivity to a large number of physiological and physical stimuli (pH, mechanical, temperature). Following our previous report describing the molecular expression of different K2P channels in the vagal sensory system, here we confirm that TREK channels are functionally expressed in neurons from the mouse nodose ganglion (mNG). Neurons were subdivided into three groups (A, Ah and C) based on their response to tetrodotoxin and capsaicin. Application of the TREK subfamily activator riluzole to isolated mNG neurons evoked a concentration-dependent outward current in the majority of cells from all the three subtypes studied. Riluzole increased membrane conductance and hyperpolarized the membrane potential by approximately 10 mV when applied to resting neurons. The resting potential was similar in all three groups, but C cells were clearly less excitable and showed smaller hyperpolarization-activated currents at -100 mV and smaller sustained currents at -30 mV. Our results indicate that the TREK subfamily of K2P channels might play an important role in the maintenance of the resting membrane potential in sensory neurons of the autonomic nervous system, suggesting its participation in the modulation of vagal reflexes

A novel role for cyclic nucleotide-gated cation channels in lung liquid homeostasis in sheep

Sheep lungs were artificially perfused in situ with warmed whole oxygenated sheep blood. The airspaces of the lungs were filled with liquid containing an impermeant tracer, to allow measurement of the rate of net transepithelial liquid movement under various conditions.Dichlorobenzamil (1.5 × 10−5m), a blocker of cyclic nucleotide-gated cation channels, inhibited the resting absorption of lung liquid in sheep aged 6 months (n = 5) (from −36.47 ± 4.62 to −4.36 ± 5.27 ml h−1, means ±s.e.m.; P < 0.005, paired t test). Amiloride (10−4 M), a blocker of epithelial sodium channels, had no additive effect to that of dichlorobenzamil.In the lungs of sheep aged 6 months (n = 4), amiloride (10−4m) partially inhibited the resting absorption of liquid (from −35.21 ± 8.57 to −11.05 ± 4.91 ml h−1; P < 0.05, one-tailed paired t test), and dichlorobenzamil (1.5 × 10−5 M) exerted an additive effect to that of amiloride resulting in secretion at +6.29 ± 3.05 ml h−1 (P < 0.01, paired t test).In the lungs of sheep aged 6 weeks (n = 3), amiloride (10−4m) also inhibited the resting absorption of liquid (from −26.36 ± 14.05 to −5.17 ± 8.27 ml h−1; P < 0.05, one-tailed paired t test); however, dichlorobenzamil (1.5 × 10−5m) did not exert an additive effect to that of amiloride.In the lungs of sheep aged 6 months (n = 4), amiloride (10−4m) partially inhibited the resting absorption of liquid (from −35.70 ± 8.58 to −6.79 ± 4.28 ml h−1; P < 0.05, paired t test), and pimozide (1.5 × 10−4m), another blocker of cyclic nucleotide-gated cation channels, also exerted an additive effect to that of amiloride, resulting in secretion of lung liquid at +15.36 ± 9.14 ml h−1 (P < 0.05, paired t test).We conclude that cyclic nucleotide-gated cation channels mediate a component of lung liquid absorption in sheep aged 6 months (but not in sheep aged 6 weeks), and that a mechanism for lung liquid secretion (present in fetuses) is retained at 6 months of age

Summer Chamber Music Festival

A faculty and guest artist recital performed at the UNT College of Music Recital Hall

PKA and Epac synergistically inhibit smooth muscle cell proliferation

Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. Cyclic AMP inhibits VSMC proliferation via mechanisms that are not fully understood. We investigated the role of PKA and Epac signalling on cAMP-induced inhibition of VSMC proliferation. cAMP-mediated growth arrest was PKA-dependent. However, selective PKA activation with 6-Benzoyl-cAMP did not inhibit VSMC proliferation, indicating a requirement for additional pathways. Epac activation using the selective cAMP analogue 8-CPT-2′-O-Me-cAMP, did not affect levels of hyperphosphorylated Retinoblastoma (Rb) protein, a marker of G1-S phase transition, or BrdU incorporation, despite activation of the Epac-effector Rap1. However, 6-Benzoyl-cAMP and 8-CPT-2′-O-Me-cAMP acted synergistically to inhibit Rb-hyperphosphorylation and BrdU incorporation, indicating that both pathways are required for growth inhibition. Consistent with this, constitutively active Epac increased Rap1 activity and synergised with 6-Benzoyl-cAMP to inhibit VSMC proliferation. PKA and Epac synergised to inhibit phosphorylation of ERK and JNK. Induction of stellate morphology, previously associated with cAMP-mediated growth arrest, was also dependent on activation of both PKA and Epac. Rap1 inhibition with Rap1GAP or siRNA silencing did not negate forskolin-induced inhibition of Rb-hyperphosphorylation, BrdU incorporation or stellate morphology. This data demonstrates for the first time that Epac synergises with PKA via a Rap1-independent mechanism to mediate cAMP-induced growth arrest in VSMC. This work highlights the role of Epac as a major player in cAMP-dependent growth arrest in VSMC