CHX10 targets a subset of photoreceptor genes

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.     

Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association

We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent-free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48-fold increase in k(cat)/K(m) (to 5.9 x 10(7)M(-1)s(-1)) with concomitant changes in the spin state equilibrium of the haem-iron due to the binding of a single detergent micelle to the protein monomer, which is atypical of P450 enzymes. This mechanism is shown to be an important determinant of the substrate specificity of CYP74A1. CYP74A1 may be suited for structural resolution of the first plant cytochrome P450 and its 9-AOS activity and behaviour in vitro has implications for its role in planta.     

Inbreeding avoidance and the evolution of gender dimorphism in Wurmbea biglandulosa (Colchicaceae)

How females establish in populations of cosexuals is central to understanding the evolution of gender dimorphism in angiosperms. Inbreeding avoidance hypotheses propose that females can establish and be maintained if cosexual fitness is reduced because they self-fertilize, and their progeny express inbreeding depression. Here we assess the role of inbreeding avoidance in maintaining sexual system variation in Wurmbea biglandulosa. We estimated costs of self-pollination, mating patterns, and inbreeding depression in gender monomorphic (cosexuals only) and dimorphic (males and females) populations. Costs of selfing, estimated from seed set of experimentally self- and cross-pollinated flowers, were severe in both males and cosexuals (inbreeding depression, sigma = 0.86). In a field experiment, intact males that could self produced fewer seeds than both emasculated males and females, whereas seed set of intact and emasculated cosexuals did not differ. Thus, pollinator-mediated selfing reduces fitness of males but not cosexuals under natural conditions. Outcrossing rates of males revealed substantial selfing (t = 0.68), whereas females and cosexuals were outcrossed (0.92 and 0.97). For males, progeny inbreeding coefficients exceeded parental coefficients (0.220 vs. 0.009), whereas for females and cosexuals these coefficients did not differ and approached zero. Differences in coefficients between males and their progeny indicate that selfed progeny express severe inbreeding depression (sigma = 0.93). Combined with inbreeding depression for seed set, cumulative sigma = 0.99, indicating that most or all selfed zygotes fail to reach reproductive maturity. We propose that present sexual system variation in W. biglandulosa is maintained by high inbreeding depression coupled with differences in selfing rates among monomorphic and dimorphic populations.     

Adaptive recovery responses to repeated forward loss of balance in older adults

Experiments designed to assess balance recovery in older adults often involve exposing participants to repeated loss of balance. The purpose of this study was to investigate the adaptive balance recovery response exhibited by older adults following repeated exposure to forward loss of balance induced by releasing participants from a static forward lean angle. Fifty-eight healthy, community-dwelling older adults, aged 65-80 years, participated in the study. Participants were instructed to attempt to recover with a single step and performed four trials at each of three lean angles. Adaptive recovery responses at four events (cable release, toe-off of the stepping foot, foot contact and maximum knee flexion angle following landing in the stepping leg) were quantified for trials performed at the intermediate lean angle using the concept of margin of stability. The antero-posterior and medio-lateral margin of stability were computed as the difference between the velocity-adjusted position of the whole body centre of mass and the corresponding anterior or lateral boundary of the base of support. Across repeated trials adaptations in reactive stepping responses were detected that resulted in improved antero-posterior stability at foot contact and maximum knee flexion angle. Improved antero-posterior stability following repeated trials was explained by more effective control of the whole body centre of mass during the reactive stepping response and not by adjustments in step timing or base of support. The observed adaptations occurred within a single testing session and need to be considered in the design of balance recovery experiments.     

Transgenic expression of an altered angiotensin type I AT1 receptor resulting in marked modulation of vascular type I collagen

The angiotensin II (AngII) type I receptor (AT1) was modified by replacing its third intracellular loop and C-terminal tail with the corresponding regions from the bradykinin B2 receptor. Transgenic mice were produced that overexpress this mutated receptor (AB3T). Considerably less collagen content in the intact aorta and in primary aortic smooth muscle cells (aSMCs) cultures was observed in the transgenic mice. On the other hand, elastin content remained unchanged as measured by Western blot, and insoluble amino acid quantitation. The contraction of isolated aortas also remained unaltered. The aSMCs derived from the transgenic mice showed a reduction in AngII responsive type I collagen production. In aSMCs from transgenic mice, the cascade of Akt to the mammalian target rapamycin (mTOR) to p70 S6 kinase (p70S6K) was not AngII activated, while in the aSMCs from wild-type (WT) mice the cascade was AngII activated. Angiotensin activation of Smad2 and Stat3 was also reduced in the AB3T aSMCs. However, no change in the effect of transforming growth factor β (TGFβ) on type I collagen production was observed. Also, the activation of ERK and JNK and G-protein linked signaling remained unaltered in response to AngII. Akt and PI3K activation inhibitors blocked AngII-stimulated type I collagen expression in WT aSMCs, whereas ERK inhibitor had no such effect. Our results point to an Akt/mTOR/p70S6K regulation of collagen production by AngII with participation of Smad2 and Stat3 cascades in this process.     

Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler''s diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K_m(NAD⁺) = 6 μm, K_m(actin) = 24 μm, and k_cat = 22 s⁻¹. VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC₅₀ = 20 μm). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.     

The 1.8 Å cholix toxin crystal structure in complex with NAD+ and evidence for a new kinetic model

Certain Vibrio cholerae strains produce cholix, a potent protein toxin that has diphthamide-specific ADP-ribosyltransferase activity against eukaryotic elongation factor 2. Here we present a 1.8 Å crystal structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide (NAD⁺). We also substituted hallmark catalytic residues by site-directed mutagenesis and analyzed both NAD⁺ binding and ADP-ribosyltransferase activity using a fluorescence-based assay. These data are the basis for a new kinetic model of cholix toxin activity. Further, the new structural data serve as a reference for continuing inhibitor development for this toxin class.     

Caspar-like gene depletion reduces Leishmania infection in sand fly host Lutzomyia longipalpis

Female phlebotomine sand flies Lutzomyia longipalpis naturally harbor populations of the medically important Leishmania infantum (syn. Leishmania chagasi) parasite in the gut, but the extent to which the parasite interacts with the immune system of the insect vector is unknown. To investigate the sand fly immune response and its interaction with the Leishmania parasite, we identified a homologue for caspar, a negative regulator of immune deficiency signaling pathway. We found that feeding antibiotics to adult female L. longipalpis resulted in an up-regulation of caspar expression relative to controls. caspar was differentially expressed when females were fed on gram-negative and gram-positive bacterial species. caspar expression was significantly down-regulated in females between 3 and 6 days after a blood feed containing Leishmania mexicana amastigotes. RNA interference was used to deplete caspar expression in female L. longipalpis, which were subsequently fed with Leishmania in a blood meal. Sand fly gut populations of both L. mexicana and L. infantum were significantly reduced in caspar-depleted females. The prevalence of L. infantum infection in the females fell from 85 to 45%. Our results provide the first insight into the operation of immune homeostasis in phlebotomine sand flies during the growth of bacterial and Leishmania populations in the digestive tract. We have demonstrated that the activation of the sand fly immune system, via depletion of a single gene, can lead to the abortion of Leishmania development and the disruption of transmission by the phlebotomine sand fly.