Methyl β-lactoside: 600-MHz H- and 75-MHz C-n.m.r. studies of H- and C-enriched compounds

Using UDP-d-galactose : 2-acetamido-2-deoxy-d-glucose 4-β-d-galactosyltransferase (EC 2.4.1.22), several methyl β-lactosides have been prepared with ²H- and/or ¹³C-enrichment at specific sites to facilitate study by ¹³C (75 MHz) and ¹H (600 MHz) n.m.r. spectroscopy. ¹³C-Chemical shift assignments were verified and the ¹H-spectrum of β-lactoside was fully assigned. Sites of enrichment were selected to permit all of the potential three-bond C-C and C-H couplings through the glycosidic bond to be obtained. Replacement of H-3 of the d-glucose residue of methyl β-lactoside with ²H allowed resolution of C-1–H-4′ coupling in the 600-MHz ¹H-spectrum. Single or multiple ¹³C-enrichment at C-1, C-2, C-3, C-1′, C-3′, and/or C-4′ in the disaccharide allowed observation of intra- and inter-residue couplings. ¹³C-Spin-lattice relaxation-times (T₁) are interpreted in terms of molecular motion in solution. The data suggest that methyl β-lactoside has an extended conformation with little rotation about the glycosidic bond. Inter-residue couplings are best explained by tortion angles of φ ~ 40° and ψ ~ 15°, indicating that the conformations of β-lactoside in solution and in the crystal are similar.     

Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

R67 dihydrofolate reductase (R67 DHFR) is a plasmid‐encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry‐related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N‐termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N‐terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4‐ and 5‐fluoroindole labeled protein and severe line broadening for 6‐ and 7‐fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the ¹⁹F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.     

Distinct acyl protein transferases and thioesterases control surface expression of calcium-activated potassium channels

Protein palmitoylation is rapidly emerging as an important determinant in the regulation of ion channels, including large conductance calcium-activated potassium (BK) channels. However, the enzymes that control channel palmitoylation are largely unknown. Indeed, although palmitoylation is the only reversible lipid modification of proteins, acyl thioesterases that control ion channel depalmitoylation have not been identified. Here, we demonstrate that palmitoylation of the intracellular S0-S1 loop of BK channels is controlled by two of the 23 mammalian palmitoyl-transferases, zDHHC22 and zDHHC23. Palmitoylation by these acyl transferases is essential for efficient cell surface expression of BK channels. In contrast, depalmitoylation is controlled by the cytosolic thioesterase APT1 (LYPLA1), but not APT2 (LYPLA2). In addition, we identify a splice variant of LYPLAL1, a homolog with ～30% identity to APT1, that also controls BK channel depalmitoylation. Thus, both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Because depalmitoylated BK channels are retarded in the trans-Golgi network, reversible protein palmitoylation provides a critical checkpoint to regulate exit from the trans-Golgi network and thus control BK channel cell surface expression.     

Conservation in a cup of water: estimating biodiversity and population abundance from environmental DNA

Three mantras often guide species and ecosystem management: (i) for preventing invasions by harmful species, ‘early detection and rapid response’; (ii) for conserving imperilled native species, ‘protection of biodiversity hotspots’; and (iii) for assessing biosecurity risk, ‘an ounce of prevention equals a pound of cure.’ However, these and other management goals are elusive when traditional sampling tools (e.g. netting, traps, electrofishing, visual surveys) have poor detection limits, are too slow or are not feasible. One visionary solution is to use an organism’s DNA in the environment (eDNA), rather than the organism itself, as the target of detection. In this issue of Molecular Ecology, Thomsen et al. (2012) provide new evidence demonstrating the feasibility of this approach, showing that eDNA is an accurate indicator of the presence of an impressively diverse set of six aquatic or amphibious taxa including invertebrates, amphibians, a fish and a mammal in a wide range of freshwater habitats. They are also the first to demonstrate that the abundance of eDNA, as measured by qPCR, correlates positively with population abundance estimated with traditional tools. Finally, Thomsen et al. (2012) demonstrate that next‐generation sequencing of eDNA can quantify species richness. Overall, Thomsen et al. (2012) provide a revolutionary roadmap for using eDNA for detection of species, estimates of relative abundance and quantification of biodiversity.     

Stress-induced phosphorylation and proteasomal degradation of mitofusin 2 facilitates mitochondrial fragmentation and apoptosis

Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery.     

Biofeedback treatments of generalized anxiety disorder: Preliminary results

Forty-five individuals with generalized anxiety (38 with GAD as defined by DSM-III) were randomized to 4 treatment conditions or a waiting list control. Patients received 8 sessions of either frontal EMG biofeedback, biofeedback to increase EEG alpha, biofeedback to decrease EEG alpha, or a pseudomeditation control condition. All treated subjects showed significant reductions in STAI-Trait Anxiety and psychophysiologic symptoms on the Psychosomatic Symptom Checklist. Only alpha-increase biofeedback subjects showed significant reductions in heart rate reactivity to stressors at a separate psychophysiological testing session. Decreased self-report of anxiety was maintained at 6 weeks posttreatment.     

