Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox

Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.     

Differences in crystal and solution structures of the cytolethal distending toxin B subunit: Relevance to nuclear translocation and functional activation

Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly233-Asp242). Residues Leu186-Thr209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined alpha-helix and beta-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions.     

Very low additive genetic variance and evolutionary potential in multiple populations of two rainforest Drosophila species

Most quantitative traits are thought to exhibit high levels of genetic variance and evolutionary potential. However, this conclusion may be biased by a lack of studies on nonmodel organisms and may not generalize to restricted species. A recent study on a single, southern population of the rainforest-restricted Drosophila birchii failed to find significant additive genetic variance for the desiccation resistance trait; however, it is unclear whether this pattern extends to other D. birchii populations or to other rainforest species. Here we use an animal model design to show very low levels of additive genetic variance for desiccation resistance in multiple populations of two highly sensitive rainforest species of Drosophila from tropical northeastern Australia. In contrast, relatively high levels of genetic variance were found for morphological traits in all populations of the species tested. This indicates limited evolutionary potential for evolving increased desiccation resistance in these rainforest restricted species.     

Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens

A key target of many intracellular pathogens is the macrophage. Although macrophages can generate antimicrobial activity, neutrophils have been shown to have a key role in host defense, presumably by their preformed granules containing antimicrobial agents. Yet the mechanism by which neutrophils can mediate antimicrobial activity against intracellular pathogens such as Mycobacterium tuberculosis has been a long-standing enigma. We demonstrate that apoptotic neutrophils and purified granules inhibit the growth of extracellular mycobacteria. Phagocytosis of apoptotic neutrophils by macrophages results in decreased viability of intracellular M. tuberculosis. Concomitant with uptake of apoptotic neutrophils, granule contents traffic to early endosomes, and colocalize with mycobacteria. Uptake of purified granules alone decreased growth of intracellular mycobacteria. Therefore, the transfer of antimicrobial peptides from neutrophils to macrophages provides a cooperative defense strategy between innate immune cells against intracellular pathogens and may complement other pathways that involve delivery of antimicrobial peptides to macrophages.     

The effect of anthropogenic disturbance on the hydrochemical characteristics of riparian wetlands at the Middle Ebro River (NE Spain)

In natural systems, the chemistry of floodplain waters is a function of the source of the water, which is influenced by geomorphic features of riparian wetlands. However, anthropogenic disturbances may alter both geomorphic features and the natural balance of water mixing in the floodplain. The aim of this study was to classify riparian wetlands and characterize their water characteristics in one reach of the Middle Ebro River to assess the hydrochemical functioning of the system. In order to accomplish that goal, water samples were collected at 40 sampling sites during low-water conditions and two floods of different magnitude. Moreover, geomorphic characteristics of riparian wetlands were also analyzed to interpret the results at broader spatio-temporal scales. Three group of wetlands were identified using multivariate ordination: (1) major and secondary channels highly connected to the river by surface water, containing weakly ionized water with high nitrate levels during floods; (2) secondary channels and artificial ponds located in riparian forests near the river, most of which were affected by river seepage during the examined events. This type of sites had high major ions concentrations and elevated spatial variability with respect to nutrient concentrations during floods; (3) Siltated oxbow lakes, whose hydrogeochemical features seemed to be unaffected by factors related to river fluctuations. Total dissolved solids, major ion (sulfate, chloride, sodium, calcium, magnesium, and potassium) and nutrient (nitrate, ammonium and organic nitrogen, and phosphate) depended upon the relationships between surface and subsurface water flows. Seasonal changes and geomorphic characterization indicated that a strong functional dependence of floodplain wetlands close to the main river channel is established, whereas most of the floodplain area remains disconnected from river dynamics. Moreover, the effect of nitrate-enriched agricultural runoff seems to affect water quality and hydrochemical gradients of the system. Based on our results, we propose different types of actions for the management of the Ebro River flow to ensure a more natural ecological functioning of its floodplains. Handling editor: P. Viaroli     

Molecular evolution of SPARC: absence of the acidic module and expression in the endoderm of the starlet sea anemone, Nematostella vectensis

The matricellular glycoprotein SPARC is composed of three functional domains that are evolutionarily conserved in organisms ranging from nematodes to mammals: a Ca²⁺-binding glutamic acid-rich acidic domain at the N-terminus (domain I), a follistatin-like module (domain II), and an extracellular Ca²⁺-binding (EC) module that contains two EF-hands and two collagen-binding epitopes (domain III). We report that four SPARC orthologs (designated nvSPARC1-4) are expressed by the genome of the starlet anemone Nematostella vectensis, a diploblastic basal cnidarian composed of an ectoderm and endoderm separated by collagen-based mesoglea. We also report that domain I is absent from all N. vectensis SPARC orthologs. In situ hybridization data indicate that N. vectensis SPARC mRNAs are restricted to the endoderm during post-gastrula development. The absence of the Ca²⁺-binding N-terminal domain in cnidarians and conservation of collagen-binding epitopes suggests that SPARC first evolved as a collagen-binding matricellular glycoprotein, an interaction likely to be dependent on the binding of Ca²⁺-ions to the two EF-hands in the EC domain. We propose that further Ca²⁺-dependent activities emerged with the acquisition of an acidic N-terminal module in triplobastic organisms.     

