Yuri Ikeda

Involvement of vanilloid receptor VR1 and prostanoids in the acid-induced writhing responses of mice.

We found that intraperitoneal injection of organic acids, such as propionic and lactic acid, are able to develop writhing responses in mice similarly as that of acetic acid. These acid-induced writhing reactions were significantly attenuated by capsazepine, a VR1 receptor-specific antagonist, but the phenylbenzoquinone-induced one was not, suggesting that the acids but not phenylbenzoquinone activate the VR1 receptor, which is involved in polymodal pain perception. Hoe 140, a bradykinin B2 receptor antagonist, also suppressed the acid-induced writhing response. Furthermore, these writhing responses were significantly suppressed after neonatal treatment with capsaicin, which treatment is known to destroy peripheral sensory afferent C-fibers. Capsazepine and Hoe 140 did not further attenuate the already reduced writhing responses of capsaicin-treated mice, suggesting that the acids stimulate the VR1 and the bradykinin B2 receptor in the pathway comprising sensory afferent C-fibers. On the other hand, indomethacin further significantly suppressed the writhing number of the capsaicin-treated animals, suggesting that the acid-induced pain perception requires prostanoid receptors not only in the pathway via capsaicin-sensitive C-fibers but also in other sensory pathways. These results provide the first evidence for the involvement of the vanilloid receptor in the acid-induced inflammatory pain perception via sensory C-fibers in addition to the known mediators bradykinin, neurokinins, and prostanoids.

Major roles of prostanoid receptors IP and EP3 in endotoxin-induced enhancement of pain perception

To know the roles of prostaglandin I (IP) and prostaglandin E (EP) receptors in pain perception, we compared the acetic acid-induced writhing response in mice deficient in prostaglandin receptors, i.e. IP, EP(1,) EP(2,) EP(3,) or EP(4,) with or without lipopolysaccharide (LPS) pretreatment. Without LPS pretreatment, IP-receptor deficient mice showed a significantly smaller number of responses, as previously reported, whereas mice deficient in any of the EP-receptor subtypes showed a number of writhings similar to those of wild-type mice. When mice were pretreated with LPS for 24 hr to induce cyclooxygenase-2 expression, the wild-type as well as EP(1)-, EP(2)-, or EP(4)-receptor-deficient mice showed a similar enhanced writhing response, whereas IP- and EP(3)-receptor-deficient mice had a significantly less enhanced number of writhings. These results indicate that IP and EP(3) are the major prostaglandin receptors mediating the enhanced acetic acid-induced writhing response in mice pre-exposed to LPS, i.e. in endotoxin-enhanced inflammatory nociception.

Intrinsic prostacyclin contributes to exudation induced by bradykinin or carrageenin: A study on the paw edema induced in IP-Receptor-deficient mice

To prove that prostaglandin I2 (PGI2) is a major prostaglandin involved in bradykinin-induced exudation, we examined carrageenin- or bradykinin-induced paw edema in prostacyclin receptor-deficient mice (IPKO). Paw volume of wild-type mice (IPWT) increased gradually 5-6 hr after the carrageenin injection in a similar manner as in ICR mice, but the swelling in IPKO mice was significantly smaller (about 60% of the IPWT volume). Indomethacin, at 10 mg/kg, suppressed the swelling of the IPWT paw to the level of the non-pretreated IPKO, which was not affected by indomethacin, confirming the previous result that PGI2 is a major prostaglandin involved in the swelling. The paw edema of IPWT and IPKO was significantly attenuated by the nonpeptide bradykinin B2-receptor antagonist FR173657, at 30 mg/kg, to the same level of swelling, indicating kinin involvement. Injection of bradykinin (1.2 nmole) into the paw caused rapid edema, which peaked around 15 min in both mice. However, the edema induced in IPKO was smaller and almost at the same level as that elicited in the indomethacin-treated IPWT, suggesting that edema induced by bradykinin includes the intrinsic effect of PGI2. Concomitant injection of carbacyclin with bradykinin caused enhancement of edema in IPWT mice but not in IPKO mice, indicating that intrinsic PGI2 could cause enhancement of bradykinin- or even carrageenin-induced edema formation. These results clearly demonstrate that bradykinin released by carrageenin may be a key mediator to induce PGI2 formation, and both autacoids work together to induce enhanced inflammatory exudation.

