Ryosuke Iijima

Novel biological function of sialic acid (N-acetylneuraminic acid) as a hydrogen peroxide scavenger

We have found that N‐acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H2O2) under physiological conditions. Close investigation of this finding revealed that NANA was oxidized by an equimolar amount of H2O2 to provide its decarboxylated product, 4‐(acetylamino)‐2,4‐dideoxy‐D‐glycero‐D‐galacto‐octonic acid (ADOA). To date, there have been little data on this reaction, and its physiological significance has not been discussed. Examining the detoxification of H2O2 in cultured cells with NANA, we were able to confirm that the cell death caused by H2O2 was suppressed by NANA in a dose‐dependent manner. These results revealed a novel role for NANA as a reactive oxygen scavenger. It is known that terminal NANA residues are removed by neuraminidase and that free NANA molecules are recycled or degraded by enzymes. We propose that released monomeric NANA is the potent defense molecule against oxidative damage.

A NOVEL ANTIMICROBIAL PEPTIDE FROM THE SEA HARE DOLABELLA AURICULARIA

The sea hare Dolabella auricularia is a marine shell-less gastropod. Four cytotoxic glycoproteins named dolabellanin A, C, E and P were found in the animal previously. An antimicrobial factor was newly isolated from the sea hares body-wall including skin and mucus. This factor is a novel peptide which consists of 33 amino acid residues, and is called dolabellanin B2. Dolabellanin B2 was cytotoxically effective against some pathogenic microorganisms at a concentration of 2.5-100 microg/ml.

Suppression of anti‐Candida activity of macrophages by a quorum‐sensing molecule, farnesol, through induction of oxidative stress

Farnesol is well known as a quorum‐sensing molecule of Candida albicans. To assess the pathological function of farnesol, its effects on macrophage viability and functions including growth inhibitory activities against C. albicans were examined in vitro. Murine macrophages, when cultured in the presence of 56–112 μM of farnesol for 1–2 hr, decreased their activity inhibiting the mycelial growth of C. albicans and lost their viability. This suppression of macrophage function by farnesol was neutralized by the coexistence of the anti‐oxidants probucol and trolox. Macrophages cultured in the presence of farnesol for 2 hr displayed morphological change of nuclei and DNA fragmentation, which suggested apoptosis of the cells. Intracellular production of ROS in the farnesol‐treated macrophages was shown by fluorescence of DCFH‐DA and increase of peroxidized materials. These effects of farnesol were blocked by probucol or trolox. These results indicate that farnesol lowered viability of the murine macrophages and suppressed their anti‐Candida activity, perhaps through induction of ROS.

l-Amino acid oxidase activity of an antineoplastic factor of a marine mollusk and its relationship to cytotoxicity

Dolabellanin A (DAA), an antineoplastic protein of an ocean mollusk, was shown to have L-amino acid oxidase (LAAO, EC 1.4.3.2) activity. With the addition of DAA, a cytotoxic concentration of hydrogen peroxide was detected in the culture medium of EL-4 murine lymphoma cells. The cytotoxicity of DAA was suppressed by antioxidants, especially by catalase. The hydrogen peroxide produced by the LAAO activity was recognized to be involved in the cytotoxicity, and the cell death seemed to be due to the direct toxicity of this substance. However, the cytotoxicity of a higher concentration of DAA was only partially suppressed even when an excess amount of catalase was used. A portion of the cells that died showed features of apoptosis such as increased caspase 3 activity and DNA fragmentation. This indicated that apoptosis was induced by DAA activity but independently of the direct toxicity of hydrogen peroxide.

Sialic acid attenuates the cytotoxicity of the lipid hydroperoxides HpODE and HpETE

Reduction of peroxide molecular species is an essential function in living organisms. In previous studies, we proposed a new function for the sialic acid N-acetylneuraminic acid (Neu5Ac)--that of antioxidant/hydrogen peroxide scavenging agent. On the basis of the reaction scheme, Neu5Ac is thought to act as a general antioxidant of all hydroperoxide-type species (R-OOHs). The concentration of tert-butyl hydroperoxide (t-BuOOH) decreased after co-incubation with N-acetylneuraminic acid. Neu5Ac also decreased the R-OOH concentration in solutions of peroxylinolenic acid (13(S)-hydroperoxy-(9Z,11E)-octadecadienoic acid, HpODE) and peroxyarachidonic acid (15(S)-hydroperoxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid, HpETE)--two lipid hydroperoxides that participate in many physiological events. Moreover, the cytotoxicity of both these lipid hydroperoxides was attenuated by reaction with Neu5Ac acid. Our results suggest that N-acetylneuraminic acid is a potential antioxidant of most hydroperoxides that accumulate in organisms.

Development of a fluorescence analysis method for N-acetylneuraminic acid and its oxidized product ADOA.

