Discovery of Paralogous GnRH and Corazonin Signaling Systems in an Invertebrate Chordate.

Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive function in vertebrates. GnRH is related to the corazonin (CRZ) neuropeptide which influences metabolism and stress responses in insects. Recent evidence suggests that GnRH and CRZ are paralogous and arose by a gene duplication in a common ancestor of bilaterians. Here, we report the identification and complete characterization of the GnRH and CRZ signaling systems in the amphioxus Branchiostoma floridae. We have identified a novel GnRH peptide (YSYSYGFAP-NH2) that specifically activates two GnRH receptors and a CRZ peptide (FTYTHTW-NH2) that activates three CRZ receptors in B. floridae. The latter appear to be promiscuous, as two CRZ receptors can also be activated by GnRH in the physiological range. Hence, there is a potential for cross-talk between these closely related signaling systems. Discovery of both the GnRH and CRZ signaling systems in one of the closest living relatives of vertebrates provides a framework to discover their roles at the transition from invertebrates to vertebrates.

Fluorescence Properties of a Novel Isoquinoline Derivative Tested in an Invertebrate Chordate, Ciona intestinalis.

3-Hydroxyisoquinolines (ISOs) and their tautomeric isoquinolin-3-ones are heterocycles with attractive biological properties. Here we reported the revisited synthesis of a highly functionalized ISO that showed blue fluorescence and the characterization of its biological properties in an invertebrate animal model, the ascidian Ciona intestinalis. Larvae exposed to ISO at concentrations higher than 1 µM showed an intense fluorescence localized in the cell nuclei of all tissues. Moreover, exposure to ISO interfered with larval ability to swim; this neuromuscular effect was reversible. Overall, these results suggested that ISOs can have promising applications as novel fluorescent dyes of the cell nuclei.

The evolutionary road to invertebrate thyroid hormone signaling: Perspectives for endocrine disruption processes.

Thyroid hormones (THs) are the only iodine-containing hormones that play fundamental roles in chordates and non-chordates. The chemical nature, mode of action and the synthesis of THs are well established in mammals and other vertebrates. Although thyroid-like hormones have been detected in protostomes and non-chordate deuterostomes, TH signaling is poorly understood as compared to vertebrates, particularly in protostomes. Therefore, the central objective of this article is to review TH system components and TH-induced effects in non-vertebrate chordates, non-chordate deuterostomes and protostomes based on available genomes and functional information. To accomplish this task, we integrate here the available knowledge on the THs signaling across non-vertebrate chordates, non-chordate deuterostomes and protostomes by considering studies encompassing TH system components and physiological actions of THs. We also address the possible interactions of thyroid disrupting chemicals and their effects in protostomes and non-chordate deuterostomes. Finally, the perspectives on current and future challenges are discussed.

Selenium-loaded cellulose film derived from Styela clava tunic accelerates the healing process of cutaneous wounds in streptozotocin-induced diabetic Sprague-Dawley rats.

PURPOSE: Aims of this study is to evaluate the therapeutic effects and toxicity of Se-loaded cellulose film originated from Styela clava tunic (SeSCTF) on cutaneous wounds during diabetic conditions. MATERIALS AND METHODS: Alterations in skin regeneration, angiogenesis and toxicity were examined using streptozotocine (STZ)-induced diabetic Sprague Dawley® (SD) rats with surgical skin wounds after application of SeSCTF for 12 days. RESULTS: SCTF showed high tensile strength (1.64 MPa), low elongation (28.59%), low water vapor transmission rate (WVTR) and outstanding porous structure. Although SeSCTF application did not induce any significant alterations in glucose concentration or toxicity, wound morphology was rapidly recovered in the SeSCTF treated group relative to the gauze (GZ) and SCTF treated group. Moreover, recovery of re-epithelization, wound contraction and number of blood vessel was observed in SeSCTF treated groups when compared with all other groups. Furthermore, the SeSCTF treated group showed complete recovery of key protein expressions of the downstream signaling pathway of vascular endothelial growth factor (VEGF), angiopoietin-2/1 (Ang-2/1), the signaling pathway of insulin receptors and anti-oxidative status. CONCLUSIONS: Overall, the results of this study suggest that SeSCTF accelerates the healing process of cutaneous wounds in STZ-induced diabetic SD rats through stimulation of angiogenesis and the glucose receptor signaling pathway.

