Comparison of the Inducing Effect of Indole Compounds on Medusa Formation in Different Classes of Medusozoa.

Scyphozoa, Cubozoa and Hydrozoa are classes in the phylum Cnidaria that undergo metagenesis involving a dramatic morphological transition. In Scyphozoa and Cubozoa, when exposed to species- or strain-specific transition-inducing stimuli, asexually reproducing benthic polyps transform into sexually reproducing planktonic medusae. In Hydrozoa, exposure to species- or strainspecific transition-inducing stimuli causes formation of medusa buds in the polyp's body. In Aurelia aurita (Linnaeus, 1758) (Scyphozoa, Semaeostomeae), polyp-to-jellyfish transition is induced by some simple indole compounds. However, whether indole compounds can induce polyp-to-jellyfish transition in Cubozoa and Hydrozoa remains unknown. In the present study, we show that an indole compound, 5-methoxy-2-methylindole, induces polyp-to-jellyfish transition in Scyphozoa and Cubozoa. This inducing action suggests that the downstream steps of polyp-to-jellyfish transition are regulated by the same biochemical reactions in Scyphozoa and Cubozoa, irrespective of the type of transition-inducing environmental stimuli.

The iron content and ferritin contribution in fresh, dried, and toasted nori, Pyropia yezoensis.

Iron is one of the essential trace elements for humans. In this study, the iron contents in fresh, dried, and toasted nori (Pyropia yezoensis) were analyzed. The mean iron content of fresh, dried, and toasted nori were 19.0, 22.6, and 26.2 mg/100 g (dry weight), respectively. These values were superior to other food of plant origin. Furthermore, most of the iron in nori was maintained during processing, such as washing, drying, and toasting. Then, the form of iron in fresh, dried, and toasted nori was analyzed. As a result, an iron storage protein ferritin contributed to iron storage in raw and dried nori, although the precise rate of its contribution is yet to be determined, while ferritin protein cage was degraded in the toasted nori. It is the first report that verified the ferritin contribution to iron storage in such edible macroalgae with commercial importance.

Sensing deep extreme environments: the receptor cell types, brain centers, and multi-layer neural packaging of hydrothermal vent endemic worms.

INTRODUCTION: Deep-sea alvinellid worm species endemic to hydrothermal vents, such as Alvinella and Paralvinella, are considered to be among the most thermotolerant animals known with their adaptability to toxic heavy metals, and tolerance of highly reductive and oxidative stressful environments. Despite the number of recent studies focused on their overall transcriptomic, proteomic, and metabolic stabilities, little is known regarding their sensory receptor cells and electrically active neuro-processing centers, and how these can tolerate and function in such harsh conditions. RESULTS: We examined the extra- and intracellular organizations of the epidermal ciliated sensory cells and their higher centers in the central nervous system through immunocytochemical, ultrastructural, and neurotracing analyses. We observed that these cells were rich in mitochondria and possessed many electron-dense granules, and identified specialized glial cells and serial myelin-like repeats in the head sensory systems of Paralvinella hessleri. Additionally, we identified the major epidermal sensory pathways, in which a pair of distinct mushroom bodies-like or small interneuron clusters was observed. These sensory learning and memory systems are commonly found in insects and annelids, but the alvinellid inputs are unlikely derived from the sensory ciliary cells of the dorsal head regions. CONCLUSIONS: Our evidence provides insight into the cellular and system-wide adaptive structure used to sense, process, and combat the deep-sea hydrothermal vent environment. The alvinellid sensory cells exhibit characteristics of annelid ciliary types, and among the most unique features were the head sensory inputs and structure of the neural cell bodies of the brain, which were surrounded by multiple membranes. We speculated that such enhanced protection is required for the production of normal electrical signals, and to avoid the breakdown of the membrane surrounding metabolically fragile neurons from oxidative stress. Such pivotal acquisition is not broadly found in the all body parts, suggesting the head sensory inputs are specific, and these heterogenetic protection mechanisms may be present in alvinellid worms.

