High-Throughput Analysis of Bacterial Toxic Lipopolysaccharide in Water by Dual-Wavelength Monitoring Using a Ratiometric Fluorescent Chemosensor.

Lipopolysaccharide (LPS) is a bacterial toxin that causes fever in humans. Our small-molecule chemosensor named Zn-dpa-C2OPy shows rapid ratiometric fluorescence response to LPS in water with a detection limit of 11 pM, which is lower than that of our previously reported sensor. Spectroscopic measurements (fluorescence, absorbance, 1H NMR, and fluorescence lifetime), dynamic light scattering measurements, and transmission electron microscopy observations revealed that the fluorescence response was induced by the changes in the aggregation state via multi-point recognition of LPS through hydrophobic and electrostatic interactions, in addition to the coordination between the zinc(II)-dipicolylamine moiety of the chemosensor and the phosphate group of LPS. The proposed Zn-dpa-C2OPy chemosensor was applied to an original flow injection analysis (FIA) system with a self-developed dual-wavelength fluorophotometer, and a high throughput of 36 samples per hour was achieved. These results demonstrate the feasibility of this unique methodology combining a ratiometric fluorescent chemosensor and FIA for continuous online monitoring of LPS in water.

Plant-Specific Domains and Fragmented Sequences Imply Non-Canonical Functions in Plant Aminoacyl-tRNA Synthetases.

Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein translation. In addition, numerous aaRSs (mostly in vertebrates) have also been discovered to possess a range of non-canonical functions. Very few studies have been conducted to elucidate or characterize non-canonical functions of plant aaRSs. A genome-wide search for aaRS genes in Arabidopsis thaliana revealed a total of 59 aaRS genes. Among them, asparaginyl-tRNA synthetase (AsnRS) was found to possess a WHEP domain inserted into the catalytic domain in a plant-specific manner. This insertion was observed only in the cytosolic isoform. In addition, a long stretch of sequence that exhibited weak homology with histidine ammonia lyase (HAL) was found at the N-terminus of histidyl-tRNA synthetase (HisRS). This HAL-like domain has only been seen in plant HisRS, and only in cytosolic isoforms. Additionally, a number of genes lacking minor or major portions of the full-length aaRS sequence were found. These genes encode 14 aaRS fragments that lack key active site sequences and are likely catalytically null. These identified genes that encode plant-specific additional domains or aaRS fragment sequences are candidates for aaRSs possessing non-canonical functions.

Retinoic acid receptor agonist Am80 inhibits CXCL2 production from microglial BV-2 cells via attenuation of NF-κB signaling.

Accumulating lines of evidence suggest that retinoic acid receptor agonists such as Am80 exerts anti-inflammatory actions in the central nervous system, although detailed mechanisms of the action remain largely unknown. Our previous findings suggest that Am80 provides therapeutic effect on intracerebral hemorrhage in mice via suppression of expression of chemokine (C-X-C motif) ligand 2 (CXCL2). Here we investigated the mechanisms of inhibitory action of Am80 on expression of CXCL2 and other pro-inflammatory factors in microglial BV-2 cells. Pretreatment with Am80 markedly suppressed lipopolysaccharide (LPS)-induced expression of CXCL2 mRNA and release of CXCL2 protein. Am80 had no effect on LPS-induced activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. On the other hand, Am80 prevented LPS-induced nuclear translocation of p65 subunit of NF-κB complex. In addition, total expression levels of p65 and IκBα proteins, as well as of mRNAs encoding p65 and IκBα, were lowered by Am80. Dependence of CXCL2 expression on NF-κB was confirmed by the effect of an NF-κB inhibitor caffeic acid phenethyl ester that abolished LPS-induced CXCL2 expression. Caffeic acid phenethyl ester also abolished LPS-induced expression of inducible nitric oxide synthase, interleukin-1ß and tumor necrosis factor α, which may be relevant to the inhibitory effect of Am80 on expression of these pro-inflammatory factors. We additionally found that Am80 attenuated LPS-induced up-regulation of CD14, a co-receptor for Toll-like receptor 4 (TLR4). These results suggest that inhibitory effect on TLR4 signaling mediated by NF-κB pathway underlies the anti-inflammatory action of retinoic acid receptor agonists in microglia.