Effect of respiratory alkalosis on skeletal muscle metabolism in the dog

These experiments were conducted to determine whether changes in skeletal muscle metabolism contribute to the previously reported increase in whole-body O2 uptake (VO2) during respiratory alkalosis. The hind-limb and gastrocnemius-plantaris preparations in anesthetized and paralyzed dogs were used. VO2 of the hindlimb and gastrocnemius muscle was calculated from measurements of venous blood flow and arterial and venous O2 concentrations (Van Slyke analysis). Whole-body VO2 was measured by the open-circuit method. Minute ventilation (hence blood gases and pH) was controlled by a mechanical respirator. Whole-body, hind-limb, and gastrocnemius muscle VO2 increased 14, 19, and 20%, respectively, during alkalosis (P less than 0.05). In all experiments, arterial lactate concentration increased significantly (P less than 0.05) during alkalosis. A positive venoarterial lactate difference across muscle during alkalosis indicated that skeletal muscle is a source of the elevated blood lactate. We concluded that VO2 of resting skeletal muscle is increased during states of respiratory alkalosis and that this increase can account for much of the increase in whole-body VO2.     

Incorporating uncertainty and prior information into stable isotope mixing models

Stable isotopes are a powerful tool for ecologists, often used to assess contributions of different sources to a mixture (e.g. prey to a consumer). Mixing models use stable isotope data to estimate the contribution of sources to a mixture. Uncertainty associated with mixing models is often substantial, but has not yet been fully incorporated in models. We developed a Bayesian-mixing model that estimates probability distributions of source contributions to a mixture while explicitly accounting for uncertainty associated with multiple sources, fractionation and isotope signatures. This model also allows for optional incorporation of informative prior information in analyses. We demonstrate our model using a predator–prey case study. Accounting for uncertainty in mixing model inputs can change the variability, magnitude and rank order of estimates of prey (source) contributions to the predator (mixture). Isotope mixing models need to fully account for uncertainty in order to accurately estimate source contributions.     

Analysing ecological networks of species interactions

Network approaches to ecological questions have been increasingly used, particularly in recent decades. The abstraction of ecological systems – such as communities – through networks of interactions between their components indeed provides a way to summarize this information with single objects. The methodological framework derived from graph theory also provides numerous approaches and measures to analyze these objects and can offer new perspectives on established ecological theories as well as tools to address new challenges. However, prior to using these methods to test ecological hypotheses, it is necessary that we understand, adapt, and use them in ways that both allow us to deliver their full potential and account for their limitations. Here, we attempt to increase the accessibility of network approaches by providing a review of the tools that have been developed so far, with – what we believe to be – their appropriate uses and potential limitations. This is not an exhaustive review of all methods and metrics, but rather, an overview of tools that are robust, informative, and ecologically sound. After providing a brief presentation of species interaction networks and how to build them in order to summarize ecological information of different types, we then classify methods and metrics by the types of ecological questions that they can be used to answer from global to local scales, including methods for hypothesis testing and future perspectives. Specifically, we show how the organization of species interactions in a community yields different network structures (e.g., more or less dense, modular or nested), how different measures can be used to describe and quantify these emerging structures, and how to compare communities based on these differences in structures. Within networks, we illustrate metrics that can be used to describe and compare the functional and dynamic roles of species based on their position in the network and the organization of their interactions as well as associated new methods to test the significance of these results. Lastly, we describe potential fruitful avenues for new methodological developments to address novel ecological questions.     

Structural organization of the protein-tyrosine autokinase Wzc within Escherichia coli cells

Protein Wzc from Escherichia coli is a member of a newly defined family of protein-tyrosine autokinases that are essential for surface polysaccharide production in both Gram-negative and Gram-positive bacteria. Although the catalytic mechanism of the autophosphorylation of Wzc was recently described, the in vivo structural organization of this protein remained unclear. Here, we have determined the membrane topology of Wzc by performing translational fusions of lacZ and phoA reporter genes to the wzc gene. It has been shown that Wzc consists of two main structural domains: an N-terminal domain, bordered by two transmembrane helices, which is located in the periplasm of cells, and a C-terminal domain, harboring all phosphorylation sites of the protein, which is located in the cytoplasm. In addition, it has been demonstrated for the first time that Wzc can oligomerize in vivo to form essentially trimers and hexamers. Cross-linking experiments performed on strains expressing various domains of Wzc have shown that the cytoplasmic C-terminal domain is sufficient to generate oligomerization of Wzc. Mutant proteins, modified in either the ATP-binding site or the different phosphorylation sites, i.e. rendered unable to undergo autophosphorylation, have appeared to oligomerize into high molecular mass species identical to those formed by the wild-type protein. It was concluded that phosphorylation of Wzc is not essential to its oligomerization. These data, connected with the phosphorylation mechanism of Wzc, may be of biological significance in the regulatory role played by this kinase in polysaccharide synthesis.     

