Identification of calmodulin and MlcC as light chains for Dictyostelium myosin-I isozymes

Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.     

Ecological Effects of Live Salmon Exceed Those of Carcasses During an Annual Spawning Migration

We tested the hypothesis that the carcasses of anadromous Pacific salmon (Oncorhynchus spp.) constitute a significant source of nutrients in the nutrient-poor freshwaters where these fish migrate, spawn, senesce, and die. In a 110 m-long stream reach in Southeast Alaska, we retained nearly 3000 salmon carcasses and compared streamwater nitrogen (N), phosphorus (P), and the biomass of benthic biofilm in this reach with an upstream reference reach. The study spanned 5 months, bracketed the entire salmon run, and encompassed significant seasonal variation in abiotic stream conditions. Concentrations of dissolved and particulate N and P followed distinctly unimodal patterns through time, which tracked the abundance of live salmon, and we observed strong predictive relationships between live-salmon abundance and streamwater-nutrient concentrations. In contrast, we did not observe clear relationships between salmon carcasses and streamwater nutrients. Biofilm biomass within our study reaches seemed to more closely track the abundance of live salmon than the abundance of carcasses. The experimental retention of carcasses had a minor or undetectable influence on nutrient concentrations and biofilm within the study reach as compared to the reference reach. We conclude that physical factors such as temperature, discharge, nutrient limitation, and irradiance vary seasonally in ways that maximize the influence of nutrients provisioned by live salmon and minimize the influence of carcass-derived nutrients on the aspects of stream ecosystems that we examined. Overall, our results promote a new perspective on the ecological role of salmon in freshwaters, and contribute to a more mechanistic understanding of how migratory fishes can influence aquatic ecosystems.     

Factors affecting the length of the breeding cycle and the frequency of nest attendance by Grey Herons Ardea cinerea

An investigation of the length of the breeding cycle and of the frequency of nest visits by Grey Herons at 3 heronries with different degrees of breeding success showed that the duration of reproduction, particularly the chickrearing period, decreased and the frequency of daily nest visits increased with increasing average breeding success of a colony. Successful pairs also seemed to spend less time displaying and incubating eggs. Herons arrived at their nests most frequently during twilight, especially in the morning. The mean time absent from the nest was greatest during incubation and smallest when feeding small chicks. The total reproductive period tended to be longer for those pairs whose periods of absence from the nest were longer.     

Proprotein Convertase Subtilisin/Kexin Type 3 Promotes Adipose Tissue-Driven Macrophage Chemotaxis and Is Increased in Obesity

Background

Matrix metalloproteinase (MMP)-dependent extracellular matrix (ECM) remodeling is a key feature in cardiometabolic syndrome-associated adipogenesis and atherosclerosis. Activation of membrane-tethered (MT) 1-MMP depends on furin (PCSK3). However, the regulation and function of the natural furin-inhibitor serpinB8 and thus furin/MT1-MMP-activity in obesity-related tissue inflammation/remodeling is unknown. Here we aimed to determine the role of serpinB8/furin in obesity-associated chronic inflammation.

Methods and Results

Monocyte → macrophage transformation was characterized by decreases in serpinB8 and increases in furin/MT1-MMP. Rescue of serpinB8 by protein overexpression inhibited furin-dependent pro-MT1-MMP activation in macrophages, supporting its role as a furin-inhibitor. Obese white adipose tissue-facilitated macrophage migration was inhibited by furin- and MMP-inhibition, stressing the importance of the furin-MMP axis in fat tissue inflammation/remodeling. Monocytes from obese patients (body mass index (BMI) >30kg/m²) had higher furin, MT1-MMP, and resistin gene expression compared to normal weight individuals (BMI<25kg/m²) with significant correlations of BMI/furin and furin/MT1-MMP. In vitro, the adipocytokine resistin induced furin and MT1-MMP in mononuclear cells (MNCs), while MCP-1 had no effect.

Conclusions

Acquisition of the inflammatory macrophage phenotype is characterized by an imbalance in serpinB8/furin, leading to MT1-MMP activation, thereby enhancing migration. Increases in MT1-MMP and furin are present in MNCs from obese patients. Dissecting the regulation of furin and its inhibitor serpinB8 should facilitate targeting inflammation/remodeling in cardiometabolic diseases.     

A comparative study of type I and type II tryparedoxin peroxidases in Leishmania major

The genome of Leishmania major, the causative agent of cutaneous leishmaniasis, contains three almost identical genes encoding putative glutathione peroxidases, which differ only at their N- and C-termini. Because the gene homologues are essential in trypanosomes, they may also represent potential drug targets in Leishmania. Recombinant protein for the shortest of these showed negligible peroxidase activity with glutathione as the electron donor indicating that it is not a bone fide glutathione peroxidase. By contrast, high peroxidase activity was obtained with tryparedoxin, indicating that these proteins belong to a new class of monomeric tryparedoxin-dependent peroxidases (TDPX) distinct from the classical decameric 2-Cys peroxiredoxins (TryP). Mass spectrometry studies revealed that oxidation of TDPX1 with peroxides results in the formation of an intramolecular disulfide bridge between Cys35 and Cys83. Site-directed mutagenesis and kinetic studies showed that Cys35 is essential for peroxidase activity, whereas Cys83 is essential for reduction by tryparedoxin. Detailed kinetic studies comparing TDPX1 and TryP1 showed that both enzymes obey saturation ping-pong kinetics with respect to tryparedoxin and peroxide. Both enzymes show high affinity for tryparedoxin and broad substrate specificity for hydroperoxides. TDPX1 shows higher affinity towards hydrogen peroxide and cumene hydroperoxide than towards t-butyl hydroperoxide, whereas no specific substrate preference could be detected for TryP1. TDPX1 exhibits rate constants up to 8 x 10(4) m(-1).s(-1), whereas TryP1 exhibits higher rate constants approximately 10(6) m(-1).s(-1). All three TDPX proteins together constitute approximately 0.05% of the L. major promastigote protein content, whereas the TryPs are approximately 40 times more abundant. Possible specific functions of TDPXs are discussed.     

Impact of phagostimulants on effectiveness of OMRI‐listed insecticides used for control of spotted‐wing drosophila (Drosophila suzukii Matsumura)

Spotted‐wing drosophila, Drosophila suzukii Matsumura, is an invasive pest in the United States that causes considerable damage to fruit crops. It is responsible for many millions of dollars of revenue loss. The female D. suzukii has a heavily sclerotized ovipositor and can lay eggs in ripening or ripe fruit. The arrival of this invasive species has disrupted existing integrated pest management programmes, and growers rely on repeated insecticide applications to protect fruit. Organic growers have few chemical control options, and their reliance on spinosad increases the risk of developing insecticide resistance. We hypothesized that combining phagostimulants with insecticides would increase insecticide efficacy by prompting flies to spend more time in contact with residues. Therefore, the objective of this study was to evaluate the effectiveness of sucrose and the yeast Saccharomyces cerevisiae as phagostimulants in combination with organic biopesticides against D. suzukii in blueberries. Adding sucrose with or without yeast did not improve insecticide efficacy in terms of adult fly mortality or fruit infestation. Spinosad was very effective in all experiments, and for this product, there is little room for improvement. The phagostimulants had no effect on residual activity of any insecticide. The addition of sucrose with or without yeast did not improve the effectiveness of organic insecticides for D. suzukii. Concentrations of these phagostimulants in our experiments (0.36%) may have been too low to elicit a response. Further research is recommended to test different types and concentrations of phagostimulants.