The Pseudomonas aeruginosa quorum-sensing signal N-(3-oxododecanoyl) homoserine lactone can accelerate cutaneous wound healing through myofibroblast differentiation in rats

Quorum sensing is a cell density-dependent gene regulation system in bacteria. N-(3-oxododecanoyl) homoserine lactone (3-oxo-C12-HSL) is used in the las quorum-sensing system in Pseudomonas aeruginosa, which is an opportunistic pathogen that causes many human diseases. Although many studies have investigated the sole effects of quorum sensing on several types of mammalian cells, including lung cells, little is known about the effects of quorum sensing on the cells associated with wound healing. To better understand the mechanism of bacterial wound infection, we investigated the effects of 3-oxo-C12-HSL on cells using a rat full-thickness wound-healing model. We found that the wound contraction was significantly increased at 24 h after the administration of 3-oxo-C12-HSL to the surface of granulation tissue. Differentiation of fibroblasts to myofibroblasts was induced in the in vivo wound-healing model and was confirmed in vitro using the rat fibroblastic cell line Rat-1. Cyclooxygenase (Cox)-2 expression was also induced in Rat-1 cells by 3-oxo-C12-HSL. This finding suggested that Cox-2 upregulation may be related to the inflammatory findings in the histological examinations, in which infiltrating polymorphonuclear neutrophils were observed at the wound site. Taken together, these results imply that mammals have a potential defense system against invading pathogens by responding to the presence of 3-oxo-C12-HSL and inducing the differentiation of fibroblasts to myofibroblasts as well as inflammation for accelerating wound healing.     

Characterization of novel glycolipid antigens with an α-galactose epitope in lactobacilli detected with rabbit anti-Lactobacillus antisera and occurrence of antibodies against them in human sera

Anti-Lactobacillus johnsonii (LJ) antisera generated by immunization of rabbits with LJ reacted with glyceroglycolipids in LJ, i.e. dihexaosyl diacylglycerol (DH-DG), trihexaosyl DG (TH-DG) and tetrahexaosyl DG (TetH-DG), whose reactivities with antisera increased proportionally with longer carbohydrate chains of glycolipids. Structural analyses of glycolipids from LJ revealed that DH-DG was Galα1-2Glcα1-3'DG, and TH-DG and TetH-DG were novel derivatives of it with α-Gal at the non-reducing terminal, i.e. Galα1-6Galα1-2Glcα1-3'DG and Galα1-6Galα1-6Galα1-2Glcα1-3'DG, respectively. DH-DG was commonly present in several lactobacilli examined, but TetH-DG was restricted to LJ, L. intestinalis and L. reuteri, while the TH-DGs from L. casei were Glc1-6Galα1-2Glcα1-3'DG and an esterified derivative of it, Glc1-6Galα1-2Glc(6-fatty acid)α1-3'DG, as reported in the literature. Anti-LJ antisera reacted with TH-DG and esterified TH-DG from L. casei to lesser extents, but not at all with gentibiosyl DG from Staphylococcus epidermidis or kojibiosyl DG from Streptococcus salivalis or sphingoglycolipids containing α-Gal residues. The major molecular species of glycolipids obtained from lactobacilli were 11-octadecenoic and 11,12-methylene-octadecanoic acids-containing ones. Also, human IgM antibodies against TH-DG and TetH-DG from LJ were detected in human sera, with various antibody titres, indicating that an immune reaction to symbiotic lactobacilli occurs against their glycolipid antigens, TH-DG and TetH-DG.     

Variation in plant Toll/Interleukin-1 receptor domain protein dependence on ENHANCED DISEASE SUSCEPTIBILITY 1

Toll/Interleukin-1 receptor (TIR) domains are integral to immune systems across all kingdoms. In plants, TIRs are present in nucleotide-binding leucine-rich repeat (NLR) immune receptors, NLR-like, and TIR-only proteins. Although TIR-NLR and TIR signaling in plants require the ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) protein family, TIRs persist in species that have no EDS1 members. To assess whether particular TIR groups evolved with EDS1, we searched for TIR-EDS1 co-occurrence patterns. Using a large-scale phylogenetic analysis of TIR domains from 39 algal and land plant species, we identified 4 TIR families that are shared by several plant orders. One group occurred in TIR-NLRs of eudicots and another in TIR-NLRs across eudicots and magnoliids. Two further groups were more widespread. A conserved TIR-only group co-occurred with EDS1 and members of this group elicit EDS1-dependent cell death. In contrast, a maize (Zea mays) representative of TIR proteins with tetratricopeptide repeats was also present in species without EDS1 and induced EDS1-independent cell death. Our data provide a phylogeny-based plant TIR classification and identify TIRs that appear to have evolved with and are dependent on EDS1, while others have EDS1-independent activity.

Plant Toll/Interleukin-1 receptor domain proteins can use different mechanisms to induce cell death.     

