Regulation of exercise carbohydrate metabolism by estrogen and progesterone in women

To assess the roles of endogenous estrogen (E2) and progesterone (P4) in regulating exercise carbohydrate use, we used pharmacological suppression and replacement to create three distinct hormonal environments: baseline (B), with E2 and P4 low; estrogen only (E), with E2 high and P4 low; and estrogen/progesterone (E + P), with E2 and P4 high. Blood glucose uptake (R(d)), total carbohydrate oxidation (CHO(ox)), and estimated muscle glycogen utilization (EMGU) were assessed during 60 min of submaximal exercise by use of stable isotope dilution and indirect calorimetry in eight eumenorrheic women. Compared with B (1.26 +/- 0.04 g/min) and E + P (1.27 +/- 0.04 g/min), CHO(ox) was lower with E (1.05 +/- 0.02 g/min). Glucose R(d) tended to be lower with E and E + P relative to B. EMGU was 25% lower with E than with B or E + P. Plasma free fatty acids (FFA) were inversely related to EMGU (r(2) = 0.49). The data suggest that estrogen lowers CHO(ox) by reducing EMGU and glucose R(d). Progesterone increases EMGU but not glucose R(d). The opposing actions of E(2) and P(4) on EMGU may be mediated by their impact on FFA availability or vice versa.     

Shared molecular characteristics of successfully transformed mitochondrial genomes in <Emphasis Type="Italic">Chlamydomonas</Emphasis><Emphasis Type="Italic">reinhardtii</Emphasis>

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16). We found that these respiratory deficient strains of C. reinhardtii can be divided into two groups: strains that are constantly transformable and those could not be transformed in our experiments. All transformable mitochondrial strains were limited to the type that has a large deletion in the left arm of the genome. For these mitochondria, transformation was successful not only with purified intact mitochondrial genomes but also with DNA-constructs containing the compensating regions. In comparison, mitochondria of all the non-transformable strains have both of their genome termini intact, leading us to speculate that mitochondria lacking their left genome terminus have unstable genomes and might have a higher potential for recombination. Analysis of mitochondrial gene organization in the resulting respiratory active transformants was performed by DNA sequencing and restriction enzyme digestion. Such analysis showed that homologous recombination occurred at various regions between the mitochondrial genome and the artificial DNA-constructs. Further analysis by Southern hybridization showed that the wild-type genome rapidly replaces the respiratory deficient monomer and dimer mitochondrial genomes, while the E. coli vector region of the artificial DNA-construct likely does not remain in the mitochondria.     

Uracil-DNA glycosylase in base excision repair and adaptive immunity: species differences between man and mouse

Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung(+/+) and Ung(-/-) backcrossed mice. Interestingly, human cells displayed ～15-fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG1 activity was ～8-fold higher in mouse cells, constituting ～50% of the total U:G excision activity compared with less than 1% in human cells. In activated B cells, both UNG and SMUG1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG1 may work as a weak backup activity for UNG2 during class switch recombination in Ung(-/-) mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity.     

Priority threat management of invasive animals to protect biodiversity under climate change

Climate change is a major threat to global biodiversity, and its impacts can act synergistically to heighten the severity of other threats. Most research on projecting species range shifts under climate change has not been translated to informing priority management strategies on the ground. We develop a prioritization framework to assess strategies for managing threats to biodiversity under climate change and apply it to the management of invasive animal species across one‐sixth of the Australian continent, the Lake Eyre Basin. We collected information from key stakeholders and experts on the impacts of invasive animals on 148 of the region's most threatened species and 11 potential strategies. Assisted by models of current distributions of threatened species and their projected distributions, experts estimated the cost, feasibility, and potential benefits of each strategy for improving the persistence of threatened species with and without climate change. We discover that the relative cost‐effectiveness of invasive animal control strategies is robust to climate change, with the management of feral pigs being the highest priority for conserving threatened species overall. Complementary sets of strategies to protect as many threatened species as possible under limited budgets change when climate change is considered, with additional strategies required to avoid impending extinctions from the region. Overall, we find that the ranking of strategies by cost‐effectiveness was relatively unaffected by including climate change into decision‐making, even though the benefits of the strategies were lower. Future climate conditions and impacts on range shifts become most important to consider when designing comprehensive management plans for the control of invasive animals under limited budgets to maximize the number of threatened species that can be protected.     

Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.     

