Amplification immunoassay for the determination of Hepatitis B surface antigen

A sensitive sandwich immunoassay for the determination of Hepatitis B surface antigen (HBs) was developed, using a cascade system of Limulus amebocyte lysate as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to anti-HBs antibody. Anti-HBs antibody was adsorbed to polystyrene beads. First, HBs were reacted to solid phase anti-HBs antibody (a-HBs). After the reaction, the beads were rinsed, and were then reacted with a-HBs-LPS. Then, LPS activity specifically bound to the beads was measured. HBs could be measured in the range of 10(-10)-10(-12) g/mL.     

Structure of the O-polysaccharide of <Emphasis Type="Italic">Erwinia carotovora</Emphasis> ssp. <Emphasis Type="Italic">atroseptica</Emphasis> GSPB 9205 containing a new higher branched monosaccharide

An O-polysaccharide was prepared by mild acid hydrolysis of a lipopolysaccharide of Erwinia carotovora ssp. atroseptica GSPB 9205 and was found to contain a new higher branched monosaccharide, viz., 3,6,8-trideoxy-4-C-(R-1-hydroxyethyl)-D-gulo-octose, which we called erwiniose. The structure of the pentasaccharide repeating unit of the O-polysaccharide was established by monosaccharide analysis, including the determination of absolute configurations, methylation analysis, O-deacetylation, Smith degradation, and one- and two-dimensional NMR spectroscopy. The configuration of erwiniose was determined based on the coupling constants of vicinal protons combined with the nuclear Overhauser effect data and the results of periodate oxidation of the polysaccharide followed by reduction of the resultant 4-keto sugar (the C(1)-C(4)-C(1′)-C(2′) fragment) to give 3,6-dideoxy-D-ribo-hexose(paratose) and oxidation of 3-hydroxybutyraldehyde (the C(5)-C(8) fragment) to give R-3-hydroxybutyric acid. Dedicated to Academician N. K. Kochetkov on the occasion of his 90th birthday. __________ Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1239–1244, May, 2005.     

The effect of covalent immobilization of sialic acid on the removal of lipopolysaccharide and reactive oxygen species for polyethylene terephthalate

Polyethylene terephthalate (PET) was aminolyzed with 1,6‐diaminohexane (DAH) and then sialic acid (NANA) was immobilized via amidation onto the surface. The surface concentration of NANA was determined by 2‐thiobarbituric acid (TBA) test. The hemocompatibility of the resulting PET fabrics was evaluated based on complete blood count (CBC), coagulating times, and protein adsorption. The ability to remove lipopolysaccharide (LPS) was also determined. In addition, the effect of contacting NANA‐immobilizing PET on the suppression of reactive oxygen species (ROS) production was measured by the chemiluminescence (CL) method. The results show that by immobilizing NANA onto PET, the adhesion of platelet (PLt) was reduced, and oxidative stress was suppressed. The level of LPS was also greatly reduced. Copyright © 2010 John Wiley & Sons, Ltd.     

The gram-negative outer membrane modeler: Automated building of lipopolysaccharide-rich bacterial outer membranes in four force fields

Outer membranes are a crucial component of Gram-negative bacteria, containing standard lipids in their inner leaflet, lipopolysaccharides (LPSs) in their outer leaflet, and transmembrane β-barrels known as outer membrane proteins (OMPs). OMPs regulate functions such as substrate transport and cell movement, while LPSs act as a protective barrier for bacteria and can cause toxic reactions in humans. However, the experimental study of outer membranes is challenging. Molecular dynamics simulations are often used for the computational study of membrane systems, but the preparation of complex, LPS-rich outer membranes is not straightforward. The Gram-Negative Outer Membrane Modeler (GNOMM) is an automated pipeline for preparing simulation systems of OMPs embedded in LPS-containing membranes in four different force fields. Given the physiological and clinical importance of outer membranes and their components, GNOMM can be a useful tool in the study of their structure, function, and implications in diseases. GNOMM is available at http://bioinformatics.biol.uoa.gr/GNOMM . © 2019 Wiley Periodicals, Inc.     

Scope and Limitations of 3‐Iodo‐Kdo Fluoride‐Based Glycosylation Chemistry using N‐Acetyl Glucosamine Acceptors

The ketosidic linkage of 3‐deoxy‐d‐manno‐octulosonic acid (Kdo) to lipid A constitutes a general structural feature of the bacterial lipopolysaccharide core. Glycosylation reactions of Kdo donors, however, are challenging due to the absence of a directing group at C‐3 and elimination reactions resulting in low yields and anomeric selectivities of the glycosides. While 3‐iodo‐Kdo fluoride donors showed excellent glycosyl donor properties for the assembly of Kdo oligomers, glycosylation of N‐acetyl‐glucosamine derivatives was not straightforward. Specifically, oxazoline formation of a β‐anomeric methyl glycoside, as well as iodonium ion transfer to an allylic aglycon was found. In addition, dehalogenation of the directing group by hydrogen atom transfer proved to be incompatible with free hydroxyl groups next to benzyl groups. In contrast, glycosylation of a suitably protected methyl 2‐acetamido‐2‐deoxy‐α‐d‐glucopyranoside derivative and subsequent deiodination proceeded in excellent yields and α‐specificity, and allowed for subsequent 4‐O‐phosphorylation. This way, the disaccharides α‐Kdo‐(2→6)‐α‐GlcNAcOMe and α‐Kdo‐(2→6)‐α‐GlcNAcOMe‐4‐phosphate were obtained in good overall yields.     

