Downregulation of Wnt-Mediated ROS generation is causally implicated in leprechaunism

Although mutations in the insulin receptor have been causally implicated with leprechaunsim, the full pathophysiology of the syndrome cannot be accounted for by malfunction of this gene alone. We sought to characterize a connection between Wnt-mediated cell signaling and the production of reactive oxygen species (ROS) which revealed a novel mechanistic basis for understanding the pathogenesis of leprechaunism. To identify candidate genes involved in this process, a PCR-based subtractive hybridization was performed. Candidate genes were examined for interaction with the Wnt signaling pathway and ROS generation. We found that Dickkopf 1 (Dkk1), a Wnt inhibitor, is overexpressed in skin fibroblast cells derived from three leprechaunism patients and that the cells showed an impaired response to Wnt2 in terms of β-catenin-Tcf activation. Knockdown of Dkk1 in the patient cell lines rescued Wnt2-mediated Tcf activation. Concerted action of Wnt2 and knockdown of Dkk1 resulted in enhanced Nox4 expression and PDGF-induced ROS generation compared to parental patient cells. Furthermore, we found that NFATc2 was activated in response to Wnt2 stimulation and directly activates Nox4 expression. These data show a crosstalk between Wnt and ROS pathways which in turn provides new mechanistic insights at the molecular level into the pathogenesis of leprechaunism.     

The differential affinity of the usher for chaperone–subunit complexes is required for assembly of complete pili

Attachment to host cells via adhesive surface structures is a prerequisite for the pathogenesis of many bacteria. Uropathogenic Escherichia coli assemble P and type 1 pili for attachment to the host urothelium. Assembly of these pili requires the conserved chaperone/usher pathway, in which a periplasmic chaperone controls the folding of pilus subunits and an outer membrane usher provides a platform for pilus assembly and secretion. The usher has differential affinity for pilus subunits, with highest affinity for the tip‐localized adhesin. Here, we identify residues F21 and R652 of the P pilus usher PapC as functioning in the differential affinity of the usher. R652 is important for high‐affinity binding to the adhesin whereas F21 is important for limiting affinity for the PapA major rod subunit. PapC mutants in these residues are specifically defective for pilus assembly in the presence of PapA, demonstrating that differential affinity of the usher is required for assembly of complete pili. Analysis of PapG deletion mutants demonstrated that the adhesin is not required to initiate P pilus biogenesis. Thus, the differential affinity of the usher may be critical to ensure assembly of functional pilus fibres.     

HDAC1 and HDAC3 underlie dynamic H3K9 acetylation during embryonic neurogenesis and in schizophrenia‐like animals

Although histone acetylation is one of the most widely studied epigenetic modifications, there is still a lack of information regarding how the acetylome is regulated during brain development and pathophysiological processes. We demonstrate that the embryonic brain (E15) is characterized by an increase in H3K9 acetylation as well as decreases in the levels of HDAC1 and HDAC3. Moreover, experimental induction of H3K9 hyperacetylation led to the overexpression of NCAM in the embryonic cortex and depletion of Sox2 in the subventricular ependyma, which mimicked the differentiation processes. Inducing differentiation in HDAC1‐deficient mouse ESCs resulted in early H3K9 deacetylation, Sox2 downregulation, and enhanced astrogliogenesis, whereas neuro‐differentiation was almost suppressed. Neuro‐differentiation of (wt) ESCs was characterized by H3K9 hyperacetylation that was associated with HDAC1 and HDAC3 depletion. Conversely, the hippocampi of schizophrenia‐like animals showed H3K9 deacetylation that was regulated by an increase in both HDAC1 and HDAC3. The hippocampi of schizophrenia‐like brains that were treated with the cannabinoid receptor‐1 inverse antagonist AM251 expressed H3K9ac at the level observed in normal brains. Together, the results indicate that co‐regulation of H3K9ac by HDAC1 and HDAC3 is important to both embryonic brain development and neuro‐differentiation as well as the pathophysiology of a schizophrenia‐like phenotype.     

Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates

Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.     

Strategic positioning of Beauveria bassiana sensu lato JEF-410 in management of poultry red mite,Dermanyssus gallinae (Mesostigmata: Dermanyssidae)

BioControl - The red mite, Dermanyssus gallinae (Mesostigmata: Dermanyssidae), is one of the most serious pests in chicken farming and causes serious economic losses. Overuse of chemical acaricides...     

Immunohistochemical visualisation of the enteric nervous system architecture in the germ-free piglets

Journal of Molecular Histology - The enteric nervous system (ENS), considered as separate branch of the autonomic nervous system, is located throughout the length of the gastrointestinal tract as a...