Vocal divergence is concordant with genomic evidence for strong reproductive isolation in grasshopper mice (Onychomys)

Behavioral barriers to gene flow often evolve faster than intrinsic incompatibilities and can eliminate the opportunity for hybridization between interfertile species. While acoustic signal divergence is a common driver of premating isolation in birds and insects, its contribution to speciation in mammals is less studied. Here we characterize the incidence of, and potential barriers to, hybridization among three closely related species of grasshopper mice (genus Onychomys). All three species use long‐distance acoustic signals to attract and localize mates; Onychomys arenicola and Onychomys torridus are acoustically similar and morphologically cryptic whereas Onychomys leucogaster is larger and acoustically distinct. We used genotyping‐by‐sequencing (GBS) to test for evidence of introgression in 227 mice from allopatric and sympatric localities in the western United States and northern Mexico. We conducted laboratory mating trials for all species pairs to assess reproductive compatibility, and recorded vocalizations from O. arenicola and O. torridus in sympatry and allopatry to test for evidence of acoustic character displacement. Hybridization was rare in nature and, contrary to prior evidence for O. torridus/O. arenicola hybrids, only involved O. leucogaster and O. arenicola. In contrast, laboratory crosses between O. torridus and O. arenicola produced litters whereas O. leucogaster and O. arenicola crosses did not. Call fundamental frequency in O. torridus and O. arenicola was indistinguishable in allopatry but significantly differentiated in sympatry, a pattern consistent with reproductive character displacement. These results suggest that assortative mating based on a long‐distance signal is an important isolating mechanism between O. torridus and O. arenicola and highlight the importance of behavioral barriers in determining the permeability of species boundaries.     

NMR assignments of 1H, 13C and 15N spectra of methionine sulfoxide reductase B1 from Mus musculus

Isotopically labeled, ¹⁵N and ¹⁵N/¹³C forms of recombinant methionine-r-sulfoxide reductase 1 (MsrB1, SelR) from Mus musculus were produced, in which catalytic selenocysteine was replaced with cysteine. We report here the ¹H, ¹⁵N and ¹³C NMR assignment of the reduced form of this mammalian protein.     

Simultaneous determination of 6 beta-blockers, 3 calcium-channel antagonists, 4 angiotensin-II antagonists and 1 antiarrhythmic drug in post-mortem whole blood by automated solid phase extraction and liquid chromatography mass spectrometry. Method development and robustness testing by experimental design

A method for the simultaneous determination of the beta-blockers atenolol, sotalol, metoprolol, bisoprolol, propranolol and carvedilol, the calcium-channel antagonists diltiazem, amlodipine and verapamil, the angiotensin-II antagonists losartan, irbesartan, valsartan and telmisartan, and the antiarrhythmic drug flecainide, in whole blood samples from forensic autopsies was developed. Sample clean-up was achieved by precipitation and solid phase extraction (SPE) with a mixed-mode column. Quantification was performed by reversed phase high performance liquid chromatography with positive electrospray ionization mass spectrometric detection (HPLC-MS). The method has been developed and robustness tested by systematically searching for satisfactory conditions using experimental designs including factorial and response surface designs. With the exception of amlodipine, the concentration limit of quantification (cLOQ) covered low therapeutic concentration levels for all the compounds. Within assay precisions and accuracies (bias) were 3.4-21% RSD and from -24 to 21% for the concentration range 1.00-5.00 microM, respectively. Between assay precisions were 4.4-28% RSD for the concentration range from 0.1 to 5 microM and recoveries varied from 9 to 103%. The method is used for determination of cardiovascular drugs in post-mortem whole blood samples from forensic autopsy cases.     

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Expression level and agonist-binding affect the turnover, ubiquitination and complex formation of peroxisome proliferator activated receptor beta

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to fatty acid ligands. Their regulation by post-translational modifications has been reported but is poorly understood. In the present study, we investigated whether ligand binding affects the turnover and ubiquitination of the PPARbeta subtype (also known as PPARdelta). Our data show that the ubiquitination and degradation of PPARbeta is not significantly influenced by the synthetic agonist GW501516 under conditions of moderate PPARbeta expression. By contrast, the overexpression of PPARbeta dramatically enhanced its degradation concomitant with its polyubiquitination and the formation of high molecular mass complexes containing multiple, presumably oligomerized PPARbeta molecules that lacked stoichiometical amounts of the obligatory PPARbeta dimerization partner, retinoid X receptor. The formation of these apparently aberrant complexes, as well as the ubiquitination and destabilization of PPARbeta, were strongly inhibited by GW501516. Our findings suggest that PPARbeta is subject to complex post-translational regulatory mechanisms that partly may serve to safeguard the cell against deregulated PPARbeta expression. Furthermore, our data have important implications regarding the widespread use of overexpression systems to evaluate the function and regulation of PPARs.