The phytosphingosine-CD300b interaction promotes zymosan-induced, nitric oxide-dependent neutrophil recruitment.

Zymosan is a glucan that is a component of the yeast cell wall. Here, we determined the mechanisms underlying the zymosan-induced accumulation of neutrophils in mice. Loss of the receptor CD300b reduced the number of neutrophils recruited to dorsal air pouches in response to zymosan, but not in response to lipopolysaccharide (LPS), a bacterial membrane component recognized by Toll-like receptor 4 (TLR4). An inhibitor of nitric oxide (NO) synthesis reduced the number of neutrophils in the zymosan-treated air pouches of wild-type mice to an amount comparable to that in CD300b-/- mice. Treatment with clodronate liposomes decreased the number of NO-producing, CD300b+ inflammatory dendritic cells (DCs) in wild-type mice, thus decreasing NO production and neutrophil recruitment. Similarly, CD300b deficiency decreased the NO-dependent recruitment of neutrophils to zymosan-treated joint cavities, thus ameliorating subsequent arthritis. We identified phytosphingosine, a lipid component of zymosan, as a potential ligand of CD300b. Phytosphingosine stimulated NO production in inflammatory DCs and promoted neutrophil recruitment in a CD300b-dependent manner. Together, these results suggest that the phytosphingosine-CD300b interaction promotes zymosan-dependent neutrophil accumulation by inducing NO production by inflammatory DCs and that CD300b may contribute to antifungal immunity.

Pharmacokinetic parameters explain the therapeutic activity of antimicrobial agents in a silkworm infection model.

Poor pharmacokinetic parameters are a major reason for the lack of therapeutic activity of some drug candidates. Determining the pharmacokinetic parameters of drug candidates at an early stage of development requires an inexpensive animal model with few associated ethical issues. In this study, we used the silkworm infection model to perform structure-activity relationship studies of an antimicrobial agent, GPI0039, a novel nitrofuran dichloro-benzyl ester, and successfully identified compound 5, a nitrothiophene dichloro-benzyl ester, as a potent antimicrobial agent with superior therapeutic activity in the silkworm infection model. Further, we compared the pharmacokinetic parameters of compound 5 with a nitrothiophene benzyl ester lacking chlorine, compound 7, that exerted similar antimicrobial activity but had less therapeutic activity in silkworms, and examined the metabolism of these antimicrobial agents in human liver fractions in vitro. Compound 5 had appropriate pharmacokinetic parameters, such as an adequate half-life, slow clearance, large area under the curve, low volume of distribution, and long mean residence time, compared with compound 7, and was slowly metabolized by human liver fractions. These findings suggest that the therapeutic effectiveness of an antimicrobial agent in the silkworms reflects appropriate pharmacokinetic properties.

Structural analysis of an innate immunostimulant from broccoli, Brassica oleracea var. italica.

Vegetables are eaten as part of a healthy diet throughout the world, and some are also applied topically as a traditional medicine. We evaluated the innate immunostimulating activities of hot water extracts of various vegetables using the silkworm muscle contraction assay system, and found that broccoli, Brassica oleracea var. italica, contains a strong innate immunostimulant. We purified the innate immunostimulant from broccoli, and characterized the chemical structure by chemical analyses and NMR spectroscopy. The innate immunostimulant comprised galacturonic acid, galactose, glucose, arabinose, and rhamnose, and had a pectic-like polysaccharide structure. To determine the structural motif involved in the innate immunostimulating activity, we modified the structure by chemical and enzymatic treatment, and found that the activity was attenuated by pectinase digestion. These findings suggest that a pectic-like polysaccharide purified from broccoli has innate immune-stimulating activity, for which the polygalacturonic acid structure is necessary.

Characterization of the chemical structure and innate immune-stimulating activity of an extracellular polysaccharide from Rhizobium sp. strain M2 screened using a silkworm muscle contraction assay.

We screened innate immunostimulant-producing bacteria using a silkworm muscle contraction assay, and isolated Rhizobium sp. strain M2 from soil. We purified the innate immunostimulant from strain M2, and characterized the chemical structure by nuclear magnetic resonance spectroscopy and chemical analyses. The innate immunostimulant (M2 EPS) comprised glucose, galactose, pyruvic acid, and succinic acid with a molar ratio of 6.8:1.0:0.9:0.4, and had a succinoglycan-like high molecular-weight heteropolysaccharide structure. To determine the structural motif involved in the innate immunostimulating activity, we modified the M2 EPS structure chemically, and found that the activity was increased by removal of the succinic and pyruvic acid substitutions. Strong acid hydrolysis completely inactivated the M2 EPS. Unmasking of the ß-1,3/6-glucan structure of the side-chain by deacylation and depyruvylation may enhance the innate immune-stimulating activity of M2 EPS. These findings suggest that the succinoglycan-like polysaccharide purified from strain M2 has innate immune-stimulating activity, and its glycan structure is necessary for the activity.

[Identification of novel therapeutically effective antibiotics using silkworm infection model].

Most antibiotics obtained by in vitro screening with antibacterial activity have inappropriate properties as medicines due to their toxicity and pharmacodynamics in animal bodies. Thus, evaluation of the therapeutic effects of these samples using animal models is essential in the crude stage. Mammals are not suitable for therapeutic evaluation of a large number of samples due to high costs and ethical issues. We propose the use of silkworms (Bombyx mori) as model animals for screening therapeutically effective antibiotics. Silkworms are infected by various pathogenic bacteria and are effectively treated with similar ED(50) values of clinically used antibiotics. Furthermore, the drug metabolism pathways, such as cytochrome P450 and conjugation systems, are similar between silkworms and mammals. Silkworms have many advantages compared with other infection models, such as their 1) low cost, 2) few associated ethical problems, 3) adequate body size for easily handling, and 4) easier separation of organs and hemolymph. These features of the silkworm allow for efficient screening of therapeutically effective antibiotics. In this review, we discuss the advantages of the silkworm model in the early stages of drug development and the screening results of some antibiotics using the silkworm infection model.