Characterization of paramyosin and thin filaments in the smooth muscle of acorn worm, a member of hemichordates.

Paramyosin is a myosin-binding protein characteristic of invertebrate animals, while troponin is a Ca2+-dependent regulator of muscle contraction. Both proteins are widely distributed in protostomes, while in deuterostomes, their distribution is limited; namely, presence of paramyosin and absence of troponin are common features in echinoderm muscles, while muscles of chordates contain troponin but lack paramyosin. In this study, we examined the muscle of a hemichordate, acorn worm, to clarify whether this animal is like echinoderms or like the other deuterostome animals. We found a 100-kDa protein in the smooth muscle of acorn worm. This protein was identified with paramyosin, since the purified protein formed paracrystals with a constant axial periodicity in the presence of divalent cations as paramyosin of other animals, showed ability to interact with myosin and shared common antigenicity with echinoderm paramyosin. On the other hand, troponin band was not detected in isolated thin filaments, and the filaments increased myosin-ATPase activity in a Ca2+-independent manner. The results indicate that troponin is lacking in thin filaments of acorn worm muscle just as in those of echinoderms. The muscle of hemichordate acorn worm is quite similar to echinoderm muscles, but different from chordate muscles.

FGF signaling repertoire of the indirect developing hemichordate Ptychodera flava.

Fibroblast growth factors (FGFs) are a group of ligands that play multiple roles during development by transducing signals through FGF receptors (FGFRs) to downstream factors. At least 22 FGF ligands and 4 receptors have been identified in vertebrates, while six to eight FGF ligands and a single FGFR are present in invertebrate chordates, such as tunicates and amphioxus. The chordate FGFs can be categorized into at least seven subfamilies, and the members of which expanded during the evolution of early vertebrates. In contrast, only one FGF and two FGFRs have been found in sea urchins. Thus, it is unclear whether the FGF subfamilies duplicated in the lineage leading to the chordates, or sea urchins lost several fgf genes. Analyses of the FGF signaling repertoire in hemichordates, which together with echinoderms form the closest group to the chordates, may provide insights into the evolution of FGF signaling in deuterostomes. In this study, we identified five FGFs and three FGFRs from Ptychodera flava, an indirect-developing hemichordate acorn worm. Phylogenetic analyses revealed that hemichordates possess a conserved FGF8/17/18 in addition to several putative hemichordate-specific FGFs. Analyses of sequence similarity and protein domain organizations suggested that the sea urchin and hemichordate FGFRs arose from independent lineage-specific duplications. Furthermore, the acorn worm fgf and fgfr genes were demonstrated to be expressed during P. flava embryogenesis. These results set the foundations for further functional studies of FGF signaling in hemichordates and provided insights into the evolutionary history of the FGF repertoire.

Structural and functional analysis of the amphioxus IGFBP gene uncovers ancient origin of IGF-independent functions.

IGFs play key roles in regulating vertebrate development, growth, reproduction, and aging. In extracellular fluids, IGFs are bound and regulated by a family of IGF-binding proteins (IGFBPs). Although all known IGFBPs are secreted proteins, some are also found in the nucleus and possess IGF-independent activities. When and how these distinct modes of biological actions have evolved is unknown. In this study, we identified and analyzed an IGFBP gene from amphioxus. Amphioxus shares a common ancestor with the modern vertebrate lineage that dates back to more than 520 million years ago. The amphioxus IGFBP shares all major structural characteristics of vertebrate IGFBPs. Phylogenetic analyses place it in a basal position in the IGFBP lineage. Ligand blot analysis reveals that amphioxus IGFBP does not bind to IGF-I or -II. Changing its Phe70 into Leu, however, is sufficient to convert it into a functional IGF binder. When tested in cultured cells, amphioxus IGFBP is localized in the nucleus, and this is attributed to 2 redundant nuclear localization sequences in its L domain. Furthermore, the amphioxus IGFBP N-terminal domain has strong transcriptional activation activity. Forced expression of amphioxus IGFBP in zebrafish embryos results in dorsalized phenotypes. This action requires nuclear localization. These results suggest that the nuclear localization and transcription activation activity of IGFBPs are ancient functions and the IGF-binding function may have been acquired by opportunistic gain-of-functional mutations later in evolution.

