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.

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.

A rod domain sequence in segment 1B triggers dimerisation of the two small Branchiostoma IF proteins B2 and A3.

Previously, we cloned two Branchiostoma IF proteins A3 and B2 and demonstrated that both can form heteropolymeric IF based on a coiled coil dimer consisting of one B2 and one A3 polypeptide. In this study we continued in the characterisation of the B2/A3 heterodimer by searching for the sequences that play an important role in the triggering of the B2/A3 heterodimer. Using a series of deletion and chimeric B2, A3 and B1 constructs and the overlay assay as a tool, we were able to identify a part of the B2 sequence (segment 1A, linker L1 and the N-terminal part of segment 1B) which retains the ability of the full length protein B2 to specifically recognize A3 in blot overlays. Moreover, inspection of this A3-competent B2 fragment identified a short sequence in segment 1B which shares with the currently known trigger-like motif of cortexillin and other coiled coil proteins potential to form multiple inter-chain ionic interactions. Thus, a common and essential feature of trigger sequences with different primary structures found so far in IF and other coiled coil proteins seems to be their ability to form multiple inter-chain ionic interactions which brings the chains close to one another and allows coiled coil formation to propagate accordingly.

A novel serpin with antithrombin-like activity in Branchiostoma japonicum: implications for the presence of a primitive coagulation system.

Serine protease inhibitors, or serpins, are a group of widely distributed proteins with similar structures that use conformational change to inhibit proteases. Antithrombin (AT) is a member of the serine protease inhibitor superfamily and a major coagulation inhibitor in all vertebrates, but its evolutionary origin remains elusive. In this study we isolated for the first time a cDNA encoding an antithrombin homolog, BjATl, from the protochordate Branchiostoma japonicum. The deduced protein BjATl consisted of 338 amino acids sharing 36.7% to 41.1% identity to known vertebrate ATs. BjATl contains a potential N-linked glycosylation site, two potential heparin binding sites and the reactive center loop with the absolutely conserved sequence Gly-Arg-Ser; all of these are features characteristic of ATs. All three phylogenetic trees constructed using Neighbor-Joining, Maximum-Likelihood and Bayesian-Inference methods also placed BjATl together with ATs. Moreover, BjATl expressed in yeast cells was able to inhibit bovine thrombin activity by forming a SDS-stable BjATl-thrombin complex. It also displays a concentration-dependent inhibition of thrombin that is accelerated by heparin. Furthermore, BjATl was predominantly expressed in the hepatic caecum and hind-gut, agreeing with the expression pattern of AT in mammalian species. All these data clearly demonstrate that BjATl is an ortholog of vertebrate ATs, suggesting that a primitive coagulation system emerged in the protochordate.

Amphioxus IGF-like peptide induces mouse muscle cell development via binding to IGF receptors and activating MAPK and PI3K/Akt signaling pathways.

Insulin-like growth factors (IGFs) are identified in all vertebrates. An insulin/IGF hybrid polypeptide has also been identified in protochordate amphioxus. However, whether this hybrid polypeptide functions as vertebrate IGFs remains unknown. Here we established a primary culture system of mouse muscle satellite cells as an in vitro model to investigate the effects of amphioxus IGF-like molecule on muscle cell development. Like human IGF, recombinant IGF-like molecule was able to stimulate the proliferation of mouse muscle cells. Besides, it was able to bind to the cells and the partially purified IGF receptors from mouse muscle cells. Moreover, recombinant IGF-like molecule was capable of activating MAPK and PI3K/Akt pathways by stimulating phosphorylation of MAPK and Akt. This interaction of amphioxus IGF-like molecule with mammalian (mouse) IGF receptors and its induction of similar downstream signaling pathways add substantially to the hypothesis of the presence of IGF signaling in the common ancestor of protochordate/vertebrates.

Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae.