Jellyfish mucin may have potential disease-modifying effects on osteoarthritis.

BACKGROUND: We aimed to study the effects of intra-articular injection of jellyfish mucin (qniumucin) on articular cartilage degeneration in a model of osteoarthritis (OA) created in rabbit knees by resection of the anterior cruciate ligament. Qniumucin was extracted from Aurelia aurita (moon jellyfish) and Stomolophus nomurai (Nomura's jellyfish) and purified by ion exchange chromatography. The OA model used 36 knees in 18 Japanese white rabbits. Purified qniumucin extracts from S. nomurai or A. aurita were used at 1 mg/ml. Rabbits were divided into four groups: a control (C) group injected with saline; a hyaluronic acid (HA)-only group (H group); two qniumucin-only groups (M groups); and two qniumucin + HA groups (MH groups). One milligram of each solution was injected intra-articularly once a week for 5 consecutive weeks, starting from 4 weeks after surgery. Ten weeks after surgery, the articular cartilage was evaluated macroscopically and histologically. RESULTS: In the C and M groups, macroscopic cartilage defects extended to the subchondral bone medially and laterally. When the H and both MH groups were compared, only minor cartilage degeneration was observed in groups treated with qniumucin in contrast to the group without qniumucin. Histologically, densely safranin-O-stained cartilage layers were observed in the H and two MH groups, but cartilage was strongly maintained in both MH groups. CONCLUSION: At the concentrations of qniumucin used in this study, injection together with HA inhibited articular cartilage degeneration in this model of OA.

Structural analysis of O-glycans of mucin from jellyfish (Aurelia aurita) containing 2-aminoethylphosphonate.

The structure of O-glycan in qniumucin (Q-mucin), which is a novel mucin extracted from jellyfish, was analyzed by a combination of NMR and ESI-MS/MS. A previously unidentified monosaccharide involved in the glycan chains was determined to be N-acetylgalactosamine (GalNAc) substituted by 2-aminoethylphosphonate (AEP) at the C-6. The O-glycans in Q-mucin from Aurelia aurita were proved to be mainly composed of three monosaccharides: GalNAc, AEP-(O-->6)-GalNAc, and P-6-GalNAc. To the best of our knowledge, this is the first example of an O-glycan structure of glycoproteins containing AEP. This exceptionally simple structure of Q-mucin and its potential use in material science and technology are revealed.

NMR study on a novel mucin from jellyfish in natural abundance, Qniumucin from Aurelia aurita.

A novel mucin (qniumucin), which we recently discovered in jellyfish, was investigated by several NMR techniques. Almost all the peaks in the (13)C and proton NMR spectra were satisfactorily assigned to the amino acids in the main chain and to the bridging GalNAc, the major sugar in the saccharide branches. The amino acid sequence in the tandem repeat part (-VVETTAAP-) was reconfirmed by the cross-peaks between alpha protons and carbonyl carbons in the HMBC spectrum. A connectivity analysis around the O-glycoside bond (GalNAc-Thr) was also performed, and detailed information on the local configuration was obtained by the DPFGSE-NOE-HSD technique. The strategy and the results described in this paper can be extended to the structural analysis of general O-glycan chains, which are more complex than the present mucin. NMR analyses reveal the simple structure of qniumucin extracted by the present protocol, and the homogeneity and purity of qniumucin are probably the result of it being extracted from jellyfish, a primitive animal.

Extracellular polysaccharides of Rhodococcus rhodochrous S-2 stimulate the degradation of aromatic components in crude oil by indigenous marine bacteria.

Rhodococcus rhodochrous S-2 produces extracellular polysaccharides (S-2 EPS) containing D-glucose, D-galactose, D-mannose, D-glucuronic acid, and lipids, which is important to the tolerance of this strain to an aromatic fraction of (AF) Arabian light crude oil (N. Iwabuchi, N. Sunairi, H. Anzai, M. Nakajima, and S. Harayama, Appl. Environ. Microbiol. 66:5073-5077, 2000). In the present study, we examined the effects of S-2 EPS on the growth of indigenous marine bacteria on AF. Indigenous bacteria did not grow significantly in seawater containing AF even when nitrogen, phosphorus, and iron nutrients were supplemented. The addition of S-2 EPS to seawater containing nutrients and AF resulted in the emulsification of AF, promotion of the growth of indigenous bacteria, and enhancement of the degradation of AF by the bacteria. PCR-denaturing gradient gel electrophoresis analyses show that addition of S-2 EPS to the seawater containing nutrients and AF changed the composition of the bacterial populations in the seawater and that bacteria closely related to the genus Cycloclasticus became the major population. These results suggest that Cycloclasticus was responsible for the degradation of hydrocarbons in AF. The effects of 15 synthetic surfactants on the degradation of AF by indigenous marine bacteria were also examined, but enhancement of the degradation of AF was not significant. S-2 EPS was hence the most effective of the surfactants tested in promoting the biodegradation of AF and may thus be an attractive agent to use in the bioremediation of oil-contaminated marine environments.