A high-throughput screen for inhibitors of the prolyl isomerase, Pin1, identifies a seaweed polyphenol that reduces adipose cell differentiation.

The peptidyl prolyl cis/trans isomerase Pin1 enhances the uptake of triglycerides and the differentiation of fibroblasts into adipose cells in response to insulin stimulation. Pin1 downregulation could be a potential approach to prevent and treat obesity-related disorders. In order to identify an inhibitor of Pin1 that exhibited minimal cytotoxicity, we established a high-throughput screen for Pin1 inhibitors and used this method to identify an inhibitor from 1,056 crude fractions of two natural product libraries. The candidate, a phlorotannin called 974-B, was isolated from the seaweed, Ecklonia kurome. 974-B inhibited the differentiation of mouse embryonic fibroblasts and 3T3-L1 cells into adipose cells without inducing cytotoxicity. We discovered the Pin1 inhibitor, 974-B, from the seaweed, E. kurome, and showed that it blocks the differentiation of fibroblasts into adipose cells, suggesting that 974-B could be a lead drug candidate for obesity-related disorders.

The diversity of shell matrix proteins: genome-wide investigation of the pearl oyster, Pinctada fucata.

In molluscs, shell matrix proteins are associated with biomineralization, a biologically controlled process that involves nucleation and growth of calcium carbonate crystals. Identification and characterization of shell matrix proteins are important for better understanding of the adaptive radiation of a large variety of molluscs. We searched the draft genome sequence of the pearl oyster Pinctada fucata and annotated 30 different kinds of shell matrix proteins. Of these, we could identified Perlucin, ependymin-related protein and SPARC as common genes shared by bivalves and gastropods; however, most gastropod shell matrix proteins were not found in the P. fucata genome. Glycinerich proteins were conserved in the genus Pinctada. Another important finding with regard to these annotated genes was that numerous shell matrix proteins are encoded by more than one gene; e.g., three ACCBP-like proteins, three CaLPs, five chitin synthase-like proteins, two N16 proteins (pearlins), 10 N19 proteins, two nacreins, four Pifs, nine shematrins, two prismalin-14 proteins, and 21 tyrosinases. This diversity of shell matrix proteins may be implicated in the morphological diversity of mollusc shells. The annotated genes reported here can be searched in P. fucata gene models version 1.1 and genome assembly version 1.0 ( http://marinegenomics.oist.jp/pinctada_fucata ). These genes should provide a useful resource for studies of the genetic basis of biomineralization and evaluation of the role of shell matrix proteins as an evolutionary toolkit among the molluscs.

Isolation and structural determination of two novel phlorotannins from the brown alga Ecklonia kurome Okamura, and their radical scavenging activities.

Two novel phlorotannins with a molecular weight of 974, temporarily named 974-A and 974-B, were isolated from the polyphenol powder prepared from the edible marine brown alga Ecklonia kurome Okamura, and their chemical structures were determined by spectroscopic method. The isolated yield of the total of 974-A and 974-B was approximately 4% (w/w) from the polyphenol powder. In 974-A, the carbon at the C2' position in the A ring of phlorofucofuroeckol-A forms a C-C bond with the carbon at the C2″ position of the C ring of triphloretol-B, while in 974-B, phlorofucofuroeckol-B and triphloretol-B form a C-C bond in the same manner as in 974-A. These structures were supported by high resolution-MS/MS data. To evaluate the antioxidant activities, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and intracellular radical scavenging assay, using 2',7'-dichlorofluorescin diacetate (DCFH-DA), were performed for 974-A, 974-B, and four known phlorotannins. The results of the DPPH assay showed that the IC(50) values of 974-A, 974-B, phlorofucofuroeckol-A, and dieckol were significantly smaller than those of phlorofucofuroeckol-B, phloroglucinol, α-tocopherol, and ascorbic acid. Furthermore, the DCFH-DA assay suggested that 974-A, 974-B, and dieckol reduce intracellular reactive oxygen species most strongly among the tested compounds.

Identification and characterization of a ferritin gene and its product from the multicellular green alga Ulva pertusa.

