Specific Inter-residue Interactions as Determinants of Human Monoacylglycerol Lipase Catalytic Competency: A ROLE FOR GLOBAL CONFORMATIONAL CHANGES*

The serine hydrolase monoacylglycerol lipase (MGL) functions as the main metabolizing enzyme of 2-arachidonoyl glycerol, an endocannabinoid signaling lipid whose elevation through genetic or pharmacological MGL ablation exerts therapeutic effects in various preclinical disease models. To inform structure-based MGL inhibitor design, we report the direct NMR detection of a reversible equilibrium between active and inactive states of human MGL (hMGL) that is slow on the NMR time scale and can be modulated in a controlled manner by pH, temperature, and select point mutations. Kinetic measurements revealed that hMGL substrate turnover is rate-limited across this equilibrium. We identify a network of aromatic interactions and hydrogen bonds that regulates hMGL active-inactive state interconversion. The data highlight specific inter-residue interactions within hMGL modulating the enzymes function and implicate transitions between active (open) and inactive (closed) states of the hMGL lid domain in controlling substrate access to the enzymes active site.     

Investigating thermal acclimation effects before and after a cold shock in Drosophila melanogaster using behavioural assays

Acclimation to environmental change can impose both costs and benefits to organisms. In this study we explored to what extent locomotor behaviour of Drosophila melanogaster is influenced by developmental temperature and adult temperature in both the laboratory and the field. Following development at 15, 25, or 31 °C, adult flies were tested for locomotor activity at all developmental temperatures in the laboratory before and after exposure to a cold shock and in the field for their ability to locate resources after a cold shock. Both test (15, 25, and 31 °C) and developmental temperatures strongly affected locomoter activity, with flies developed at 25 °C having the highest activity at all three test temperatures before the cold shock. After the cold shock flies developed at 15 °C had higher activity compared with flies developed at 25 and 31 °C when tested at 15 and 25 °C, and flies developed at 25 °C had the highest activity when tested at 31 °C. Furthermore, flies developed at 31 °C showed longer recovery times following the cold shock at test temperatures of 15 and 25 °C. However, flies acclimated at 15 °C during development did not recover faster at 15 and 25 °C compared with flies developed at 25 °C. There were no significant correlations between recovery time and locomotor activity at any of the test temperatures. Flies developed at 15 °C and exposed to a cold shock before release in the field were much more successful in locating a resource at low field temperatures compared with flies developed at 25 and 31 °C. Our results provide support for both the beneficial acclimation hypothesis and the optimal developmental temperature hypothesis, but the results are highly context dependent and change with the temperature experienced by the individual during its lifetime.     

Haplotyping cryptic Adélie penguin taxa using low-cost,high-resolution melt curves

Distinguishing morphologically cryptic taxa, by definition, requires genetic data such as DNA sequences. However, DNA sequences may not be obtained easily for taxa from remote sites. Here we provide the details of a high-resolution melt-curve-based method using taxon-specific primers that can distinguish two taxa of Adélie penguins, and that will be usable in Antarctica when combined with some of the newly developed field-deployable thermal cyclers. We suggest that the wider adoption of field-deployable polymerase-chain-reaction-based techniques will enable faster assignation of haplotype to individuals in situ, and so allow the targeting of observations and sample collection to specimens relevant to the research question. Targeting individuals will also reduce the need to repeatedly handle animals and reduce the time and travel required to complete field work.     

The pectic disaccharides lepidimoic acid and β-d-xylopyranosyl-(1→3)-d-galacturonic acid occur in cress-seed exudate but lack allelochemical activity

Background and aims Cress-seed (Lepidium sativum) exudate exerts an allelochemical effect, promoting excessive hypocotyl elongation and inhibiting root growth in neighbouring Amaranthus caudatus seedlings. We investigated acidic disaccharides present in cress-seed exudate, testing the proposal that the allelochemical is an oligosaccharin—lepidimoic acid (LMA; 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnose).Methods Cress-seed exudate was variously treated [heating, ethanolic precipitation, solvent partitioning, high-voltage paper electrophoresis and gel-permeation chromatography (GPC)], and the products were bioassayed for effects on dark-grown Amaranthus seedlings. Two acidic disaccharides, including LMA, were isolated and characterized by electrophoresis, thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy, and then bioassayed.Key Results Cress-seed exudate contained low-M_r, hydrophilic, heat-stable material that strongly promoted Amaranthus hypocotyl elongation and inhibited root growth, but that separated from LMA on electrophoresis and GPC. Cress-seed exudate contained ∼250 µm LMA, whose TLC and electrophoretic mobilities, susceptibility to mild acid hydrolysis and NMR spectra are reported. A second acidic disaccharide, present at ∼120 µm, was similarly characterized, and shown to be β-d-xylopyranosyl-(1→3)-d-galacturonic acid (Xyl→GalA), a repeat unit of xylogalacturonan. Purified LMA and Xyl→GalA when applied at 360 and 740 µm, respectively, only slightly promoted Amaranthus hypocotyl growth, but equally promoted root growth and thus had no effect on the hypocotyl:root ratio, unlike total cress-seed exudate.Conclusions LMA is present in cress seeds, probably formed by rhamnogalacturonan lyase action on rhamnogalacturonan-I during seed development. Our results contradict the hypothesis that LMA is a cress allelochemical that appreciably perturbs the growth of potentially competing seedlings. Since LMA and Xyl→GalA slightly promoted both hypocotyl and root elongation, their effect could be nutritional. We conclude that rhamnogalacturonan-I and xylogalacturonan (pectin domains) are not sources of oligosaccharins with allelochemical activity, and the biological roles (if any) of the disaccharides derived from them are unknown. The main allelochemical principle in cress-seed exudate remains to be identified.     

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC¹]-Gm; [TOAC¹, Ser^2,6,11,15]-Gm; [Trp⁷]-Gm; [Ser^2,6,11,15, Trp⁷]-Gm; [Trp⁹]-Gm; and [Ser^2,6,11,15, Trp⁹]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a β-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC¹]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.     

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: Differential scanning calorimetric and Fourier transform infrared spectroscopic studies

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into α-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D₂O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1 > aurein 1.2 > citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1 > aurein 1.2 ≅ citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.     

Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of alpha-synuclein

Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of alpha-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-alpha-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of alpha-synuclein.     

Dynamical stress characterization and energy evaluation of single cardiac myocyte actuating on flexible substrate

Contractility estimation of cardiac myocyte is important for power evaluation of the cell in heart performance. In this paper, we used digital image correlation (DIC) method to obtain dynamic deformation field of the flexible substrate distributively actuated by single cardiac myocyte, which resulted in the dynamic history of traction forces of the cell during contraction-relaxation cycles. Furthermore, the resultant work and power of the single neonatal cardiac myocyte was evaluated to show the energy characteristics of the cells and the responses to the stiffness variation of the substrate. The method provides a useful tool to study behaviors of the cardiac myocytes interacted with the substrates.