Tunic morphology and viral surveillance in diseased Korean ascidians: Soft tunic syndrome in the edible ascidian, Halocynthia roretzi (Drasche), in aquaculture.

'Soft tunic syndrome' causes mass mortality in the edible ascidian Halocynthia roretzi in Korean and Japanese aquaculture. In histopathological comparison, there were no specific differences between diseased specimens from Korea and Japan, indicating that soft tunic syndrome occurring in Korea and Japan is the same disease. No bacterial or protozoan cells were microscopically detected in either healthy or diseased tunics suggesting they are not the direct causes of soft tunic syndrome. Attempts were made to isolate virus from affected ascidians taking into account temperature conditions in which soft tunic syndrome is most prevalent in the field. However, no viruses were isolated from diseased or non-diseased specimens using chinook salmon embryo (CHSE-214), flounder fin (FFN) or epithelioma papillosum cyprini (EPC) cell lines.

Morphological characterization of the tunic in the edible ascidian, Halocynthia roretzi (Drasche), with remarks on 'soft tunic syndrome' in aquaculture.

'Soft tunic syndrome' is a serious problem in the aquaculture of the edible ascidian, Halocynthia roretzi (Drasche), and often leads to mass mortality. Here, we describe the tunic morphology of intact and diseased ascidians to reveal structural differences between them. Morphologically, diseased tunics are not very different from intact tunics, although the former are thinner and softer than the latter. While several types of cells are distributed in the tunic, the cell types and their cytomorphologies were almost identical in both groups. As bacterial/protozoan cells were not found in either intact or diseased tunics, they are not the direct cause of soft tunic syndrome. The most remarkable difference was in the bundles of tunic fibres that compose the tunic matrix; in intact tunics, the thick bundles interlace to form a firm matrix, whereas in soft tunics, the tunic fibres do not form thick bundles. Furthermore, areas of low fibre density were found in diseased tunics. Therefore, soft tunic syndrome probably causes inhibition of bundle formation and degradation of tunic bundles, creating areas of low fibre density, although the causes remain unknown.

An ancient lectin-dependent complement system in an ascidian: novel lectin isolated from the plasma of the solitary ascidian, Halocynthia roretzi.

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway. To elucidate the origin and evolution of MBL, MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of approximately 36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive complement system, consisting of the lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.

Cloning and characterization of integrin alpha subunits from the solitary ascidian, Halocynthia roretzi.

Recent molecular and biochemical analysis has revealed the presence of an opsonic complement system in the solitary ascidian, Halocynthia roretzi, composed of at least C3, two mannan binding protein-associated serine proteases, and factor B. To elucidate further the structure and function of this apparently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are members of the leukocyte integrin family in mammals. Using degenerate primers, we isolated two integrin alpha subunits (alpha(Hr1) and alpha(Hr2)) from the hemocyte mRNA of H. roretzi, by RT-PCR, and the entire coding sequence of alpha(Hr1) was determined from cDNA clones. alpha(Hr1) contains an I domain, the inserted domain characteristic of a subset of mammalian alpha subunits, including the leukocyte integrin family. A phylogenetic tree constructed for the alpha subunits also supports the ancestral position of alpha(Hr1) in the monophyletic cluster of I domain-containing alpha integrins. The alpha(Hr1) gene shows hemocyte-specific expression on Northern blot analysis. Western blot analysis and immunocytochemical staining of the hemocytes of H. roretzi using anti-alpha(Hr1) Ab showed that alpha(Hr1) subunits exist on the surface of a subpopulation of phagocytic hemocytes. Furthermore, anti-alpha(Hr1) Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, of yeast cells by ascidian hemocytes. These observations strongly suggest that alpha(Hr1) constitutes an integrin molecule on the hemocytes of H. roretzi that functions as an ancestral form of CR3 and CR4 and mediates phagocytosis in the primitive complement system of the ascidian.

Involvement of tyrosine kinase and phosphatidylinositol 3-kinase in phagocytosis by ascidian hemocytes.

