Ninjurin1 Assembles Into a Homomeric Protein Complex Maintained by N-linked Glycosylation.

Ninjurin1 (Ninj1) is a cell surface protein known as a homophilic adhesion molecule. Previous studies have shown a trans-interaction of Ninj1 between immune cells and endothelial cells; however, little is known about Ninj1 modification and structure in the cis-interaction. We showed that Ninj1 assembles into a homomeric complex via a cis-interaction mediated by the intracellular region and N-glycosylation at Asn60 . We identified cis-interaction between Ninj1 proteins using CFP- and YFP-tagged Ninj1 by Förster resonance energy transfer using a confocal microscope and fluorescence-activated cell sorter. We further observed the Ninj1 homomeric complexes composed of two to six monomeric Ninj1 molecules by a formaldehyde cross-linking assay. Co-immunoprecipitation assays with epitope-tagged truncated Ninj1 suggested that the intracellular region encompassing Leu101 -Ala110 participates in Ninj1 homomer assembly. Ninj1 N-glycosylation was characterized by treatment of tunicamycin and substitution of Asn to Gln or Ala. Fluorescence-activated cell sorting-based Förster resonance energy transfer assays further demonstrated that N-glycosylation is indispensable for the Ninj1 cis-interaction, and a formaldehyde cross-linking assay confirmed that interruption of N-glycosylation by Asn substitution disrupted Ninj1 homomeric complex formation. In silico analysis revealed that Ninj1 is highly conserved in vertebrates and that the conserved sequence contains an N-glycosylation motif and cis-interacting intracellular region, which participate in Ninj1 homomer assembly. Taken together, these data show that Ninj1 assembles into a homomeric protein complex and that N-glycosylation is a prerequisite for Ninj1 homomer assembly. J. Cell. Biochem. 118: 2219-2230, 2017. © 2017 Wiley Periodicals, Inc.

Ninjurin1 regulates lipopolysaccharide-induced inflammation through direct binding.

Ninjurin1 is a transmembrane protein involved in macrophage migration and adhesion during inflammation. It was recently reported that repression of Ninjurin1 attenuated the lipopolysaccharide (LPS)-induced inflammatory response in macrophages; however, the precise mechanism by which Ninjurin1 modulates LPS-induced inflammation remains poorly understood. In the present study, we found that the interaction between Ninjurin1 and LPS contributed to the LPS-induced inflammatory response. Notably, pull-down assays using lysates from HEK293T cells transfected with human or mouse Ninjurin1 and biotinylated LPS (LPS-biotin) showed that LPS directly bound Ninjurin1. Subsequently, LPS binding assays with various truncated forms of Ninjurin1 protein revealed that amino acids (aa) 81-100 of Ninjurin1 were required for LPS binding. In addition, knockdown experiments using Ninj1 siRNA resulted in decreased nitric oxide (NO) and tumor necrosis factor-α (TNFα) secretion upon LPS treatment in Raw264.7 cells. Collectively, our results suggest that Ninjurin1 regulates the LPS-induced inflammatory response through its direct binding to LPS, thus, identifying Ninjurin1 as a putative target for the treatment of inflammatory diseases, such as sepsis and inflammation-associated carcinogenesis.

Characterization of white and black deposits on the surface of Korean stone cultural heritages.

White and black deposits have been frequently observed on the surface of Korean stone cultural heritages, and they are considered as damage factors in both conservation and esthetic points of view. In order to set up the appropriate conservation remedy, it is important to know their origins, characteristics, and compositions. In this study, optical and scanning electron microscopes (SEMs) equipped with an energy-dispersive spectrometer (EDS) and X-ray diffractometer were employed to determine the white and black deposits. It was found that both deposits consisted mainly of calcium carbonate (calcite) and calcium sulfate (gypsum). The calcite and gypsum were characterized by bladed, rhombohedral, tabular, and amorphous morphologies under a SEM. The black deposit was not only composed of calcite or gypsum, but also accompanied amorphous and irregular matrix. SEM-EDS analysis revealed an abundance of silicon, aluminum, iron, phosphorus, and carbon on the matrix, which were major elements of soil, atmospheric deposits, and organisms. The white deposit, on the other hand, barely contained those coloring substances. These salts and deposited substances were caused by chemical reaction and physical adhesion between rock-forming minerals, lime mortar, sulfur in polluted air environment, soil dust, and microorganisms.

Enhanced emission and two-photon absorption cross-section by nanoaggregation of a cyano-substituted stilbene derivative.

This study reports the synthesis and the investigation of optical behaviors of a stilbene derivative, (Z)-2,3-bis[4-(N-4-(diphenylamino)styryl)phenyl]acrylonitrile (StCN). The nanoaggregates of StCN in 50% water/DMF have exhibit an increased fluorescence intensity and a blue shift with emission maximum wavelength with respect to StCN in 100% N, N-dimethylformamide (DMF) solution. This fluorescence enhancement can be attributed to the formation of J-aggregates. The cyano group prevents coplanarization of both arms and intermolecular face-to-face orientation leading to the formation of J-aggregates preventing the solvent relaxation. A strong fluorescence bathochromic solvatochromism is indicative of a large dipole moment in solution. Also, the two-photon absorption cross-section of nanoaggregates has been increased 1.5 times compared to that in DMF solution at the maximum wavelength.