The origin of germline cells was a crucial step in animal evolution. Therefore, in both developmental biology and evolutionary biology, the mechanisms of germline specification have been extensively studied over the past two centuries. However, in many animals, the process of germline specification remains unclear. Here, we show that in the cephalochordate amphioxus Branchiostoma floridae, the germ cell-specific molecular markers Vasa and Nanos become localized to the vegetal pole cytoplasm during oogenesis and are inherited asymmetrically by a single blastomere during cleavage. After gastrulation, this founder cell gives rise to a cluster of progeny that display typical characters of primordial germ cells (PGCs). Blastomeres separated at the two-cell stage grow into twin embryos, but one of the twins fails to develop this Vasa-positive cell population, suggesting that the vegetal pole cytoplasm is required for the formation of putative PGCs in amphioxus embryos. Contrary to the hypothesis that cephalochordates may form their PGCs by epigenesis, our data strongly support a preformation mode of germ cell specification in amphioxus. In addition to the early localization of their maternal transcripts in the putative PGCs, amphioxus Vasa and Nanos are also expressed zygotically in the tail bud, which is the posterior growth zone of amphioxus. Thus, in addition to PGC specification, amphioxus Vasa and Nanos may also function in highly proliferating somatic stem cells.

Conserved eukaryotic fusogens can fuse viral envelopes to cells.

Caenorhabditis elegans proteins AFF-1 and EFF-1 [C. elegans fusion family (CeFF) proteins] are essential for developmental cell-to-cell fusion and can merge insect cells. To study the structure and function of AFF-1, we constructed vesicular stomatitis virus (VSV) displaying AFF-1 on the viral envelope, substituting the native fusogen VSV glycoprotein. Electron microscopy and tomography revealed that AFF-1 formed distinct supercomplexes resembling pentameric and hexameric "flowers" on pseudoviruses. Viruses carrying AFF-1 infected mammalian cells only when CeFFs were on the target cell surface. Furthermore, we identified fusion family (FF) proteins within and beyond nematodes, and divergent members from the human parasitic nematode Trichinella spiralis and the chordate Branchiostoma floridae could also fuse mammalian cells. Thus, FF proteins are part of an ancient family of cellular fusogens that can promote fusion when expressed on a viral particle.

Structural and functional insights into the ligand-binding domain of a nonduplicated retinoid X nuclear receptor from the invertebrate chordate amphioxus.

Retinoid X nuclear receptors (RXRs), as well as their insect orthologue, ultraspiracle protein (USP), play an important role in the transcription regulation mediated by the nuclear receptors as the common partner of many other nuclear receptors. Phylogenetic and structural studies have shown that the several evolutionary shifts have modified the ligand binding ability of RXRs. To understand the vertebrate-specific character of RXRs, we have studied the RXR ligand-binding domain of the cephalochordate amphioxus (Branchiostoma floridae), an invertebrate chordate that predates the genome duplication that produced the three vertebrates RXRs (alpha, beta, and gamma). Here we report the crystal structure of a novel apotetramer conformation of the AmphiRXR ligand-binding domain, which shows some similarity with the structures of the arthropods RXR/USPs. AmphiRXR adopts an apo antagonist conformation with a peculiar conformation of helix H11 filling the binding pocket. In contrast to the arthropods RXR/USPs, which cannot be activated by any RXR ligands, our functional data show that AmphiRXR, like the vertebrates/mollusk RXRs, is able to bind and be activated by RXR ligands but less efficiently than vertebrate RXRs. Our data suggest that amphioxus RXR is, functionally, an intermediate between arthropods RXR/USPs and vertebrate RXRs.

A family of GFP-like proteins with different spectral properties in lancelet Branchiostoma floridae.