It has been proposed that protein tyrosine phosphorylation plays important roles in signal transduction in mammalian T- and B-cells and monocytes. During our investigations on the ascidian host defense system, we have shown that the monoclonal antibody A74 strongly inhibits both phagocytosis of sheep red blood cells (SRBCs) by hemocytes and hemocyte aggregation, and that the A74 antigen protein has two immunoreceptor tyrosine-based activation motifs and several other motifs that are thought to function in signal transduction in mammals. In this study, we found that the A74 antibody strongly inhibited phagocytosis by ascidian hemocytes of yeast cells, as strongly as that of SRBCs, but not that of latex beads. We also found that herbimycin A and an erbstatin analog, tyrosine kinase inhibitors, and wortmannin, a specific inhibitor for phosphatidylinositol 3-kinase (PI3-kinase), inhibited the phagocytosis of yeast cells. We investigated which hemocyte proteins were specifically tyrosine-phosphorylated during phagocytosis by ascidian hemocytes and found that a protein with a molecular mass of 100 kDa was specifically tyrosine-phosphorylated upon phagocytosis; its tyrosine phosphorylation was inhibited by the A74 antibody. These results strongly suggest that both tyrosine kinase and PI3-kinase play important roles in phagocytosis by ascidian hemocytes.

Opsonin-independent and -dependent Phagocytosis in the Ascidian Halocynthia roretzi: Galactose-specific Lectin and Complement C3 Function as Target-dependent Opsonins.

To clarify the molecular mechanisms of phagocytosis, we have been preparing monoclonal antibodies that inhibit phagocytosis by the hemocytes of the ascidian Halocynthia roretzi. A monoclonal antibody, RA5, inhibited the phagocytosis of non-treated sheep red blood cells (SRBCs) and yeast cells. It was demonstrated that the phagocytosis by the hemocytes was enhanced by pretreatment of target cells, SRBCs or yeast cells, with H. roretzi plasma. However, the RA5 antibody was unable to inhibit the phagocytosis of plasma-treated target cells. These results strongly suggest that the molecule recognized with the RA5 antibody is involved in the opsonin-independent phagocytosis. Western blot analysis showed that this antibody recognized a 200 kDa protein in H. roretzi hemocytes. On the other hand, flow cytometry analyses showed that a galactose-specific lectin (Gal-lectin) and complement C3 (AsC3), present in H. roretzi plasma, can bind to SRBCs and yeast cells, respectively, to enhance the phagocytosis of the respective target cells. Thus, H. roretzi hemocytes undergo opsonin-independent and -dependent phagocytosis, and Gal-lectin and AsC3 both function in the opsonin-dependent phagocytosis.

Opsonic complement system of the solitary ascidian, Halocynthia roretzi.

To elucidate the molecular architecture and function of the possibly primitive complement system of the solitary ascidian. Halochynthia roretzi, cDNA clones for the third component (C3) and mannose-binding lectin (MBL)-associated serine protease (MASP) were isolated from the hepatopancreas cDNA library. The deduced primary structure of ascidian C3 (AsC3) shows overall similarity to mammalian C3 including a typical thioester site. Two distinct ascidian MASPs, termed AsMASPa and AsMASPb, have the same domain structure as mammalian Clr/ Cls/MASP-1/MASP-2. Both of them show a closer similarity to mammalian MASP-1 than to mammalian Clr/Cls/ MASP-2. Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and an antibody against AsC3 inhibits this opsonic activity. These results indicate that the lectin-dependent, opsonic complement system was present prior to the emergence of the vertebrates and well ahead of the establishment of adaptive immunity.

Complement systems in invertebrates. The ancient alternative and lectin pathways.