The In Vitro Anti-Inflammatory Activities of Galangin and Quercetin towards the LPS-Injured Rat Intestinal Epithelial (IEC-6) Cells as Affected by Heat Treatment

Flavonols possess several beneficial bioactivities in vitro and in vivo. In this study, two flavonols galangin and quercetin with or without heat treatment (100 °C for 15–30 min) were assessed for their anti-inflammatory activities in lipopolysaccharide (LPS)-stimulated rat intestinal epithelial (IEC-6) cells and whether the heat treatment caused activity changes. The flavonol dosages of 2.5–20 μmol/L had no cytotoxicity on the cells but could enhance cell viability (especially using 5 μmol/L flavonol dosage). The flavonols could decrease the production of prostaglandin E₂ and three pro-inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α, and simultaneously promote the production of two anti-inflammatory cytokines IL-10 and transforming growth factor-β. The Western-blot results verified that the flavonols could suppress the LPS-induced expression of TLR4 and phosphorylated IκBα and p65, while the molecular docking results also illustrated that the flavonols could bind with TLR4 and NF-κB to yield energy decreases of −(21.9–28.6) kJ/mol. Furthermore, an inhibitor BAY 11-7082 blocked the NF-κB signaling pathway by inhibiting the expression of phosphorylated IκBα/p65 and thus mediated the production of IL-6/IL-10 as the flavonols did, which confirmed the assessed anti-inflammatory effect of the flavonols. Consistently, galangin had higher anti-inflammatory activity than quercetin, while the heated flavonols (especially those with longer heat time) were less active than the unheated counterparts to exert these target anti-inflammatory effects. It is highlighted that the flavonols could antagonize the LPS-caused IEC-6 cells inflammation via suppressing TLR4/NF-κB activation, but heat treatment of the flavonols led to reduced anti-inflammatory efficacy.     

5,2′-Dibromo-2,4′,5′-trihydroxydiphenylmethanone Inhibits LPS-Induced Vascular Inflammation by Targeting the Cav1 Protein

Vascular inflammation is directly responsible for atherosclerosis. 5,2′-Dibromo-2,4′,5′-trihydroxydiphenylmethanone (TDD), a synthetic bromophenol derivative, exhibits anti-atherosclerosis and anti-inflammatory effects. However, the underlying pathways are not yet clear. In this study, we first examined the effects of TDD on toll-like receptor-4 (TLR4) activity, the signaling receptor for lipopolysaccharide (LPS), and found that TDD does not inhibit LPS-induced TLR4 expression in EA.hy926 cells and the vascular wall in vivo. Next, we investigated the global protein alterations and the mechanisms underlying the action of TDD in LPS-treated EA.hy926 cells using an isobaric tag for the relative and absolute quantification technique. Western blot analysis revealed that TDD inhibited NF-κB activation by regulating the phosphorylation and subsequent degradation IκBα. Among the differentially expressed proteins, TDD concentration-dependently inhibited Caveolin 1(Cav1) expression. The interaction between Cav1 and TDD was determined by using biolayer interference assay, UV-vis absorption spectra, fluorescence spectrum, and molecular docking. We found that TDD can directly bind to Cav1 through hydrogen bonds and van der Waals forces. In conclusion, our results showed that TDD inhibited LPS-induced vascular inflammation and the NF-κB signaling pathway by specifically targeting the Cav1 protein. TDD may be a novel anti-inflammatory compound, especially for the treatment of atherosclerosis.     

Onion (Allium cepa L.)-Derived Nanoparticles Inhibited LPS-Induced Nitrate Production,However, Their Intracellular Incorporation by Endocytosis Was Not Involved in This Effect on RAW264 Cells

The aim of this study was to evaluate the involvement of nanoparticles prepared from Allium cepa L. as anti-inflammatory agents. In the present study, we identified nanoparticles from Allium cepa L. using the ultracentrifugation exosome purification method. The nanoparticles were referred to as 17,000× g and 200,000× g precipitates, and they contained quercetins, proteins, lipids, and small-sized RNA. The nanoparticles inhibited nitric oxide production from lipopolysaccharide (LPS)-stimulated RAW264 cells without cytotoxic properties. Cellular incorporation was confirmed by laser microscopic observation after PKH26 staining. The inhibition of caveolae-dependent endocytosis and macropinocytosis significantly prevented the incorporation of the nanoparticles but had no effect on the inhibition of nitric oxide in RAW264 cells. Collectively, the identified nanoparticles were capable of inhibiting the LPS response via extracellular mechanisms. Taken together, the way of consuming Allium cepa L. without collapsing the nanoparticles is expected to provide an efficient anti-inflammatory effect.