N-acetylneuraminic acid (NANA) consumes toxic hydrogen peroxide (H2O2) under physiological conditions and is oxidized by an equimolar amount of H2O2 to yield its decarboxylated product 4-(acetylamino)-2,4-dideoxy-d-glycero-d-galacto-octonic acid (ADOA). Highly sensitive analytical methods are required to detect ADOA in the human body. We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED) to enable their fluorometric detection, and developed a method using HPLC with fluorometric detection (HPLC-FD) for the simultaneous determination of the derivatized NANA and ADOA. The derivatized NANA and ADOA were separated by a hydrophilic interaction liquid chromatography (HILIC) column using an H2O/CH3CN/HCOOH (10/90/0.35) mobile phase. Fluorescence was monitored at excitation and emission wavelengths of 450nm and 560nm, respectively. Both intra- and inter-day (n=6) repeat determinations of the DBD-ED-derivatized NANA and ADOA gave relative standard deviations of less than 5%. The calibration curves for standard solutions of DBD-ED-derivatized NANA and ADOA were linear over the ranges from 576fmol to 2.0nmol and 556fmol to 2.0nmol, respectively. The method developed was highly specific and sensitive for NANA and ADOA. The presence of ADOA in biological samples was revealed for the first time using this method.

Sensitive Analysis of Sialic Acid and Related Compound by Hydrophilic Interaction Liquid Chromatography Using Fluorescence Detection after Derivatization with DBD-PZ

N-Acetylneuraminic acid (NANA) has been reported to react with hydrogen peroxide in vitro to produce 4-(acetylamino)-2,4-dideoxy-D-glycero-D-galacto-octonic acid (ADOA). We labeled NANA and ADOA with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) for simultaneous detection. The derivatized NANA and ADOA were separated using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection. The calibration curves of DBD-PZ-derivatized NANA and ADOA showed good linearity in the range of 221 fmol to 1.5 nmol, and 44 fmol to 1.5 nmol, respectively. This analytical method has high specificity and is useful for the detection of NANA and ADOA in saliva and serum.

Report on the 25th Annual Meeting of the Japanese Association for Developmental and Comparative Immunology (JADCI), August 26 (Monday) to 28 (Wednesday), 2013, the 50th Year Commemorative Building of Okayama University of Science, Okayama 700-0005, Japan (Local Organizer, Nobuhiko Asada).

Officer Ryosuke Iijima⇑ Faculty of Pharmaceutical Sciences, Teikyo University, Itabashi 173-8605, Tokyo, Japan ⇑ Corresponding author. Tel.: +81 339648257; fax: +81 339648257. E-mail addresses: ryo-iiji@pharm.teikyo-u.ac.jp JADCI Office and Secretary/Treasurer Shoichiro Kurata Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan ⇑ Tel.: +81 92 642 2894. E-mail addresses: jadci2office@google.com Available online 21 March 2014

Report on the 22nd annual meeting of the Japanese Association for Developmental and Comparative Immunology (JADCI), August 2–4, 2010, Nishijin Plaza, Kyushu University, Fukuoka (Local Organizer: Shun-ichiro Kawabata, Kyushu University)

The 22nd annual meeting of the Japanese Association for Develpmental and Comparative Immunology (JADCI) was held on ugust 2–4, 2010, at Nishijin Plaza of Kyushu University, Fukuoka. bout one hundred and fifty scientists and graduate students ttended this meeting and discussed the host defense mechanisms f invertebrates and vertebrates. The meeting featured a special ecture, the Furuta Award lecture, and a symposium. In this year he main theme for the special lecture and the symposium was Comparative Biology on the Regulation of Immune Responses.” In ddition, twenty-five general presentations were made orally and ollowed by active discussion. The special lecture: Dr. Kozo Fujisaki (Department of Frontier eterinary Medicine, Kagoshima University). A cysteine proteases hat exerts a killing effect against the midgut-stage Babesia parsites in Haemaphysalis ticks. He introduced the molecular and reverse genetic characteriation of a multifunctional cysteine protease, longipain, from the abesial parasite vector tick Haemaphysalis longicornis. Longipain s a papain-family cysteine protease and is mainly expressed in the idgut epithelium. It specifically localizes at lysosomal vacuoles nd is released into the lumen. Its expression is up-regulated by ost blood feeding. Longipain hydrolysis occurs over a broad range f pH and temperature. Longipain dose-dependently kills tickorne Babesia parasites, and its babesiacidal effect occurs via speific adherence to the parasite membranes. Longipain is involved in he digestion of the host blood meal, and longipain exerts a killing ffect against the midgut-stage Babesia parasites in ticks. Longipain s essential for tick survival, and may have a role in controlling the ransmission of tick-transmittable Babesia parasites. Furuta Award 2010: Drs. Hiroaki Suetake and Yuzuru Suzuki Fisheries Laboratory, Graduate School of Agricultural and Life Scinces, the University of Tokyo; Present address for HS, Faculty of Marine ioscience, Fukui Prefectural University). Defense mechanisms of sh: Impacts of fugu genome analysis on fish immunology. Unlike avian and mammalian CD4, fugu CD4 lacks the Cys pair f the first Ig-like domain, but has a unique possible disulfide bond n the third domain. The fugu CD4 gene is composed of 12 exons, iffering from other CD4 genes, but showing conserved synteny and any conserved sequence motifs in the promoter region. The fugu D4 gene is expressed predominantly in lymphoid tissues, and can e expressed on the surface of cells via transfection. There are three embers of the B7 family in fugu: B7-H1/DC, B7-H3, and B7-H4. The hree fugu B7s are expressed on the surface of blood monocytes. he B7+ monocytes, which are composed of at least two distinct