Application of a Recombinant Three-Factor Chromogenic Reagent, PyroSmart, for Bacterial Endotoxins Test Filed in the Pharmacopeias.

Assays using lysate reagents prepared from horseshoe crab hemocyte extract (limulus amoebocyte lysate, LAL) are commonly and widely used to detect and measure endotoxin in parenteral drugs and medical devices. However, lysate reagents suffer from lot-to-lot variations leading to possible fluctuations in testing. Also, this continued usage of lysate reagents leads to the possible decline of the horseshoe crab population.

Retraction Note to: Changes of Absorptive and Secretory Transporting System of (1 → 3) β-D-glucan Based on Efflux Transporter in Indomethacin-induced Rat.

The Editor-in-Chief has retracted this article [1] based on an investigation by the Ministry of Education, Culture, Sports, Science and Technology, Japan, which found that the article contained overlap with a previously published article by Kalitsky-Szirtes J, et al. [2].

Genetic engineering approach to develop next-generation reagents for endotoxin quantification.

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent.

Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade.

Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to β-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B.

The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.

Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form.

Changes of Absorptive and Secretory Transporting System of (1 → 3) β-D-glucan Based on Efflux Transporter in Indomethacin-induced Rat.

Infection and inflammation suppress the expression and activity of several drug transporters in liver. In the intestine, P-glycoprotein (P-gp/MDR1), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) are important barriers to the absorption of many clinically important drugs. The expression and activity of these proteins were examined under inflammation.

Toll-like receptor 2 heterodimers, TLR2/6 and TLR2/1 induce prostaglandin E production by osteoblasts, osteoclast formation and inflammatory periodontitis.

TLR2 forms heterodimers with TLR1 and TLR6, and regulates host defense mechanisms against pathogens. We examined the role of TLR2 heterodimer signaling in osteoclast formation and inflammatory periodontitis. In co-cultures of mouse bone marrow cells and osteoblasts, a TLR2/6 ligand (diacylated lipopeptide designed from Gram-positive bacteria) markedly induced osteoclast formation.

Soluble cell wall beta-glucan of Candida induces/enhances apoptosis and oxidative stress in murine lung.

The bioactivity of cell wall component(s) of fungi has not been fully investigated, especially in vivo. We have successfully extracted a soluble cell wall beta-glucan from C. albicans (CSBG), and shown that pulmonary exposure to CSBG induces lung inflammation in mice.

Candida soluble cell wall beta-glucan facilitates ovalbumin-induced allergic airway inflammation in mice: Possible role of antigen-presenting cells.

Background: Although fungi have been implicated as initiating/deteriorating factors for allergic asthma, their contributing components have not been fully elucidated. We previously isolated soluble beta-glucan from Candida albicans (CSBG) (Ohno et al., 2007).

Nonlinear intestinal absorption of (1-->3)-beta-D-glucan caused by absorptive and secretory transporting system.

The mechanism of the nonlinear concentration dependence of intestinal absorption of (1-->3)-beta-D-glucan was studied using in situ rat intestinal perfusion, as well as the in vitro Ussing-type chamber method mounted with rat intestinal tissue. The intestinal absorption rate constant of a (1-->3)-beta-D-glucan, laminaran, evaluated by the loop method increased significantly with increasing concentration of laminaran up to 0.5 muM in a nonlinear fashion and tended to decrease at higher concentrations.

Pulmonary exposure to diesel exhaust particles enhances coagulatory disturbance with endothelial damage and systemic inflammation related to lung inflammation.

Pulmonary exposure to diesel exhaust particles (DEP) enhances lung inflammation related to bacterial endotoxin (lipopolysaccharide [LPS]) in mice. Severe lung inflammation can reportedly induce coagulatory abnormalities and systemic inflammation. This study examined the effects of components of DEP on lung inflammation, pulmonary permeability, coagulatory changes, systemic inflammatory response, and lung-to-systemic translocation of LPS in a murine model of lung inflammation.

[Evaluation of an improved pretreatment method for the measurement of (1-->3)-beta-D-glucan in blood samples].

Measurement of blood (1-->3)-beta-D-glucan is useful for early diagnosis and follow-up of the therapeutic process of deep seated mycoses. The Fungitec G test MK (Seikagaku Corp., Tokyo) kit using alkaline-pretreatment followed by chromogenic kinetic assay has been widely used in Japan because of its high sensitivity and easy handling of a large number of samples.

Pulmonary toxicity induced by intratracheal instillation of Asian yellow dust (Kosa) in mice.

Asian yellow dust (Kosa) causes adverse respiratory health effects in humans. The objective of this study was to clarify the lung toxicity of Kosa. ICR mice (5 weeks of age) were administered intratracheally with Kosa samples-two samples from Maowusu desert and Shapotou desert, one sample consisted of Shapotou Kosa plus sulfate, and natural Asian dust (NAD) from the atmosphere of Beijing-at doses of 0.