Testing the impact of calibration on molecular divergence times using a fossil-rich group: the case of Nothofagus (Fagales)

Although temporal calibration is widely recognized as critical for obtaining accurate divergence-time estimates using molecular dating methods, few studies have evaluated the variation resulting from different calibration strategies. Depending on the information available, researchers have often used primary calibrations from the fossil record or secondary calibrations from previous molecular dating studies. In analyses of flowering plants, primary calibration data can be obtained from macro- and mesofossils (e.g., leaves, flowers, and fruits) or microfossils (e.g., pollen). Fossil data can vary substantially in accuracy and precision, presenting a difficult choice when selecting appropriate calibrations. Here, we test the impact of eight plausible calibration scenarios for Nothofagus (Nothofagaceae, Fagales), a plant genus with a particularly rich and well-studied fossil record. To do so, we reviewed the phylogenetic placement and geochronology of 38 fossil taxa of Nothofagus and other Fagales, and we identified minimum age constraints for up to 18 nodes of the phylogeny of Fagales. Molecular dating analyses were conducted for each scenario using maximum likelihood (RAxML + r8s) and Bayesian (BEAST) approaches on sequence data from six regions of the chloroplast and nuclear genomes. Using either ingroup or outgroup constraints, or both, led to similar age estimates, except near strongly influential calibration nodes. Using "early but risky" fossil constraints in addition to "safe but late" constraints, or using assumptions of vicariance instead of fossil constraints, led to older age estimates. In contrast, using secondary calibration points yielded drastically younger age estimates. This empirical study highlights the critical influence of calibration on molecular dating analyses. Even in a best-case situation, with many thoroughly vetted fossils available, substantial uncertainties can remain in the estimates of divergence times. For example, our estimates for the crown group age of Nothofagus varied from 13 to 113 Ma across our full range of calibration scenarios. We suggest that increased background research should be made at all stages of the calibration process to reduce errors wherever possible, from verifying the geochronological data on the fossils to critical reassessment of their phylogenetic position.     

Connectivity of Caribbean coral populations: complementary insights from empirical and modelled gene flow

Understanding patterns of connectivity among populations of marine organisms is essential for the development of realistic, spatially explicit models of population dynamics. Two approaches, empirical genetic patterns and oceanographic dispersal modelling, have been used to estimate levels of evolutionary connectivity among marine populations but rarely have their potentially complementary insights been combined. Here, a spatially realistic Lagrangian model of larval dispersal and a theoretical genetic model are integrated with the most extensive study of gene flow in a Caribbean marine organism. The 871 genets collected from 26 sites spread over the wider Caribbean subsampled 45.8% of the 1900 potential unique genets in the model. At a coarse scale, significant consensus between modelled estimates of genetic structure and empirical genetic data for populations of the reef-building coral Montastraea annularis is observed. However, modelled and empirical data differ in their estimates of connectivity among northern Mesoamerican reefs indicating that processes other than dispersal may dominate here. Further, the geographic location and porosity of the previously described east-west barrier to gene flow in the Caribbean is refined. A multi-prong approach, integrating genetic data and spatially realistic models of larval dispersal and genetic projection, provides complementary insights into the processes underpinning population connectivity in marine invertebrates on evolutionary timescales.     

Social behavior of Matschie's tree kangaroos (Dendrolagus matschiei) and its implications for captive management

Social behavior was studied in four (one male and three females) adult, captivebred Matschie's tree kangaroos (Dendrolagus matschiei) over a 124 day span in a large, naturalistic exhibit at the Woodland Park Zoological Gardens, Seattle, WA. A relatively high rate of social interaction occurred (15.2 per hour), over half of which consisted of approaches and nose contacts. The male initiated 54.1% of all social behaviors. Females tended to respond aggressively toward the male (avoid, bite, cuff, or swipe). The male initiated more affiliative behaviors and the females initiated more agonistic behaviors toward both the male and other females. A clear-cut dominance hierarchy could not be determined; reversals (i.e., cases in which the “subordinate” supplanted the “dominant”) occurred 43% of the time overall. Non-aggressive contact behavior consisted primarily of olfactory examination (nose contact). Allogrooming was extremely rare. The high level of agonism and lack of consistent association among individuals suggests that this species may be solitary in the wild. Two joeys were found dead on the floor during the course of the study, and based on previous findings, infanticide or behavioral stress was suspected to be the cause. Successful reproduction did not occur until females were isolated by removing other conspecifics from the enclosure.