Remarkable similarities between the hemichordate (Saccoglossus kowalevskii) and vertebrate GPCR repertoire.

Saccoglossus kowalevskii (the acorn worm) is a hemichordate belonging to the superphylum of deuterostome bilateral animals. Hemichordates are sister group to echinoderms, and closely related to chordates. S. kowalevskii has chordate like morphological traits and serves as an important model organism, helping developmental biologists to understand the evolution of the central nervous system (CNS). Despite being such an important model organism, the signalling system repertoire of the largest family of integral transmembrane receptor proteins, G protein-coupled receptors (GPCRs) is largely unknown in S. kowalevskii. Here, we identified 260 unique GPCRs and classified as many as 257 of them into five main mammalian GPCR families; Glutamate (23), Rhodopsin (212), Adhesion (18), Frizzled (3) and Secretin (1). Despite having a diffuse nervous system, the acorn worm contains well conserved orthologues for human Adhesion and Glutamate family members, with a similar N-terminal domain architecture. This is particularly true for genes involved in CNS development and regulation in vertebrates. The average sequence identity between the GPCR orthologues in human and S. kowalevskii is around 47%, and this is same as observed in couple of the closest vertebrate relatives, Ciona intestinalis (41%) and Branchiostoma floridae (~47%). The Rhodopsin family has fewer members than vertebrates and lacks clear homologues for 6 of the 13 subgroups, including olfactory, chemokine, prostaglandin, purine, melanocyte concentrating hormone receptors and MAS-related receptors. However, the peptide and somatostatin binding receptors have expanded locally in the acorn worm. Overall, this study is the first large scale analysis of a major signalling gene superfamily in the hemichordate lineage. The establishment of orthologue relationships with genes involved in neurotransmission and development of the CNS in vertebrates provides a foundation for understanding the evolution of signal transduction and allows for further investigation of the hemichordate neurobiology.

Global view of the evolution and diversity of metazoan neuropeptide signaling.

Neuropeptides are signaling molecules that commonly act via G protein-coupled receptors (GPCRs) and are generated in neurons by proneuropeptide (pNP) cleavage. Present in both cnidarians and bilaterians, neuropeptides represent an ancient and widespread mode of neuronal communication. Due to the inherent difficulties of analyzing highly diverse and repetitive pNPs, the relationships among different families are often elusive. Using similarity-based clustering and sensitive similarity searches, I obtained a global view of metazoan pNP diversity and evolution. Clustering revealed a large and diffuse network of sequences connected by significant sequence similarity encompassing one-quarter of all families. pNPs belonging to this cluster were also identified in the early-branching neuronless animal Trichoplax adhaerens. Clustering of neuropeptide GPCRs identified several orthology groups and allowed the reconstruction of the phyletic distribution of receptor families. GPCR phyletic distribution closely paralleled that of pNPs, indicating extensive conservation and long-term coevolution of receptor-ligand pairs. Receptor orthology and intermediate sequences also revealed the homology of pNPs so far considered unrelated, including allatotropin and orexin. These findings, together with the identification of deuterostome achatin and luqin and protostome opioid pNPs, extended the neuropeptide complement of the urbilaterian. Several pNPs were also identified from the hemichordate Saccoglossus kowalevskii and the cephalochordate Branchiostoma floridae, elucidating pNP evolution in deuterostomes. Receptor-ligand conservation also allowed ligand predictions for many uncharacterized GPCRs from nonmodel species. The reconstruction of the neuropeptide-signaling repertoire at deep nodes of the animal phylogeny allowed the formulation of a testable scenario of the evolution of animal neuroendocrine systems.

An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri.

