Wild Populations of Triatoma infestans Are Highly Connected to Intra-Peridomestic Conspecific Populations in the Bolivian Andes

Triatoma infestans, the major vector of Chagas disease south of the Amazon in South America, has a large distribution of wild populations, contrary to what has previously been stated. These populations have been suspected of being the source of reinfestation of human habitats and could impede the full success of vector control campaigns. This study examined gene flow between intra-peridomestic populations and wild populations collected in the surround areas in three Andean localities in Bolivia. The populations were defined according to temporal, ecological, and spatial criteria. After DNA extraction from the legs of each insect, the samples were analyzed using seven microsatellite markers. First, the analysis of molecular variance (AMOVA) detected an absence of differentiation between wild and intra-peridomestic populations, although strong structuring was observed between the populations within each environment. Then for some populations, the Bayesian method of assignment to inferred populations showed very similar assignment patterns of the members of wild or intra-peridomestic populations in each locality. Finally, the detection of the first-generation migrants within the different populations provided evidence of insect displacement from the wild to the intra-peridomestic environment. This result indicates that, after control campaigns in the Andes, controlling this new paradigm of vector transmission risk stemming from the invasion of human habitats by wild populations of T. infestans requires long-term maintenance of public monitoring to keep the risk at a minimal level. Since wild populations of T. infestans have also been detected elsewhere in Argentina, Paraguay, and Chile, there is an urgent need to take these populations into account in future monitoring of Chagas disease transmission.     

Size and metabolic properties of fibers in rat fast-twitch muscles after hindlimb suspension

Hindlimb suspension (HS) results in whole muscle atrophic and metabolic changes that vary in magnitude in different hindlimb muscles. The present study was designed to investigate these effects in single fibers. Fiber type and size and the activities of two metabolic marker enzymes were determined in a deep (close to the bone) and a superficial (away from the bone) region of the medial gastrocnemius (MG) and the tibialis anterior (TA) of control (CON) and 28-day HS adult female rats. Fibers were classified as dark or light adenosinetriphosphatase (ATPase) based on their qualitative staining reaction for myosin ATPase following alkaline preincubation. Fiber area and succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities were determined in tissue sections by use of an image analysis system. After 28 days of HS, the mean body weights of the CON and HS were similar. MG atrophied 28%, whereas TA weight was maintained in the HS. Both dark and light ATPase fibers in the deep region of the MG had smaller cross-sectional areas following HS, with the atrophic response being approximately twice as great in the light ATPase fibers. No significant changes in fiber type composition in either muscle or in fiber sizes in the superficial region of the MG or in either region of the TA were observed. Mean SDH activities of both fiber types were significantly lower in the MG and TA following HS. In contrast, mean GPD activities were either increased or maintained in light and dark ATPase fibers of both muscles in HS. Changes in SDH and GPD activity could not be directly linked to changes in fiber cross-sectional area. In summary, these data suggest an independence of the mechanisms determining muscle fiber size and metabolic adaptations associated with HS.     

A note on the complexity of finding and enumerating elementary modes

In the context of the study into elementary modes of metabolic networks, we prove two complexity results. Enumerating elementary modes containing a specific reaction is hard in an enumeration complexity sense. The decision problem if there exists an elementary mode containing two specific reactions is NP-complete. The complexity of enumerating all elementary modes remains open.     

Toxoplasma gondii grown in human cells uses GalNAc-containing glycosylphosphatidylinositol precursors to anchor surface antigens while the immunogenic Glc-GalNAc-containing precursors remain free at the parasite cell surface

Toxoplasma gondii is a ubiquitous parasite that infects nearly all warm-blooded animals. Developmental switching in T. gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for the disease propagation after alteration of the immune status of the carrier. The redifferentiation event is characterized by an over expression of a tachyzoite specific set of glycosylphosphatidylinositol anchored surface antigens and free GPIs. T. gondii grown in animal cells uses two glycosylphosphatidylinositol precursors to anchor the parasite surface proteins. The first form has an N-acetylgalactosamine residue bound to a conserved three-mannosyl core glycan, while the second structure contains an additional terminal glucose linked to the N-acetylgalactosamine side branch. Sera from persons infected with T. gondii reacted only with the glucose-N-acetylgalactosamine-containing structure. Here we report that T. gondii cultured in human cells uses predominantly the N-acetylgalactosamine-containing structure to anchor the parasite surface antigens. On the other hand, glycosylphosphatidylinositol structures having an additional terminal glucose are found exclusively on the parasite cell surface as free glycolipids participating in the production of cytokines that are implicated in the pathogenesis of T. gondii. We also provide evidence that such free glycosylphosphatidylinositols are restricted mainly to the lipid microdomains in the parasite cell surface membrane and mostly associated with proteins involved in the parasite motility as well as invasion of the host cell.     