The complement system in higher vertebrates is composed of about thirty proteins that function in three activation cascades and converge in a single terminal pathway. It is believed that these cascades, as they function in the higher vertebrates, evolved from a few ancestral genes through a combination of gene duplications and divergences plus pathway duplication (perhaps as a result of genome duplication). Evidence of this evolutionary history is based on sequence analysis of complement components from animals in the vertebrate lineage. There are fewer components and reduced or absent pathways in lower vertebrates compared to mammals. Modern examples of the putatively ancestral complement system have been identified in sea urchins and tunicates, members of the echinoderm phylum and the protochordate subphylum, which are sister groups to the vertebrates. Thus far, this simpler system is composed of homologues of C3, factor B, and mannose binding protein associated serine protease suggesting the presence of simpler alternative and lectin pathways. Additional components are predicted to be present. A complete analysis of this invertebrate defense system, which evolved before the invention of rearranging genes, will provide keys to the primitive beginnings of innate immunity in the deuterostome lineage of animals.

Primary structure and function of superoxide dismutase from the ascidian Halocynthia roretzi.

A protein with a molecular weight of 17K, immunoreactive with the S-1B2 antibody, has been isolated from hemocytes of Halocynthia roretzi. Its amino acid sequence has been determined by sequential Edman degradation analysis of peptide fragments derived from proteolytic fragmentation. The 17K protein is a single chain protein consisting of 151 amino acids with an acylated N-terminal serine. A comparison of the amino acid sequence of H. roretzi 17K protein with those of other proteins reveals that the 17K protein is Cu,Zn-SOD. The protein was found to have a KCN-inhibited SOD activity. Cu,Zn-SOD has been purified from H. roretzi plasma. The molecular weight is 17K and the activity is inhibited with KCN and diethyldithiocarbamate. It has been demonstrated that it can enhance phagocytosis by H. roretzi hemocytes. Thus, plasma Cu,Zn-SOD plays a role in H. roretzi as a defense molecule.

A divergent non-classical class I gene conserved in salmonids.

Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UAC32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the alpha 3 domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2-6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14-16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene.

A unique primary structure, cDNA cloning and function of a galactose-specific lectin from ascidian plasma.

The complete amino acid sequence of a galactose-specific lectin from the plasma of the ascidian Halocynthia roretzi has been determined by sequential Edman degradation analysis of peptide fragments derived by proteolytic fragmentation and chemical cleavage of the reductive S-pyridylethylated lectin. Peptide fragments were separated by reverse-phase HPLC. The N-terminal and C-terminal amino acid sequences were determined by Edman degradation and enzymatic digestion. The H. roretzi plasma lectin is a single-chain protein consisting of 327 amino acids and four disulfide bonds, one of which was found to be cross-linked intramolecularly. A comparison of the amino acid sequence of the H. roretzi plasma lectin with the sequences of other proteins reveals that the H. roretzi lectin has a structure consisting of a twice-repeated sequence, a fibrinogen-related sequence and a C-type lectin-homologous sequence. The above amino acid sequence was verified by cDNA cloning of this lectin. Three cDNA clones that have single ORFs encoding the lectin precursor were isolated from an H. roretzi hepatopancreas cDNA library. The deduced amino acid sequences in the three cDNA clones contain the same sequence of the mature lectin molecule and the same putative signal sequence. In addition, it was demonstrated that this lectin can enhance phagocytosis by H. roretzi hemocytes. Thus, the plasma lectin is constructed into an oligomer structure via intermolecular disulfide bonds and plays a role in the biological defense of H. roretzi as a defense molecule.

Opsonic complement component C3 in the solitary ascidian, Halocynthia roretzi.

The recent identification of two mannose-binding lectin-associated serine protease clones from Halocynthia roretzi, an ascidian, suggested the presence of a complement system in urochordates. To elucidate the structure and function of this possibly primitive complement system, we have isolated cDNA clones for ascidian C3 (AsC3) and purified AsC3 protein from body fluid. The deduced primary structure of AsC3 shows overall similarity to mammalian C3, including a typical thioester site with the His residue required for nucleophilic activation of the thioester. AsC3 has a two-subunit chain structure, and the alpha-chain is cleaved at a specific site near to the N terminus upon activation. Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and Ab against AsC3 inhibits this opsonic activity. These results indicate that the complement system played a pivotal role in innate immunity by enhancing phagocytosis before the emergence of the vertebrates and well ahead of the establishment of adaptive immunity, which is believed to have occurred at about the time of the appearance of cartilaginous fish.