Iron is an essential element for virtually all kingdoms of life, and especially for primary producers in ocean ecosystems. To date, the molecular mechanism of iron utilization by macroalgae remains largely unknown. To elucidate the strategy of iron acquisition and storage in macroalgae, we focused on the function of the iron storage protein ferritin in the sea lettuce, Ulva pertusa, which has abundant iron content. Judging from the primary structure, U. pertusa ferritin (UpFer) can be classified as a land-plant-type ferritin, which is usually found in plastids. The gene of UpFer was expressed in the peripheral, central and rhizoid parts. Western blot analysis showed that UpFER was present and functioned in processed 26- and 22-kDa forms. Furthermore, recombinant UpFER had iron incorporation activity comparable to other ferritins. These results suggest that ferritin also functions as an iron storage protein as in unicellular algae and land plants.

A comparative study of cellulase and hemicellulase activities of brackish water clam Corbicula japonica with those of other marine Veneroida bivalves.

Corbicula japonica is a typical brackish water bivalve species belonging to the order Veneroida, and it is the most important inland fishery resource in Japan. Corbicula japonica has been suggested to assimilate organic matter from terrestrial plants, unlike Ruditapes philippinarum and Mactra veneriformis, which selectively assimilate organic matter of marine origin. This led us to hypothesize that C. japonica, despite being a suspension feeder, could assimilate cellulosic materials derived from terrestrial plants. In the present study, we measured cellulase and hemicellulase activities in the crystalline styles of C. japonica and other commercially important Veneroida bivalve species in Japan: Ruditapes philippinarum, Meretrix lamarckii and Meretrix lusoria. Corbicula japonica demonstrated notably higher cellulase, xylanase and beta-mannanase activities than the other marine bivalves, suggesting that this species possesses a far greater biochemical capacity to break down the structural polysaccharides of plant cell walls than the other species. In contrast, the beta-1,3-glucanase and pectinase activities of C. japonica were similar to or even lower than those of the others. This is possibly due to the presence of these polysaccharides in the cell walls of diatoms, a principal food of most marine bivalves. Although direct evidence is lacking, the high cellulase, xylanase and beta-mannanase activities of C. japonica may result from adaptation to an upstream estuarine environment where phytoplankton and diatoms are scarce, but plant-derived substances are abundant.

Identification of possible hypoxia sensor for behavioral responses in a marine annelid, Capitella teleta.

Hypoxia often occurs in summer and causes deleterious effects on marine benthic animals. A marine annelid, Capitella teleta, is tolerant to hypoxia, as shown by the fact that it inhabits organically polluted areas, where severe hypoxia is often observed. To understand how this species adapts to the environment, we focused on its hypoxia sensor, and we showed that TRPAbasal was a possible contributor to hypoxia detection in C. teleta To examine the involvement of TRPA1 in the response of C. teleta to hypoxia, we exposed C. teleta to hypoxic water with or without a TRPA1-specific inhibitor, A-967079. Hypoxic stimulation induced escape behavior in C. teleta from the sediment, and this behavior was suppressed by the inhibitor. The cloned TRPA gene from C. teleta was phylogenetically categorized into TRPAbasal, and contains an oxygen-dependent degradation domain, which is important for the detection of hypoxia. Whole-mount in situ hybridization analysis showed that the gene was transcribed in the prostomium, where sensing functions are localized. These results suggested that the worm has a hypoxia-sensing system possibly utilizing CtTRPAbasal, and this system contributes to expanding the organism's niches in hypoxic environments by detecting whether hypoxia exceeds a level that would imperil its survival.

Wetland environmental bioreactor system contributes to the decomposition of cellulose.