BACKGROUND: Members of the green fluorescent protein (GFP) family share sequence similarity and the 11-stranded beta-barrel fold. Fluorescence or bright coloration, observed in many members of this family, is enabled by the intrinsic properties of the polypeptide chain itself, without the requirement for cofactors. Amino acid sequence of fluorescent proteins can be altered by genetic engineering to produce variants with different spectral properties, suitable for direct visualization of molecular and cellular processes. Naturally occurring GFP-like proteins include fluorescent proteins from cnidarians of the Hydrozoa and Anthozoa classes, and from copepods of the Pontellidae family, as well as non-fluorescent proteins from Anthozoa. Recently, an mRNA encoding a fluorescent GFP-like protein AmphiGFP, related to GFP from Pontellidae, has been isolated from the lancelet Branchiostoma floridae, a cephalochordate (Deheyn et al., Biol Bull, 2007 213:95). RESULTS: We report that the nearly-completely sequenced genome of Branchiostoma floridae encodes at least 12 GFP-like proteins. The evidence for expression of six of these genes can be found in the EST databases. Phylogenetic analysis suggests that a gene encoding a GFP-like protein was present in the common ancestor of Cnidaria and Bilateria. We synthesized and expressed two of the lancelet GFP-like proteins in mammalian cells and in bacteria. One protein, which we called LanFP1, exhibits bright green fluorescence in both systems. The other protein, LanFP2, is identical to AmphiGFP in amino acid sequence and is moderately fluorescent. Live imaging of the adult animals revealed bright green fluorescence at the anterior end and in the basal region of the oral cirri, as well as weaker green signals throughout the body of the animal. In addition, red fluorescence was observed in oral cirri, extending to the tips. CONCLUSION: GFP-like proteins may have been present in the primitive Metazoa. Their evolutionary history includes losses in several metazoan lineages and expansion in cephalochordates that resulted in the largest repertoire of GFP-like proteins known thus far in a single organism. Lancelet expresses several of its GFP-like proteins, which appear to have distinct spectral properties and perhaps diverse functions. REVIEWERS: This article was reviewed by Shamil Sunyaev, Mikhail Matz (nominated by I. King Jordan) and L. Aravind.

The cephalostatin way of apoptosis.

The cephalostatins, bis-steroidal natural products from the marine tube worm Cephalodiscus gilchristi, were isolated by Dr. G. R. Pettit and his group. These compounds show a unique cytotoxicity profile in the in vitro screen of the National Cancer Institute, suggesting a novel mechanism of action. Indeed, cephalostatin 1 ( 1) is an extremely powerful agent that acts via an unusual apoptosis pathway. It induces selective Smac/DIABLO, but no cytochrome c release from mitochondria. Nevertheless, caspase-9 is required for apoptosis induction. Interestingly, caspase-9 is activated without the participation of the apoptosome, leading to the question of its mechanism of activation. We found that endoplasmic reticulum stress-associated caspase-4 contributes to nonclassical cephalostatin-mediated caspase-9 activation, additionally pointing out the unusual pathway used by this substance. Cephalostatin 1 ( 1), therefore, provides a very good tool to discover novel apoptotic pathways, which might be important in the understanding and treatment of chemo-resistant cancer.

Review of cytotoxic cephalostatins and ritterazines: isolation and synthesis.

The cephalostatins and ritterazines comprise a family of structurally related natural products reported by Professors G. R. Pettit and N. Fusetani from 1988 -1998. Isolated from the invertebrate marine chordates Cephalodiscus gilchristi and Ritterella tokioka, the cephalostatins and ritterazines exhibit potent cytotoxicity toward the murine P388 lymphocytic leukemia cell line. In fact, cephalostatin 1 ( 1, ED 50 0.1-0.001 pM) proved to be one of the most powerful cancer cell growth inhibitors ever tested by the U.S. National Cancer Institute. The ritterazines and cephalostatins share many common structural features in which two highly oxygenated steroidal units with side chains forming either 5/5 or 5/6 spiroketals are fused via a pyrazine core. Professor P. L. Fuchs and colleagues reported the total syntheses of 1, cephalostatins 7 ( 7), and 12 ( 12), ritterazines K ( 30) and M ( 32), and cytotoxic analogues. The synthesis of 1, described in 1998, required 65 synthetic operations to complete.

N-methyl-D-aspartic acid (NMDA) in the nervous system of the amphioxus Branchiostoma lanceolatum.

BACKGROUND: NMDA (N-methyl-D-aspartic acid) is a widely known agonist for a class of glutamate receptors, the NMDA type. Synthetic NMDA elicits very strong activity for the induction of hypothalamic factors and hypophyseal hormones in mammals. Moreover, endogenous NMDA has been found in rat, where it has a role in the induction of GnRH (Gonadotropin Releasing Hormone) in the hypothalamus, and of LH (Luteinizing Hormone) and PRL (Prolactin) in the pituitary gland. RESULTS: In this study we show evidence for the occurrence of endogenous NMDA in the amphioxus Branchiostoma lanceolatum. A relatively high concentration of NMDA occurs in the nervous system of this species (3.08 +/- 0.37 nmol/g tissue in the nerve cord and 10.52 +/- 1.41 nmol/g tissue in the cephalic vesicle). As in rat, in amphioxus NMDA is also biosynthesized from D-aspartic acid (D-Asp) by a NMDA synthase (also called D-aspartate methyl transferase). CONCLUSION: Given the simplicity of the amphioxus nervous and endocrine systems compared to mammalian, the discovery of NMDA in this protochordate is important to gain insights into the role of endogenous NMDA in the nervous and endocrine systems of metazoans and particularly in the chordate lineage.