Evidence for bradykinin mediation of carrageenin-induced inflammatory pain: a study using kininogen-deficient Brown Norway Katholiek rats

Inflammatory pain was induced following an intradermal injection of carrageenin into rat paws, and the hyperalgesia was measured in terms of withdrawal time following thermal pain stimulation of the inflamed paw. This hyperalgesia was significantly less in kininogen-deficient Brown Norway (B/N)-Katholiek rats, which also showed less swelling in carrageenin-induced paw edema, than in normal B/N-Kitasato rats at 1 approximately 4 hr after the carrageenin injection (at the early phase). However, 24 hr after the injection, hyperalgesia and the swelling volume of the kininogen-deficient rats were almost the same as those in normal rats. The bradykinin B2 receptor antagonist FR173657, (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide, attenuated the carrageenin-induced swelling and hyperalgesia of the normal rats at the early phase to almost the levels of the B/N-Katholiek rats. Pretreatment with indomethacin, a cyclooxygenase inhibitor, also inhibited the carrageenin-induced responses significantly in normal rats. These results indicate that bradykinin, acting on the B2 receptor, is the main mediator at the early phase of inflammatory pain of carrageenin edema and that prostaglandins, produced by cyclooxygenase, potentiate the effects of bradykinin.

Taxonomic profiles in metagenomic analyses of free-living microbial communities in the Ofunato Bay

The Ofunato Bay in Iwate Prefecture, Japan is a deep coastal bay located at the center of the Sanriku Rias Coast and considered an economically and environmentally important asset. Here, we describe the first whole genome sequencing (WGS) study on the microbial community of the bay, where surface water samples were collected from three stations along its length to cover the entire bay; we preliminarily sequenced a 0.2 μm filter fraction among sequentially size-fractionated samples of 20.0, 5.0, 0.8 and 0.2 μm filters, targeting the free-living fraction only. From the 0.27-0.34 Gb WGS library, 0.9 × 106-1.2 × 106 reads from three sampling stations revealed 29 bacterial phyla (~80% of assigned reads), 3 archaeal phyla (~4%) and 59 eukaryotic phyla (~15%). Microbial diversity obtained from the WGS approach was compared with 16S rRNA gene results by mining WGS metagenomes, and we found similar estimates. The most frequently recovered bacterial sequences were Proteobacteria, predominantly comprised of 18.0-19.6% Planktomarina (Family Rhodobacteraceae) and 13.7-17.5% Candidatus Pelagibacter (Family Pelagibacterales). Other dominant bacterial genera, including Polaribacter (3.5-6.1%), Flavobacterium (1.8-2.6%), Sphingobacterium (1.4-1.6%) and Cellulophaga (1.4-2.0%), were members of Bacteroidetes and likely associated with the degradation and turnover of organic matter. The Marine Group I Archaea Nitrosopumilus was also detected. Remarkably, eukaryotic green alga Bathycoccus, Ostreococcus and Micromonas accounted for 8.8-15.2%, 3.6-4.9% and 2.1-3.1% of total read counts, respectively, highlighting their potential roles in the phytoplankton bloom after winter mixing.

Repercussions of the Great East Japan Earthquake tsunami on ellipsoidal Alexandrium cysts (Dinophyceae) in Ofunato Bay, Japan

Shellfish aquaculture in Ofunato Bay, Northeast Japan, was seriously damaged by a tsunami generated by the Great East Japan Earthquake, March 11th, 2011, accompanied by paralytic shellfish poisoning (PSP) outbreaks caused by Alexandrium tamarense (Dinophyceae). To understand longer future trends of PSP, an investigation was made of the historical occurrence and causes of Alexandrium outbreaks after the tsunami. Vertical distributions of Alexandrium cysts in two sediment-cores from Ofunato Bay revealed that the sediments above ca. 25 cm were eroded, re-suspended and re-deposited, and they included unusually abundant Alexandrium cysts. This abundance of cysts was due to re-deposition of older sediments by the tsunami. The first Ofunato Bay PSP incident was in 1961 after the Chilean Earthquake tsunami and was probably caused by similar unusual blooms of Alexandrium germinated from older sediments as the Great East Japan tsunami, together with nutrient enrichment because of population increase at the start of shellfish aquaculture.