Recently, numerous species of aquatic invertebrates inhabiting wetlands have been shown to possess endogenous cellulase, following the discovery that termites have cellulase genes encoded in their own genome rather than relying on symbiotic bacteria for decomposing cellulose. Wetlands have been empirically shown to play an important role in the decomposition of land-originating hard-to-degrade polysaccharides such as cellulose. However, the mechanism that connects the cellulase producer and the wetlands remains unknown, which makes it very difficult to evaluate the ecological function of wetlands. Here we found that a macrobenthic bivalve, Corbicula japonica, secretes its cellulase to the wetland sediment. Secreted cellulases are immobilized in the components of the sediment. Moreover, adding cellulose or glucose to C. japonica could trigger its cellulase secretion level. These findings suggest a novel wetland cellulose decomposition mechanism. The decomposition ability of wetlands was previously ascribed only to microbes and/or invertebrates that contain cellulases. Our findings suggest that benthic animals supply wetlands with their enzymes as decomposition agents, while wetland sediments serve as immobilization scaffolds for the enzymes. This system, which was named by us an "environmental bioreactor system," could provide a key function in wetlands.

Possible role of a taurine transporter in the deep-sea mussel Bathymodiolus septemdierum in adaptation to hydrothermal vents.

Various invertebrates inhabiting hydrothermal vents possess sulfur-oxidizing bacteria in their tissues; however, the mechanisms by which toxic sulfides are delivered to these endosymbionts remain unknown. Recently, detoxification of sulfides using thiotaurine, a sulfur-containing amino acid, has been suggested. In this study, we propose the involvement of a taurine transporter in sulfide detoxification in the deep-sea mussel Bathymodiolus septemdierum by demonstrating: (i) the abundance of its mRNA in the gill; (ii) its activity under a wide range of salinities; (iii) its low Michaelis constant value in taurine transportation; and (iv) its affinity for thiotaurine and the thiotaurine precursor, hypotaurine.

Identification of receptors of main sex-pheromone components of three Lepidopteran species.

Male moths discriminate conspecific female-emitted sex pheromones. Although the chemical components of sex pheromones have been identified in more than 500 moth species, only three components in Bombyx mori and Heliothis virescens have had their receptors identified. Here we report the identification of receptors for the main sex-pheromone components in three moth species, Plutella xylostella, Mythimna separata and Diaphania indica. We cloned putative sex-pheromone receptor genes PxOR1, MsOR1 and DiOR1 from P. xylostella, M. separata and D. indica, respectively. Each of the three genes was exclusively expressed with an Or83b orthologous gene in male olfactory receptor neurons (ORNs) that are surrounded by supporting cells expressing pheromone-binding-protein (PBP) genes. By two-electrode voltage-clamp recording, we tested the ligand specificity of Xenopus oocytes co-expressing PxOR1, MsOR1 or DiOR1 with an OR83b family protein. Among the seven sex-pheromone components of the three moth species, the oocytes dose-dependently responded only to the main sex-pheromone component of the corresponding moth species. In our study, PBPs were not essential for ligand specificity of the receptors. On the phylogenetic tree of insect olfactory receptors, the six sex-pheromone receptors identified in the present and previous studies are grouped in the same subfamily but have no relation with the taxonomy of moths. It is most likely that sex-pheromone receptors have randomly evolved from ancestral sex-pheromone receptors before the speciation of moths and that their ligand specificity was modified by mutations of local amino acid sequences after speciation.

Immunohistochemical, in situ hybridization and biochemical studies on endogenous cellulase of Corbicula japonica.

We previously reported on the endogenous cellulase gene of Corbicula japonica, CjCel9A. In this study, the tissue localization of the mRNA and translated products of CjCel9A was investigated in order to understand how this gene is physiologically involved in cellulose decomposition by C. japonica. Antiserum against recombinant CjCel9A protein was prepared. Multiple bands were observed mainly on western blot analysis of the crystalline style, and the band sizes partially corresponded to the active bands detected using zymographic analysis. In situ hybridization and immunohistochemical analyses clarified the exclusive production and secretion of this cellulase by the secretory cells localized in the epithelium of the digestive tubules in the digestive gland. These data strongly support our previous assumption that the endogenous cellulase of C. japonica is produced in the digestive gland and transported to the crystalline style to act as a component of its cellulolytic activity.

Highly sensitive avoidance plays a key role in sensory adaptation to deep-sea hydrothermal vent environments.