Ascidian phenoloxidase: its release from hemocytes, isolation, characterization and physiological roles.

Hemocytes of the solitary ascidian Halocynthia roretzi released phenoloxidase in response to sheep red blood cells and yeast cells but not to latex beads. Phenoloxidase was also released from the hemocytes by treatments with zymosan and lipopolysaccharides but not with beta 1-3 glucan. EDTA scarcely inhibited the activity of phenoloxidase but inhibited the release of the enzyme. Phenoloxidase was purified from H. roretzi hemocytes by SP-Sephadex chromatography and Sephadex G-100 gel filtration. The molecular weight of the purified enzyme was estimated to be 62,000. Phenoloxidase activity was strongly inhibited by diethyldithiocarbamate, phenylthiourea and reducing agents. H. roretzi phenoloxidase was characterized as a metalloenzyme that required copper ions for the expression of full activity. The phenoloxidase showed antibacterial activity in the presence of L-(3,4-dihydroxy)-phenylalanine and H. roretzi plasma. Thus, it can be concluded that phenoloxidase released from H. roretzi hemocytes functions as a humoral factor in the defense system of H. roretzi.

Ancient origin of the complement lectin pathway revealed by molecular cloning of mannan binding protein-associated serine protease from a urochordate, the Japanese ascidian, Halocynthia roretzi.

Recent identification of a C3-like gene in sea urchins revealed the presence of a complement system in invertebrates. To elucidate further the components and function of the pre-vertebrate complement system, we attempted to isolate an ascidian (urochordata) C3 convertase. After identification of C3 cDNA from Halocynthia roretzi, a Japanese ascidian, reverse transcriptase-PCR amplification of hepatopancreas RNA was performed using primers encoding highly conserved amino acid sequences of the vertebrate Bf and C2 serine protease domain. Two candidate sequences were identified, and the corresponding cDNA clones were isolated from a hepatopancreas library. Surprisingly, neither clone is related to Bf/C2 but rather share the same domain structure of mammalian C1r/C1s/MASP (mannan binding protein-associated serine protease), and are more related evolutionarily to mammalian MASP than to mammalian C1r or C1s. The identification of the tunicate MASP clones, amplified with primers designed to amplify Bf or C2, suggests that the lectin pathway antedated the classical and alternative pathways of complement activation.

Cloning and tyrosine phosphorylation of a novel invertebrate immunocyte protein containing immunoreceptor tyrosine-based activation motifs.

Immunoreceptor tyrosine-based activation motif (ITAM) plays an important role in signal transduction through mammalian T-cell and B-cell antigen receptors and Fc receptors. The ITAM has been found only in vertebrate immunocytes. Ascidians are intriguing invertebrates from the viewpoint of the evolution of immune systems because they are considered to be ancestors of the vertebrates. We have previously shown that the monoclonal antibody A74 inhibits cellular defense reactions of the ascidian. In the present studies, we found that the A74 antigen protein has two ITAMs and several motifs that are proposed to function in signal transduction. The A74 protein is tyrosine-phosphorylated and associated with other proteins in the initial stages of cellular defense reactions. The ITAMs of the A74 protein are tyrosine-phosphorylated by a c-Src kinase in vitro. The A74 protein provides a key to the understanding of the origin of vertebrate immune systems.

Characterization of novel metallo-proteases released from ascidian hemocytes by treatment with calcium ionophore.

We have previously demonstrated that calcium ionophore induced the release of a novel metallo-protease from hemocytes of a solitary ascidian, Halocynthia roretzi. Here, we isolated the enzymes, PI and PII, from the culture media of H. roretzi hemocytes, which had been treated with calcium ionophore, A23187. The purification procedure included hydrophobic and anion-exchange chromatographies, and gel filtration. The molecular weights of the enzymes were estimated to be 11,000 by gel filtration, but the apparent sedimentation coefficients were 5.0 S, which suggests that the H. roretzi enzymes are of larger proteins with molecular weights of 80,000-90,000. The most susceptible substrate was succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, and the optimum pH was 8.0, in either case of PI or PII. The activities of PI and PII enzymes were strongly inhibited by metal-chelating agents and propioxatin A, but not by phosphoramidon, a typical metallo-protease inhibitor. Zinc and calcium ions were found to be essential for the maximum expression of protease activity in both enzymes. Thus, the isolated enzymes are characterized as phosphoramidon-insensitive metallo-proteases, which are inhibited by propioxatin A. Extracellular roles of these enzymes were also discussed.