The notochordal sheath in amphioxus--an ultrastructural and histochemical study.

The notochord and notochordal sheath of 10 adult amphioxus were investigated ultrastructurally and histochemically. The notochord in amphioxus consists of parallel notochordal cells (plates) and each plate consists of parallel thicker and thinner fibrils and numerous profiles of smooth endoplasmic reticulum situated just beneath the cell membrane. Histochemical staining shows that the notochordal plates resemble neither the connective tissue notochordal sheath nor the typical muscular structure myotomes. The notochordal sheath has a complex three-layered organization with the outer, middle and inner layer The outer and middle layer are composed of collagen fibers of different thickness and course, that correspond to collagen type I and collagen type III in vertebrates, respectively, and the inner layer is amorphous, resembles basal lamina, and is closely attached to the notochord by hemidesmosome junctions. These results confirm the presence of collagen fibers and absence of elastic fibers in amphioxus.

Expression of a novel somite-formation-related gene, AmphiSom, during amphioxus development.

A novel gene, AmphiSom, was identified in amphioxus Branchiostoma belcheri tsingtauense. Its sequence and developmental expression pattern were determined. AmphiSom transcripts were first detected in the presomitic mesoderm at the late gastrula stage and reached the highest level in the forming and nascent somites in neurulae. However, the expression of AmphiSom was rapidly down-regulated after somites were formed. It was maintained in the most anterior somite and most posterior somite at neurula stages. By 48 h, AmphiSom transcripts were detected only in the developing tail bud but were no longer detected in 72-h larva. Our data demonstrated that the AmphiSom gene is expressed during the development of somites in amphioxus and could play a role in somite formation.

Tetrabromohydroquinone and riboflavin are possibly responsible for green luminescence in the luminous acorn worm, Ptychodera flava.

2,3,5,6-Tetrabromohydroquinone was isolated as a luminous substance from Ptychodera flava. This compound emitted light after addition of hydrogen peroxide under basic conditions. Since hydroquinone had no fluorescence, further investigation by spectral analysis revealed that riboflavin was the only possible light emitter having green fluorescence. In the presence of both tetrabromohydroquinone and riboflavin under a basic condition containing 70% 1,4-dioxane, green light emission was observed following the addition of hydrogen peroxide. We succeeded in recording the same emission spectrum as that in the bioluminescence caused by the addition of aqueous diluted hydrogen peroxide solution in P. flava.

Isolation of brominated quinones showing chemiluminescence activity from luminous acorn worm, Ptychodera flava.

Luminous acorn worm, Ptychodera flava emits green light by stimulating with diluted hydrogen peroxide. We have recently reported isolation and structure determination of 2,3,5,6-tetrabromohydroquinone as a luminous substance and riboflavin as a possible light emitter. There are three other luminous substances in the extracts from P. flava, so here we report the isolation and structure determination of other luminous substances as 2,3,5-tribromohydroquinone, tetrabromo-1,4-benzoquinone, and 2,3,5-tribromo-6-(2,3,5-tribromo-4-hydroxy-phenoxy)-benzene-1,4-diol. Besides, this is the first report of isolation of tetrabromo-1,4-benzoquinone from acorn worm. Structure-activity relationship of chemiluminescence activity of halogenated quinone derivatives reveals that a highly halogen substitution and 1,4-quinone skeleton are important for high chemiluminescence activity.

Regioselective syntheses of 2,3,4-tribromopyrrole and 2,3,5-tribromopyrrole.

2,3,4-Tribromopyrrole (1) and 2,3,5-tribromopyrrole (2) were each synthesized from pyrrole. Spectral data and antifeedant effects for synthetic 1 and the antipredatory chemical defense compound of the marine hemichordate Saccoglossus kowalevskii were in agreement, confirming the structure of the deterrent natural product as 1. Spectral data for 2 differed from synthetic and natural 1.