Metabolic network visualization eliminating node redundance and preserving metabolic pathways

Background  

The tools that are available to draw and to manipulate the representations of metabolism are usually restricted to metabolic pathways. This limitation becomes problematic when studying processes that span several pathways. The various attempts that have been made to draw genome-scale metabolic networks are confronted with two shortcomings: 1- they do not use contextual information which leads to dense, hard to interpret drawings, 2- they impose to fit to very constrained standards, which implies, in particular, duplicating nodes making topological analysis considerably more difficult.     

Genetic background determines metabolic phenotypes in the mouse

To evaluate the contribution of genetic background to phenotypic variation, we compared a large range of biochemical and metabolic parameters at different ages of four inbred mice strains, C57BL/6J, 129SvPas, C3HeB/FeJ, and Balb/cByJ. Our results demonstrate that important metabolic, hematologic, and biochemical differences exist between these different inbred strains. Most of these differences are gender independent and are maintained or accentuated throughout life. It is therefore imperative that the genetic background is carefully defined in phenotypic studies. Our results also argue that certain backgrounds are more suited to study a given physiologic phenomenon, as distinct mouse strains have a different propensity to develop particular biochemical, hematologic, and metabolic abnormalities. These genetic differences can furthermore be exploited to identify new genes/proteins that contribute to phenotypic abnormalities. The choice of the genetic background in which to generate and analyze genetically engineered mutant mice is important as it is, together with environmental factors, one of the most important contributors to the variability of phenotypic results.     

Vinculin Is a Dually Regulated Actin Filament Barbed End-capping and Side-binding Protein

The focal adhesion protein vinculin is an actin-binding protein involved in the mechanical coupling between the actin cytoskeleton and the extracellular matrix. An autoinhibitory interaction between the N-terminal head (Vh) and the C-terminal tail (Vt) of vinculin masks an actin filament side-binding domain in Vt. The binding of several proteins to Vh disrupts this intramolecular interaction and exposes the actin filament side-binding domain. Here, by combining kinetic assays and microscopy observations, we show that Vt inhibits actin polymerization by blocking the barbed ends of actin filaments. In low salt conditions, Vt nucleates actin filaments capped at their barbed ends. We determined that the interaction between vinculin and the barbed end is characterized by slow association and dissociation rate constants. This barbed end capping activity requires C-terminal amino acids of Vt that are dispensable for actin filament side binding. Like the side-binding domain, the capping domain of vinculin is masked by an autoinhibitory interaction between Vh and Vt. In contrast to the side-binding domain, the capping domain is not unmasked by the binding of a talin domain to Vh and requires the dissociation of an additional autoinhibitory interaction. Finally, we show that vinculin and the formin mDia1, which is involved in the processive elongation of actin filaments in focal adhesions, compete for actin filament barbed ends.     

TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton

Subversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections. Both pathogens translocate the trans-membrane receptor protein-translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton. While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir(EPEC) requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir(EHEC) is not phosphorylated and utilizes an unidentified linker. Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells. A tccP mutant did not affect Tir translocation and focusing but failed to recruit alpha-actinin, Arp3, N-WASP and actin to the site of bacterial adhesion. When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line. TccP has a similar biological activity on infected human intestinal explants ex vivo. Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro. These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.     

Posttranslational Modifications and β/γ Chain Associations of Human Laminin α1 and Laminin α5 Chains: Purification of Laminin-3 from Placenta

Laminins assemble into trimers composed of α, β, and γ chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin α1 and α5 chains. Comparative pulse–chase experiments and deglycosylation studies in JAR cells established that the M_r 360,000 laminin α1 chain is glycosylated into a mature M_r 400,000 band while the M_r 370,000 laminin α5 chain is glycosylated into a M_r 390,000 form that upon secretion is further processed into a M_r 380,000 form. Hence, despite the shorter peptide length of α1 chain in comparison with the α5 chain, secreted α1 assumes a larger size in SDS–PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin α1 and laminin α5 chains in laminin-1 and laminin-10. In placenta laminin α1 chain (M_r 400,000) and laminin α5 chain (M_r 380,000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin α1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin β2 chains. Surprisingly, a fraction of the laminin α1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin β1–β3 chains, possibly pointing to an unexpected complexity in the chain composition of α1-containing laminin isoforms.     

The depletion of F1 subunit ε in yeast leads to an uncoupled respiratory phenotype that is rescued by mutations in the proton-translocating subunits of F0

The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F₁ and the proton translocation channel in F₀ for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F₀-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F₀ subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F₀.