Amphioxus is a promising model animal for evolutionary and developmental studies. However, as an emerging model organism, amphioxus lacks most molecular techniques applied in other well-developed model animals. Microinjection is a powerful technique for gene manipulation, and thus it undoubtedly is one of useful approaches in the studies of gene function and embryonic development. Although the method has been exploited in Florida and European amphioxus, it still remains to be optimized and introduced into other amphioxus species. In order to introduce the technique into our lab, we followed and optimized the previous description and successfully performed microinjection on unfertilized eggs of Asian amphioxus Branchiostoma belcheri. We made six solutions for practice: 200 mM KCl, 100 ng/µl actin-LacZ or 100 ng/µl actin-RFP vectors, LacZ or RFP vectors without promoter and RFP capped mRNA. More than 99.2 % of eggs injected with KCl were able to be fertilized, 94.3 % of them could hatch normally and 55.9 % survived until 2-day larvae, all of which were nearly equivalent to those obtained from normally fertilized eggs. Embryos injected with two plasmid constructs also showed very high fertilizing and hatching ratios, but normally developing ratios were slightly lower than that of KCl injection. Of those injected embryos, 91.8 % expressed exogenous gene LacZ and 80.5 % exhibited foreign RFP expression, which were driven by a promoter from amphioxus ß-actin gene. The data indicated a successful modified microinjection method for the unfertilized eggs of Asian amphioxus, and those modifications improved the feasibility and efficiency of microinjection on amphioxus.

Characterization and comparative analyses of two amphioxus intelectins involved in the innate immune response.

Intelectin is a new type of soluble galactofuranose-binding lectin involved in innate immunity. Here we report another intelectin homolog, AmphiITLN239631, obtained from amphioxus, the transitional form between vertebrates and invertebrates. AmphiITLN239631 encoded 396 amino acids with a highly conserved fibrinogen-related domain (FReD), An intelectin domain and a putative Collagen domain. AmphiITLN239631 was ubiquitously expressed in all tissues we tested and transcripts in skin increased after challenge of both Escherichia coli and Staphylococcus aureus, although in different levels. Recombinant AmphiITLN239631 expressed in E. coli system could agglutinate both Gram-positive and Gram-negative bacteria in a calcium independent manner. Furthermore, recombinant protein was able to bind to lipopolysaccharide (LPS) and peptidoglycan (PGN), the major components of Gram-positive and Gram-negative bacteria cell walls, respectively. We also compared AmphiITLN239631 with previously identified AmphiITLN71469 and found that their tissue specificities, expression patterns upon bacteria challenge, and polysaccharide-binding affinities etc vary considerably. Our results could provide insight into the evolution and function of the intelectin family.

Cephalostatins and ritterazines.

This review article is a tribute to the numerous chemists whose relentless effort for the last quarter of a century resulted in the isolation, identification, and finally the chemical synthesis of a family of bis-steroidal pyrazine alkaloids of marine origin. In the task of defeating cancer, the search for bioactive substances among the naturally occurring compounds is, without any doubt, a preferential approach. The remarkable contribution of Petitt, Fusetani, and their coworkers allowed to discover this family of marine alkaloids that emerge as potential therapeutic anticancer agents, although there is still a long way to go. The challenging and dangerous task of collecting living organisms from deep-waters was followed by a laborious isolation, elucidation of the complicated structures and biological tests. The outcome of this paramount effort was the identification of 45 compounds that stand, to date, as some of the most potent anticancer agents. The intriguing structures of the isolated alkaloids drew the attention of synthetic chemists, valiant enough to undertake the challenging task of synthesizing some of the most active members of the family. Fuchs, Heathcock, Winterfeldt, Suarez, Shair, and their associates pioneered in the establishment of feasible synthetic routes for the preparation of some of the naturally occurring compounds and a large number of synthetic analogs, allowing to establish SAR criteria that have guided the design of new synthetic analogs. Numerous analogs have been prepared to investigate the mechanism of action of bis-steroidal pyrazines, e.g. cephalostatin analogs bearing a strained spiroketal moiety. However, the mechanism of action and the biological target of these compounds remain far from being understood. Therefore, the rational design of simpler, yet highly active analogs seems at the current stage elusive. It is still 1 to clear why these compounds need to be dimeric to show high biological activity. Furthermore, it is not known whether the central pyrazine ring is simply a linker or has some additional function. This could be tested by examining the biological activity of steroidal dimers with other linkers, e.g. with a benzene ring. Such analogs have been actually prepared but without functional groups necessary for biological activity. The clinical trials of cephalostatins have got stuck due to a shortage of material. There is an urgent need to provide highly active, yet not too complex analogs, which could be available in substantial amounts for advanced pharmacological studies.