CD83 expression is a sensitive marker of activation required for B cell and CD4+ T cell longevity in vivo

CD83 is a surface marker that differentiates immature and mature human dendritic cell populations. Thymic epithelial cell expression of CD83 is also necessary for efficient CD4+ T cell development in mice. The altered phenotypes of peripheral B and CD4+ T cells, and the reduction of peripheral CD4+ T cells in CD83-/- mice, suggest additional functions for CD83. To assess this, a panel of mAbs was generated to characterize mouse CD83 expression by peripheral leukocytes. As in humans, activation of conventional and plasmacytoid murine dendritic cell subsets led to rapid up-regulation of CD83 surface expression in mice. In primary and secondary lymphoid compartments, a subset of B cells expressed low-level CD83, while CD83 was not detected on resting T cells. However, CD83 was prominently up-regulated on the majority of spleen B and T cells within hours of activation in vitro. In vivo, a low dose of hen egg lysozyme (1 microg) induced significant CD83 but not CD69 expression by Ag-specific B cells within 4 h of Ag challenge. Although B cell development appeared normal in CD83-/- mice, B and CD4+ T cell expression of CD83 was required for lymphocyte longevity in adoptive transfer experiments. Thus, the restricted expression pattern of CD83, its rapid induction following B cell and T cell activation, and its requirement for B cell and CD4+ T cell longevity demonstrate that CD83 is a functionally significant and sensitive marker of early lymphocyte activation in vivo.     

Mutants of circadian-associated PRR genes display a novel and visible phenotype as to light responses during de-etiolation of Arabidopsis thaliana seedlings

In Arabidopsis thaliana, it is currently accepted that certain mutants with lesions in clock-associated genes commonly display hallmarked phenotypes with regard to three characteristic biological events: (i) altered rhythmic expression of circadian-controlled genes, (ii) changes in flowering time, and (iii) altered sensitivity to red light in elongation of hypocotyls. During the course of examination of the clock-associated mutants of PSEUDO-RESPONSE REGULATORS, PRRs, including TOC1 (PRR1), we found that they commonly show another visible phenotype of anomalous greening responses upon the onset to light exposure of etiolated seedlings. These findings are indicative of a novel link between circadian rhythms and chloroplast development.     

VIPL has sugar-binding activity specific for high-mannose-type N-glycans, and glucosylation of the alpha1,2 mannotriosyl branch blocks its binding

VIP36-like protein (VIPL) was identified as an endoplasmic reticulum (ER) resident protein with homology to VIP36, a cargo receptor involved in the transport of glycoproteins within cells. Although VIPL is structurally similar to VIP36, VIPL is thought not to be a lectin, because its sugar-binding activity has not been detected in several experiments. Here, recombinant soluble VIPL proteins (sVIPL) were expressed in Escherichia coli, biotinylated with biotin ligase and oligomerized with R-phycoerythrin (PE)-labeled streptavidin (SA). As measured with flow cytometry, PE-labeled sVIPL-SA bound to deoxymannojirimycin (DMJ)- or kifunensine (KIF)- but not to swainsonine (SW)-treated HeLaS3 cells in the presence of calcium. A surface plasmon resonance analysis showed that the avidity of sVIPL was enhanced after it formed a complex with SA. The binding of PE-labeled sVIPL-SA was abrogated by endo beta-N-acetylglucosaminidase H treatment of the DMJ- or KIF-treated cells. Competition with several high-mannose-type N-glycans inhibited VIPL binding, and indicated that VIPL recognizes the Manalpha1-2Manalpha1-2Man sequence. Glucosylation of the outer mannose residue of this portion decreased the binding. Although the biochemical characteristics of VIPL are similar to those of VIP36, the sugar-binding activity of VIPL was stronger at neutral pH, corresponding to the pH in the lumen of the ER, than under acidic conditions.     

Identification of the role of bone morphogenetic protein (BMP) and transforming growth factor‐β (TGF‐β) signaling in the trajectory of serotonergic differentiation in a rapid assay in mouse embryonic stem cells in vitro

The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock‐in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet‐1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate‐based screening and identified BMP type I receptor kinase inhibitors LDN‐193189 and DMH1 as activators of luciferase. LDN‐193189 induced ES cells to express the genes encoding Pet‐1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF‐β receptor inhibitor SB‐431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN‐193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF‐β target gene Lefty, whereas the opposite effect was observed with SB‐431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF‐β receptor signaling are implicated in serotonergic differentiation.

     

Degradation of Stop Codon Read-through Mutant Proteins via the Ubiquitin-Proteasome System Causes Hereditary Disorders

During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped, or mutated, resulting in the production of aberrant proteins with C-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46-amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system, causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In humans, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with C-terminal extension encoded by read-through mutant genes is a mechanism for loss of function resulting in hereditary disorders.