The environments around deep-sea hydrothermal vents are very harsh conditions for organisms due to the possibility of exposure to highly toxic compounds and extremely hot venting there. Despite such extreme environments, some indigenous species have thrived there. Alvinellid worms (Annelida) are among the organisms best adapted to high-temperature and oxidatively stressful venting regions. Although intensive studies of the adaptation of these worms to the environments of hydrothermal vents have been made, little is known about the worms' sensory adaptation to the severe chemical conditions there. To examine the sensitivity of the vent-endemic worm Paralvinella hessleri to low pH and oxidative stress, we determined the concentration of acetic acid and hydrogen peroxide that induced avoidance behavior of this worm, and compared these concentrations to those obtained for related species inhabiting intertidal zones, Thelepus sp. The concentrations of the chemicals that induced avoidance behavior of P. hessleri were 10-100 times lower than those for Thelepus sp. To identify the receptors for these chemicals, chemical avoidance tests were performed with the addition of ruthenium red, a blocker of transient receptor potential (TRP) channels. This treatment suppressed the chemical avoidance behavior of P. hessleri, which suggests that TRP channels are involved in the chemical avoidance behavior of this species. Our results revealed for the first time hypersensitive detection systems for acid and for oxidative stress in the vent-endemic worm P. hessleri, possibly mediated by TRP channels, suggesting that such sensory systems may have facilitated the adaptation of this organism to harsh vent environments.

Two types of phenoloxidases contribute to hemolymph PO activity in spiny Lobster.

Phenoloxidases (POs) play a crucial role in melanization of crustaceans. There are at least two types of POs characterized in crustaceans: the conventional type (POα here) that is expressed in hemocytes and POß, a secreted protein synthesized in the hepatopancreas. We investigated the source of PO activity in the hemolymph of a lobster and determined the kinetic parameters of mono- and di-PO activities. In the lobster hemolymph, POα, which formed a hexamer similar to both POß and hemocyanin, contributed to PO activity, whereas the amount of POß was low. Kinetic analyses using purified prophenoloxidase of crustaceans showed that lobster POα has a higher rate constant, while shrimp POß has higher specificity in both mono- and di-PO reactions, when tyramine and dopamine were employed as substrates. There should be at least two types of PO molecules in crustacean hemolymph, but the dominant PO molecule type varies among species.

The nuclear export signal (NES) found in the amino-terminal region of carp MEK1 and MKK6 is lacking in carp MKK4.

Carp MKK4 (cMKK4) cDNA was isolated from an ovary cDNA library. cMKK4 mRNA was ubiquitously distributed in various tissues of adult carp. Sequence analysis revealed that cMKK4 lacks a nuclear export signal sequence, unlike mammalian and frog MEKs (ERK activator) and cMKK6 (carp p38 activator), where it plays an important role in anchoring these MAP kinase activators to the cytoplasm. cMKK4 protein was found to be diffused throughout the cell, whereas cMEK1 and cMKK6 proteins were seen exclusively in the cytoplasm.

Occurrence of two distinct types of tissue inhibitor of metalloproteinases-2 in teleost fish.

We have cloned for the first time two cDNAs encoding distinct types of tissue inhibitor of metalloproteinases-2 (TIMP-2) from teleost fish, Japanese flounder, and designated these types as jfTIMP-2a and jfTIMP-2b. The open reading frames of the jfTIMP-2a and jfTIMP-2b cDNAs are composed of 663 and 657 nucleotides and 221 and 219 amino acids, respectively. Both jfTIMP-2s contain 12 cysteine residues, which might form six disulfide bonds as in other animals' TIMP-2s. The predicted full-length amino acid sequence of jfTIMP-2a has lower identity to jfTIMP-2b (63%) than to those of human (74%) and chicken (73%) TIMP-2s, but higher than to those of other human TIMPs (TIMP-1: 39%, TIMP-3: 43%, TIMP-4: 45%), indicating that jfTIMP-2a is a common TIMP-2, while jfTIMP-2b is unique to Japanese flounder. However, the C-terminal region including the last three disulfide bonds of jfTIMP-2b has higher amino acid identity to those of other animal TIMP-2s than to that of jfTIMP-2a. Reverse-transcribed polymerase chain reaction (RT-PCR) analysis showed the mRNAs of jfTIMP-2a and jfTIMP-2b to be ubiquitously expressed in all tissues examined, but with different expression patterns. These findings suggest that the two distinct jfTIMP-2s might perform different functions in teleost tissues.