Cloning, characterization and expression of tyrosinase-like gene in amphioxus Branchiostoma japonicum.

Prophenoloxidase (tyrosinase) widely distributed in invertebrates and vertebrates, and plays a crucial role in the innate immune. In the present study, the full-length cDNA of a tyrosinase-like (designated AmphiTYR) was cloned from amphioxus Branchiostoma japonicum by PCR techniques. The full-length cDNA of AmphiTYR is 2314 bp, and its predicted open-reading frame codes for a protein of 544 amino acids with a predicted molecular mass of approximately 60.9 kDa and an isoelectric point of 5.65. It has a conserved putative copper-binding domain with six histidines in tyrosinase proteins. Six potential N-linked glycosylation sites and 14 conserved cysteine residues were also predicted to be present in B. japonicum tyrosinase. Homology analysis revealed that AmphiTYR was higher similar to vertebrates tyrosinases (32.5-40.5%) than to invertebrates phenoloxidase (6.4-25.4%). In the adult, AmphiTYR mRNA was expressed in the muscle, epidermis, notochord, ovary, hepatic caecum, pharynx and gill, but not in the neural tube, intestines and testis. During the different development stages from unfertilized egg to larvae of amphioxus, AmphiTYR expressed during all the amphioxus development. These results indicated that AmphiTYR gene not only play a pivotal role in innate immune but also play an important role during embryonic development of cephalochordate amphioxus.

Amphioxus CaVPT and creatine kinase are crucial immune-related molecules in response to bacterial infection and immunization.

Although a great progress has been made, our understanding of innate immunity is incomplete. Here, we hypothesize that the innate immune response to pathogens is attributed into a group of functional proteins. The group contains information on host status post bacterial entry (infection or immunity) and bacterial species (Gram-positive or Gram-negative bacteria). Investigation of the group of proteins may result in disclosing of biomarkers identifying the status and species. For this regard, differential proteomics approach coupled with the pattern recognition methods are used to identify biomarkers from the proteins that being specifically regulated during the innate immune response of amphioxus to Gram-positive and Gram-negative bacteria with live or dead status. Four proteins, Calcium vector protein (CaVP), sarcoplasmic calcium-binding protein (SCP), CaVP-target protein (CaVPT) and creatine kinase (CK), are selected as the key biomarkers. Since immunoprotection of CaVP and SCP has been reported, the role of CaVPT and CK are further investigated. Gut CaVPT appears in dying amphioxus, whereas humoral fluid CK downregulates and gut CK keep no change in animals with immunity. The responses are stronger in Gram-negative than Gram-positive bacteria. These results indicate that CaVPT, CK, CaVP and SCP are the most important biomarkers to uncover amphioxus innate immunity to bacteria, and the approach is an efficient way to identify key biomarkers.

A novel short peptidoglycan recognition protein in amphioxus: identification, expression and bioactivity.