Seasonal changes in the communities of photosynthetic picoeukaryotes in Ofunato Bay as revealed by shotgun metagenomic sequencing

Small photosynthetic eukaryotes play important roles in oceanic food webs in coastal regions. We investigated seasonal changes in the communities of photosynthetic picoeukaryotes (PPEs) of the class Mamiellophyceae, including the genera Bathycoccus, Micromonas and Ostreococcus, in Ofunato Bay, which is located in northeastern Japan and faces the Pacific Ocean. The abundances of PPEs were assessed over a period of one year in 2015 at three sampling stations, KSt. 1 (innermost bay area), KSt. 2 (middle bay area) and KSt. 3 (bay entrance area) at depths of 1 m (KSt. 1, KSt. 2 and KSt. 3), 8 m (KSt. 1) or 10 m (KSt. 2 and KSt. 3) by employing MiSeq shotgun metagenomic sequencing. The total abundances of Bathycoccus, Ostreococcus and Micromonas were in the ranges of 42-49%, 35-49% and 13-17%, respectively. Considering all assayed sampling stations and depths, seasonal changes revealed high abundances of PPEs during the winter and summer and low abundances during late winter to early spring and late summer to early autumn. Bathycoccus was most abundant in the winter, and Ostreococcus showed a high abundance during the summer. Another genus, Micromonas, was relatively low in abundance throughout the study period. Taken together with previously suggested blooming periods of phytoplankton, as revealed by chlorophyll a concentrations in Ofunato Bay during spring and autumn, these results for PPEs suggest that greater phytoplankton blooming has a negative influence on the seasonal occurrences of PPEs in the bay.

Metagenome-based diversity analyses suggest a strong locality signal for bacterial communities associated with oyster aquaculture farms in Ofunato Bay

Ofunato Bay, in Japan, is the home of buoy-and-rope-type oyster aquaculture activities. Since the oysters filter suspended materials and excrete organic matters into the seawater, bacterial communities residing in its vicinity may show dynamic changes depending on the oyster culture activities. We employed a shotgun metagenomic technique to study bacterial communities near oyster aquaculture facilities at the center of the bay (KSt. 2) and compared the results with those of two other localities far from the station, one to the northeast (innermost bay, KSt. 1) and the other to the southwest (bay entrance, KSt. 3). Seawater samples were collected every month from January to December 2015 from the surface (1 m) and deeper (8 or 10 m) layers of the three locations, and the sequentially filtered fraction on 0.2-μm membranes was sequenced on an Illumina MiSeq system. The acquired reads were uploaded to MG-RAST for KEGG functional abundance analysis, while taxonomic analyses at the phylum and genus levels were performed using MEGAN after parsing the BLAST output. Discrimination analyses were then performed using the ROC-AUC value of the cross validation, targeting the depth (shallow or deep), locality [(KSt. 1 + KSt. 2) vs. KSt 3; (KSt. 1 + KSt. 3) vs. KSt. 2 or the (KSt. 2 + KSt. 3) vs. KSt. 1] and seasonality (12 months). The matrix discrimination analysis on the adjacent 2 continuous seasons by ROC-AUC, which was based on the datasets that originated from different depths, localities and months, showed the strongest discrimination signal on the taxonomy matrix at the phylum level for the datasets from July to August compared with those from September to June, while the KEGG matrix showed the strongest signal for the datasets from March to June compared with those from July to February. Then, the locality combination was subjected to the same ROC-AUC discrimination analysis, resulting in significant differences between KSt. 2 and KSt. 1 + KSt. 3 on the KEGG matrix. These results suggest that aquaculture activities markedly affect bacterial functions.

Seasonal changes in the abundance of bacterial genes related to dimethylsulfoniopropionate catabolism in seawater from Ofunato Bay revealed by metagenomic analysis

Ofunato Bay is located in the northeastern Pacific Ocean area of Japan, and it has the highest biodiversity of marine organisms in the world, primarily due to tidal influences from the cold Oyashio and warm Kuroshio Currents. Our previous results from performing shotgun metagenomics indicated that Candidatus Pelagibacter ubique and Planktomarina temperata were the dominant bacteria (Reza et al., 2018a, 2018b). These bacteria are reportedly able to catabolize dimethylsulfoniopropionate (DMSP) produced from phytoplankton into dimethyl sulfide (DMS) or methanethiol (MeSH). This study was focused on seasonal changes in the abundances of bacterial genes (dddP, dmdA) related to DMSP catabolism in the seawater of Ofunato Bay by BLAST+ analysis using shotgun metagenomic datasets. We found seasonal changes among the Candidatus Pelagibacter ubique strains, including those of the HTCC1062 type and the Red Sea type. A good correlation was observed between the chlorophyll a concentrations and the abundances of the catabolic genes, suggesting that the bacteria directly interact with phytoplankton in the marine material cycle system and play important roles in producing DMS and MeSH from DMSP as signaling molecules for the possible formation of the scent of the tidewater or as fish attractants.