Scallop DMT functions as a Ca2+ transporter.

We identified a DMT (divalent metal transporter) homologous protein that functions as a Ca(2+) transporter. Scallop DMT cDNA encodes a 539-amino-acid protein with 12 putative membrane-spanning domains and has a consensus transport motif in the fourth extracellular loop. Since its mRNA is significantly expressed in the gill and intestine, it is assumed that scallop DMT transports Ca(2+) from seawater by the gill and from food by the intestine. Scallop DMT lacks the iron-responsive element commonly found in iron-regulatory proteins, suggesting that it is free of the post-transcriptional regulation from intracellular Fe(2+) concentration. Scallop DMT distinctly functions as a Ca(2+) transporter unlike other DMTs, however, it also transports Fe(2+) and Cd(2+) similar to them.

Isolation and characterization of a Japanese flounder clonal line, reversed, which exhibits reversal of metamorphic left-right asymmetry.

We have isolated a clonal line reversed (rev) of homozygous Japanese flounder through gynogenesis. The homozygous offspring gynogenetically produced from rev exhibited reversal of organization of the metamorphic L/R asymmetry such as the direction of eye-migration at a high frequency (20-30%). The molecular analysis using a left-specific marker pitx2 revealed that the embryonic L/R axis was ambiguously established: in more than half of rev embryos, pitx2 was expressed bilaterally in the lateral plate mesoderm (LPM). Previous studies in other animals demonstrated that ectopic pitx2 expression in the LPM could cause laterality defects of the visceral organs. Likewise, our results using rev imply that bilateral pitx2 expression could lead to randomization of the visceral organs. Coincidence of ectopic pitx2 expression and reversal of the direction of eye-migration in the population of rev offspring suggests that the rev locus is critical in specification of both the metamorphic and the visceral L/R asymmetries. However, reversal of the sidedness of the orientation of the visceral organs was not always accompanied by reversal of the direction of metamorphic eye-migration, suggesting that different mechanisms should be involved downstream of the rev locus in directing these two phases of asymmetric morphogenesis in the Japanese flounder.

The extension peptide of plant ferritin from sea lettuce contributes to shell stability and surface hydrophobicity.

Plant ferritins have some unique structural and functional features. Most of these features can be related to the plant-specific "extension peptide" (EP), which exists in the N-terminus of the mature region of a plant ferritin. Recent crystallographic analysis of a plant ferritin revealed the structure of the EP, however, two points remain unclear: (i) whether the structures of well-conserved EP of plant ferritins are common in all plants, and (ii) whether the EP truly contributes to the shell stability of the plant ferritin oligomer. To clarify these matters, we have cloned a green-plant-type ferritin cDNA from a green alga, Ulva pertusa, and investigated its crystal structure. Ulva pertusa ferritin (UpFER) has a plant-ferritin-specific extension peptide composed of 28 amino acid residues. In the crystal structure of UpFER, the EP lay on and interacted with the neighboring threefold symmetry-related subunit. The amino acid residues involved in the interaction were very highly conserved among plant ferritins. The EPs masked the hydrophobic pockets on the ferritin shell surface by lying on them, and this made the ferritin oligomer more hydrophilic. Furthermore, differential scanning calorimetric analysis of the native and its EP-deletion mutant suggested that the EP contributed to the thermal stability of the plant ferritin shell. Thus, the shell stability and surface hydrophobicity of plant ferritin were controlled by the presence or absence of the plant-ferritin-specific EP. This regulation can account for those processes such as shell stability, degradation, and association of plant ferritin, which are significantly related to iron utilization in plants.