Peptidoglycan recognition proteins (PGRPs) are widely distributed in invertebrates and vertebrates, and structure-activity relationship of insect and mammalian PGRPs has been well characterized, but functional and structural insights into PGRPs in other species are rather limited. Here we identified a novel short PGRP gene from the amphioxus Branchiostoma japonicum, named pgrp-s, which possesses a domain combination of ChtBD1 domain-PGRP domain, which is unique to all known PGRPs. Amphioxus pgrp-s was predominantly expressed in the hepatic caecum, hind-gut and muscle in a tissue-specific manner. Recombinant PGRP-S, rPGRP-S, and truncated protein with ChtBD1 domain deleted, rP86/250, both showed affinity to Dap-type PGN, Lys-type PGN and chitin. Consistently, they were also able to bind to Escherichia coli, Staphylococcus aureus and Pichia pastoris. Moreover, both rPGRP-S and rP86/250 had amidase enzymatic activity, capable of hydrolyzing Dap-type and Lys-type PGNs. Like vertebrate PGRPs, rPGRP-S was directly microbicidal, capable of killing E. coli, S. aureus and P. pastoris, whereas rP86/250 only inhibited the growth of E. coli and S. aureus, and its anti-P. pastoris activity was significantly reduced. It is clear that neither the binding of amphioxus PGRP-S nor its amidase enzymatic activity depend on the N-terminal ChtBD1 domain, but its antifungal activity does. Collectively, these data suggested that amphioxus PGRP-S may function as a multivalent pattern recognition receptor, capable of recognizing PGN and chitin, a microbicidal agent, capable of killing bacteria such as E. coli and S. aureus and fungus like P. pastoris, and probably a PGN scavenger, capable of hydrolyzing PGN.

The function and developmental expression of alternatively spliced isoforms of amphioxus and Xenopus laevis Pax2/5/8 genes: revealing divergence at the invertebrate to vertebrate transition.

Pax genes encode highly conserved transcription factors vital for metazoan development. Pax transcripts, particularly those in Group II (Pax2/5/8), are extensively alternatively spliced. This study compares the transcriptional activation capacity and developmental stage-specific expression of major isoforms of Group II Pax proteins in amphioxus (Branchiostoma floridae) and in Xenopus laevis. The comparison reveals considerable divergence of splice forms between the lineages, with the X. laevis Group II Pax genes (Pax2, Pax5, and Pax8) possessing a greater repertoire of regulated and functionally distinct splice forms than the single amphioxus gene (Pax2/5/8). Surprisingly, some apparently conserved splice forms are expressed at quite different levels during development in the two organisms and present different capacities to activate transcription. However, despite this divergence, the combinatorial transcriptional activation capacity of the isoforms present in early X. laevis and amphioxus development are broadly similar. This suggests that the some of the conserved functional roles, implied by the expression of Group II Pax genes in homologous tissues of amphioxus and X. laevis embryos, may depend upon the combination of isoforms expressed in a particular tissue at a particular time in development. Thus, during early development, the evolutionary constraint on the net effect of several isoforms co-expressed in a given tissue may be more strict than that on specific isoforms. This flexibility may facilitate the appearance of new exons and splicing patterns in the vertebrate duplicates, leading to isoforms with subtly distinct functions critical to the subsequent development of vertebrate-specific cell types and structures.

Biomineral ultrastructure, elemental constitution and genomic analysis of biomineralization-related proteins in hemichordates.

Here, we report the discovery and characterization of biominerals in the acorn worms Saccoglossus bromophenolosus and Ptychodera flava galapagos (Phylum: Hemichordata). Using electron microscopy, X-ray microprobe analyses and confocal Raman spectroscopy, we show that hemichordate biominerals are small CaCO(3) aragonitic elements restricted to specialized epidermal structures, and in S. bromophenolosus, are apparently secreted by sclerocytes. Investigation of urchin biomineralizing proteins in the translated genome and expressed sequence tag (EST) libraries of Saccoglossus kowalevskii indicates that three members of the urchin MSP-130 family, a carbonic anhydrase and a matrix metaloprotease are present and transcribed during the development of S. kowalevskii. The SM family of proteins is absent from the hemichordate genome. These results increase the number of phyla known to biomineralize and suggest that some of the gene-regulatory 'toolkit', if not mineralized tissue themselves, may have been present in the common ancestor to hemichordates and echinoderms.

Identification, expression and bioactivity of a chitotriosidase-like homolog in amphioxus: dependence of enzymatic and antifungal activities on the chitin-binding domain.