Genetic mosaic in a marine species flock

We used molecular approaches to study the status of speciation in coral reef fishes known as hamlets (Serranidae: Hypoplectrus). Several hamlet morphospecies coexist on Caribbean reefs, and mate assortatively with respect to their strikingly distinct colour patterns. We provide evidence that, genetically, the hamlets display characteristics common in species flocks on land and in freshwaters. Substitutions within two mitochondrial DNA (mtDNA) protein-coding genes place hamlets within a monophyletic group relative to members of two related genera (Serranus and Diplectrum), and establish that the hamlet radiation must have been very recent. mtDNA distances separating hamlet morphospecies were slight (0.6 +/- 0.04%), yielding a coalescent estimate for the age of the hamlet flock of approximately 430 000 years. Morphospecies did not sort into distinct mtDNA haplotype phylogroups, and alleles at five hypervariable microsatellite loci were shared broadly across species boundaries. None the less, molecular variation was not distributed at random. Analyses of mtDNA haplotype frequencies and nested clades in haplotype networks revealed significant genetic differences between geographical regions and among colour morphospecies. We also observed significant microsatellite differentiation between geographical regions and in Puerto Rico, among colour morphospecies; the latter providing evidence for reproductive isolation between colour morphospecies at this locale. In our Panama collection, however, colour morphospecies were mostly genetically indistinguishable. This mosaic pattern of DNA differentiation implies a complex interaction between population history, mating behaviour and geography and suggests that porous boundaries separate species in this flock of brilliantly coloured coral reef fishes.     

Recombination hot spot in a 3.2-kb region of the Charcot-Marie-Tooth type 1A repeat sequences: new tools for molecular diagnosis of hereditary neuropathy with liability to pressure palsies and of Charcot-Marie-Tooth type 1A. French CMT Collaborative Research Group.

Charcot-Marie-Tooth type 1A (CMT1A) disease and hereditary neuropathy with liability to pressure palsies (HNPP) are autosomal dominant neuropathies, associated, respectively, with duplications and deletions of the same 1.5-Mb region on 17p11.2-p12. These two rearrangements are the reciprocal products of an unequal meiotic crossover between the two chromosome 17 homologues, caused by the misalignment of the CMT1A repeat sequences (CMT1A-REPs), the homologous sequences flanking the 1.5-Mb CMT1A/HNPP monomer unit. In order to map recombination breakpoints within the CMT1A-REPs, a 12.9-kb restriction map was constructed from cloned EcoRI fragments of the proximal and distal CMT1A-REPs. Only 3 of the 17 tested restriction sites were present in the proximal CMT1A-REP but absent in the distal CMT1A-REP, indicating a high degree of homology between these sequences. The rearrangements were mapped in four regions of the CMT1A-REPs by analysis of 76 CMT1A index cases and 38 HNPP patients, who where unrelated. A hot spot of crossover breakpoints, located in a 3.2-kb region, accounted for three-quarters of the rearrangements, detected after EcoRI/SacI digestion, by the presence of 3.2-kb and 7.8-kb junction fragments in CMT1A and HNPP patients, respectively. These junction fragments, which can be detected on classical Southern blots, permit molecular diagnosis. Other rearrangements can also be detected by gene dosage on the same Southern blots.     

Seasonal variations in active dispersal of natural populations of Triatoma infestans in rural north-western Argentina

The flight dispersal of Triatoma infestans Klug (Hemiptera: Reduviidae) is one of the main mechanisms determining community re-infestation after control interventions. An empirical model of flight initiation coupled with data from a longitudinal study predicted that the flight dispersal of T. infestans would peak in summer. To test this prediction, longitudinal light trap collections were conducted during 3-8 nights in March (late summer), July (winter) and November (spring) 2003, and in March 2004 in a rural community in north-west Argentina. Following each light-trapping collection date, all peridomestic sites around light traps were inspected to assess the relative abundance and nutritional status of T. infestans at each site. A total of 21 adult and five nymph T. infestans, six Triatoma guasayana Wygodzinsky & Abalos, and nine Triatoma garciabesi Carcavallo et al. were collected in 96 light-trapping nights, whereas 696 T. infestans were collected from the peridomestic sites that surrounded the light traps. The arrival of T. infestans in the light traps occurred in 64% of catch stations and peaked in the summer surveys (10-14 bugs) compared with spring and winter surveys. When winds were < 5 km/h, the arrival of adult T. infestans at the light traps was significantly associated with maximum temperature and relative humidity. This is the first field report of seasonal variations in the flight dispersal activity of T. infestans.     

Human cyclooxygenase-2 is a sequence homodimer that functions as a conformational heterodimer

Prostaglandin endoperoxide H synthases 1 and 2, also known as cyclooxygenases (COXs) 1 and 2, convert arachidonic acid (AA) to prostaglandin endoperoxide H(2). Prostaglandin endoperoxide H synthases are targets of nonspecific nonsteroidal anti-inflammatory drugs and COX-2-specific inhibitors called coxibs. PGHS-2 is a sequence homodimer. Each monomer has a peroxidase and a COX active site. We find that human PGHS-2 functions as a conformational heterodimer having a catalytic monomer (E(cat)) and an allosteric monomer (E(allo)). Heme binds tightly only to the peroxidase site of E(cat), whereas substrates, as well as certain inhibitors (e.g. celecoxib), bind the COX site of E(cat). E(cat) is regulated by E(allo) in a manner dependent on what ligand is bound to E(allo). Substrate and nonsubstrate fatty acids (FAs) and some COX inhibitors (e.g. naproxen) preferentially bind to the COX site of E(allo). AA can bind to E(cat) and E(allo), but the affinity of AA for E(allo) is 25 times that for E(cat). Palmitic acid, an efficacious stimulator of human PGHS-2, binds only E(allo) in palmitic acid/murine PGHS-2 co-crystals. Nonsubstrate FAs can potentiate or attenuate actions of COX inhibitors depending on the FA and whether the inhibitor binds E(cat) or E(allo). Our studies suggest that the concentration and composition of the free FA pool in the environment in which PGHS-2 functions in cells, the FA tone, is a key factor regulating PGHS-2 activity and its responses to COX inhibitors. We suggest that differences in FA tone occurring with different diets will likely affect both base-line prostanoid synthesis and responses to COX inhibitors.