Moose movement rates are altered by wolf presence in two ecosystems

Predators directly impact prey populations through lethal encounters, but understanding nonlethal, indirect effects is also critical because foraging animals often face trade‐offs between predator avoidance and energy intake. Quantifying these indirect effects can be difficult even when it is possible to monitor individuals that regularly interact. Our goal was to understand how movement and resource selection of a predator (wolves; Canis lupus) influence the movement behavior of a prey species (moose; Alces alces). We tested whether moose avoided areas with high predicted wolf resource use in two study areas with differing prey compositions, whether avoidance patterns varied seasonally, and whether daily activity budgets of moose and wolves aligned temporally. We deployed GPS collars on both species at two sites in northern Minnesota. We created seasonal resource selection functions (RSF) for wolves and modeled the relationship between moose first‐passage time (FPT), a method that discerns alterations in movement rates, and wolf RSF values. Larger FPT values suggest rest/foraging, whereas shorter FPT values indicate travel/fleeing. We found that the movements of moose and wolves peaked at similar times of day in both study areas. Moose FPTs were 45% lower in areas most selected for by wolves relative to those avoided. The relationship between wolf RSF and moose FPT was nonlinear and varied seasonally. Differences in FPT between low and high RSF values were greatest in winter (?82.1%) and spring (?57.6%) in northeastern Minnesota and similar for all seasons in the Voyageurs National Park ecosystem. In northeastern Minnesota, where moose comprise a larger percentage of wolf diet, the relationship between moose FPT and wolf RSF was more pronounced (ave. across seasons: ?60.1%) than the Voyageurs National Park ecosystem (?30.4%). These findings highlight the role wolves can play in determining moose behavior, whereby moose spend less time in areas with higher predicted likelihood of wolf resource selection.     

Supervillin binding to myosin II and synergism with anillin are required for cytokinesis

Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin''s role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.     

Apoptotic Regulation by MCL-1 through Heterodimerization

Myeloid cell leukemia 1 (MCL-1), an anti-apoptotic BCL-2 family member active in the preservation of mitochondrial integrity during apoptosis, has fundamental roles in development and hematopoiesis and is dysregulated in human cancers. It bears a unique, intrinsically unstructured, N-terminal sequence, which leads to its instability in cells and hinders protein production and structural characterization. Here, we present collective data from NMR spectroscopy and titration calorimetry to reveal the selectivity of MCL-1 in binding BCL-2 homology 3 (BH3) ligands of interest for mammalian biology. The N-terminal sequence weakens the BH3 interactions but does not affect selectivity. Its removal by calpain-mediated limited proteolysis results in a stable BCL-2-like core domain of MCL-1 (cMCL-1). This core is necessary and sufficient for BH3 ligand binding. Significantly, we also characterized the in vitro protein-protein interaction between cMCL-1 and activated BID by size exclusion chromatography and NMR titrations. This interaction occurs in a very slow manner in solution but is otherwise similar to the interaction between cMCL-1 and BID-BH3 peptides. We also present the solution structure of complex cMCL-1·hBID-BH3, which completes the family portrait of MCL-1 complexes and may facilitate drug discovery against human tumors.     

Hemin as a generic and potent protein misfolding inhibitor

Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer’s disease, Parkinson’s disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.     

Porphyromonas gingivalis lipopolysaccharide induces miR-146a without altering the production of inflammatory cytokines

Lipopolysaccharide (LPS) from Porphyromonas gingivalis, an oral Gram-negative bacterium, acts as a virulence factor for periodontal disease. Although P. gingivalis LPS does not induce proinflammatory cytokines as strongly as Escherichia coli LPS, it is still able to exploit negative Toll-like receptor (TLR) regulatory pathways and facilitate pathogen persistence. Recent reports suggest that microRNAs (miRNAs) are also involved in the regulation of TLR signaling. Here, we demonstrate that P. gingivalis LPS strongly induces miRNA-146a expression in THP-1 cells and THP-1-derived macrophages. However, the inhibition or overexpression of miR-146a, through the transfection of a specific inhibitor or precursor, respectively, had little effect on cytokine production in macrophages stimulated with P. gingivalis LPS. Moreover, the expression of interleukin-1 associated-kinase-1 (IRAK-1) and tumor-necrosis factor (TNF) receptor-associated factor-6 (TRAF6), potential target molecules of miR-146a, were not affected by the stimulation with P. gingivalis LPS. Because TLR signaling induces various negative regulators, these results call into question the role of miR-146a in cells stimulated with TLR ligands.