The mammalian chitinase family 18 consists of two members, chitotriosidase (ChT) and acidic chitinase (AMCase). Despite the enormous progress on mammalian ChT study, little information regarding ChT is available to date in lower animals. In this study, we identified a chitotriosidase-like gene from the amphioxus Branchiostoma japonicum, named BjChTl, which consisted of a signal peptide, a catalytic domain, a Ser/Thr-rich linker region and a chitin-binding domain (CB domain). Sequence comparison and phylogenetic analysis showed that BjChTl was the common ancestor of ChTs and AMCases, implicating that ChT and AMCase evolved from an ancient gene like BjBhTl via gene duplication. qRT-PCR analysis revealed that BjChTl was expressed in the hepatic caecum and hind gut in a tissue-specific fashion. Both chitin-binding and enzymatic activities as well as antifungal activity assays demonstrated that like human ChT, recombinant BjChTl was able to bind to chitin particles, to hydrolyze artificial chitin substrate 4-methylumbelliferyl-ß-D-N,N',N″-triacetylchitotrioside, and to inhibit the growth of the fungus Candida albicans. Surprisingly, recombinant BjChTl-CD lacking CB domain retained partial capacity to bind to chitin, but its enzymatic activity was almost completely lost. These findings suggest that the CB domain is necessary for the execution of both enzymatic and antifungal activities of recombinant BjChTl. It is also the first study showing the presence of a ChT-like homolog with both chitinolytic activity and fungistatic activity in non-vertebrate species.

The first chordate big defensin: identification, expression and bioactivity.

Defensins are broadly present in plants, invertebrates and vertebrates, but little information is available about it in amphioxus, a protochordate holding a key phylogenetic position. In this study, a big defensin cDNA was identified from the amphioxus Branchiostoma japonicum (termed Bjbd). The cDNA contained an open reading frame (ORF) of 354 bp encoding a 117 amino acid protein, which had an N-terminal signal sequence followed by a propeptide and the mature big defensin. The mature peptide had the hydrophobic region GAAAVT(A)AA at N-terminus and the consensus pattern C-X6-C-X3-C-X13(14)-C-X4-C-C at C-terminus as well as four α-helices, four ß-sheets, and three disulfide bridges (C1-C5, C2-C4 and C3-C6) in the predicted tertiary structure. This is the first big defensin gene ever identified in the phylum Chordata. Quantitative real-time PCR analysis revealed that Bjbd was constitutively expressed in most of the tissues examined, and its expression was remarkably up-regulated following the challenge with LPS, LTA, Aeromonas hydrophila and Staphylococcus aureus. Moreover, the recombinant BjBD was shown to be able to inhibit the growth of S. aureus, Escherichia coli and A. hydrophila. Taken together, these suggest that BjBD is a molecule involved in the removal of invading pathogens.

Sialome analysis of the cephalochordate Branchiostoma belcheri, a key organism for vertebrate evolution.

Sialic acid, a common terminal substitution of glycoconjugates, has been so far consistently identified in all vertebrates as well as in a growing number of bacterial species. It is assumed to be widely distributed among animal species of the deuterostome phylum, based on its identification in few echinoderm and all vertebrate species. However, whole sections of deuterostome, especially those intermediate species between invertebrates and vertebrates including cephalochordates, urochordates and hemichordates, are still unexplored in term of sialylation capacities. The discovery of functional sialic acid machinery in some of these species may shed new light onto the evolution of glycosylation capacities in deuterostome lineage. In a first approach, we investigated the sialylation pattern of a cephalocordate species, Branchiostoma belcheri, which occupies a strategic phylogenetic position to understand the transition of invertebrates toward vertebrates. Structural analysis of B. belcheri glycoconjugates established that this organism synthesizes large quantities of various sialic acids, some of which present rare or novel structures such as methylated sialic acids. These sialic acids were shown to be mainly associated with mono- and disialylated core 1-type O-glycans. Moreover, screening of the animal organs revealed the existence of exquisite tissue specificity in the distribution of sialic acids. Description of sialylation profiles was then correlated with the expression patterns of key enzymes involved in the biosynthesis of major forms of sialic acids, which provides the first complete overview of the sialylation patterns in cephalochordates.