The podosomal-adaptor protein SH3PXD2B is essential for normal postnatal development

Podosome-type adhesions are actin-based membrane protrusions involved in cell-matrix adhesion and extracellular matrix degradation. Despite growing knowledge of many proteins associated with podosome-type adhesions, much remains unknown concerning the function of podosomal proteins at the level of the whole animal. In this study, the spontaneous mouse mutant nee was used to identify a component of podosome-type adhesions that is essential for normal postnatal growth and development. Mice homozygous for the nee allele exhibited runted growth, craniofacial and skeletal abnormalities, ocular anterior segment dysgenesis, and hearing impairment. Adults also exhibited infertility and a form of lipodystrophy. Using genetic mapping and DNA sequencing, the cause of nee phenotypes was identified as a 1-bp deletion within the Sh3pxd2b gene on mouse Chromosome 11. Whereas the wild-type Sh3pxd2b gene is predicted to encode a protein with one PX domain and four SH3 domains, the nee mutation is predicted to cause a frameshift and a protein truncation altering a portion of the third SH3 domain and deleting all of the fourth SH3 domain. The SH3PXD2B protein is believed to be an important component of podosomes likely to mediate protein-protein interactions with membrane-spanning metalloproteinases. Testing this directly, SH3PXD2B localized to podosomes in constitutively active Src-transfected fibroblasts and through its last SH3 domain associated with a transmembrane member of a disintegrin and metalloproteinase family of proteins, ADAM15. These results identify SH3PXD2B as a podosomal-adaptor protein required for postnatal growth and development, particularly within physiologic contexts involving extracellular matrix regulation.     

Plasmodium falciparum: Genetic and immunogenic characterisation of the rhoptry neck protein PfRON4

The Apicomplexan parasites Toxoplasma and Plasmodium, respectively, cause toxoplasmosis and malaria in humans and although they invade different host cells they share largely conserved invasion mechanisms. Plasmodium falciparum merozoite invasion of red blood cells results from a series of co-ordinated events that comprise attachment of the merozoite, its re-orientation, release of the contents of the invasion-related apical organelles (the rhoptries and micronemes) followed by active propulsion of the merozoite into the cell via an actin-myosin motor. During this process, a tight junction between the parasite and red blood cell plasma membranes is formed and recent studies have identified rhoptry neck proteins, including PfRON4, that are specifically associated with the tight junction during invasion. Here, we report the structure of the gene that encodes PfRON4 and its apparent limited diversity amongst geographically diverse P. falciparum isolates. We also report that PfRON4 protein sequences elicit immunogenic responses in natural human malaria infections.     

Regulation of Oscillatory Contraction in Insect Flight Muscle by Troponin

Insect indirect flight muscle is activated by sinusoidal length change, which enables the muscle to work at high frequencies, and contracts isometrically in response to Ca²⁺. Indirect flight muscle has two TnC isoforms: F1 binding a single Ca²⁺ in the C-domain, and F2 binding Ca²⁺ in the N- and C-domains. Fibres substituted with F1 produce delayed force in response to a single rapid stretch, and those with F2 produce isometric force in response to Ca²⁺. We have studied the effect of TnC isoforms on oscillatory work. In native Lethocerus indicus fibres, oscillatory work was superimposed on a level of isometric force that depended on Ca²⁺ concentration. Maximum work was produced at pCa 6.1; at higher concentrations, work decreased as isometric force increased. In fibres substituted with F1 alone, work continued to rise as Ca²⁺ was increased up to pCa 4.7. Fibres substituted with various F1:F2 ratios produced maximal work at a ratio of 100:1 or 50:1; a higher proportion of F2 increased isometric force at the expense of oscillatory work. The F1:F2 ratio was 9.8:1 in native fibres, as measured by immunofluorescence, using isoform-specific antibodies. The small amount of F2 needed to restore work to levels obtained for the native fibre is likely to be due to the relative affinity of F1 and F2 for TnH, the Lethocerus homologue of TnI. Affinity of TnC isoforms for a TnI fragment of TnH was measured by isothermal titration calorimetry. The K_d was 1.01 μM for F1 binding and 22.7 nM for F2. The higher affinity of F2 can be attributed to two TnH binding sites on F2 and a single site on F1. Stretch may be sensed by an extended C-terminal domain of TnH, resulting in reversible dissociation of the inhibitory sequence from actin during the oscillatory cycle.