Unexpected beta2-microglobulin sequence diversity in individual rainbow trout.

For mammals beta2-microglobulin (beta2m), the light chain of major histocompatibility complex (MHC) class I molecules, is invariant (or highly conserved) and is encoded by a single gene unlinked to the MHC. We find that beta2m of a salmonid fish, the rainbow trout (Oncorhynchus mykiss), does not conform to the mammalian paradigm. Ten of 12 randomly selected beta2m cDNA clones from an individual fish have different nucleotide sequences. A complex restriction fragment length polymorphism pattern is observed with rainbow trout, suggesting multiple beta2m genes in the genome, in excess of the two genes expected from the ancestral salmonid tetraploidy. Additional duplication and diversification of the beta2m genes might have occurred subsequently. Variation in the beta2m cDNA sequences is mainly at sites that do not perturb the structure of the mature beta2m protein, showing that the observed diversity of the trout beta2m genes is not primarily a result of pathogen selection.

A novel membrane glycoprotein involved in ascidian hemocyte aggregation and phagocytosis.

Invertebrate hemocytes undergo several cellular defense reactions. To clarify the molecular mechanisms for cellular recognition between hemocytes and also between hemocytes and foreign materials, we established hybridoma clones producing monoclonal antibodies that inhibit hemocyte aggregation (i.e. a cellular reaction between hemocytes) in the solitary ascidian, Halocynthia roretzi. The antibody, A74, also inhibited phagocytosis of foreign materials by H. roretzi hemocytes. Immunocytochemistry of H. roretzi hemocytes using A74 antibody revealed the localization of the A74 antigen on the surface of hemocytes. The A74 antigen, which is referred to as A74 protein, was purified from a hemocyte membrane preparation by three chromatographies on phenyl-Sepharose, A74 antibody-immobilized Sepharose and Mono Q. The A74 protein was a glycoprotein with a molecular mass of 160 kDa; N-glycosidase or neuraminidase treatment resulted in a reduction of its molecular mass. The N-terminal amino acid sequence of A74 protein showed little similarity to other known proteins. Thus, the A74 protein is a novel membrane protein that plays an important role in ascidian cellular defense reactions.

Hemocyte aggregation in the solitary ascidian Halocynthia roretzi: plasma factors, magnesium ion, and Met-Lys-bradykinin induce the aggregation.

Hemocytes of the ascidian Halocynthia roretzi undergo aggregation in hemolymph that has been collected from the body through the tunic. To investigate the mechanisms involved, we first established two methods of measuring hemocyte aggregation. In one method, hemocyte aggregation was quantified by its reduction of light scattering intensity as measured with a fluorescence spectrophotometer. In the other method, the increase of transmittance accompanying aggregation was measured with an ELISA reader. We found that ascidian plasma, Mg2+, and Met-Lys-bradykinin can induce the hemocytes of H. roretzi to aggregate. The aggregation induced by any of these three substances was inhibited by EDTA, N-ethylmaleimide, and cytochalasin B. Lipopolysaccharide had little inducing effect. We also demonstrated that, when H. roretzi plasma was treated with trypsin, low molecular weight aggregation-inducing substances were produced. These results suggest that metal ions and peptide-like substances present in the hemolymph play essential roles in the progression of hemocyte aggregation of H. roretzi.

Sleep spindle frequency changes during the menstrual cycle.

Five healthy adult women aged 20 to 28 had 12-15 polysomnographic recordings, as well as daily basal body temperature and multiple LH, FSH, estrogen and progesterone measurements taken during a single menstrual cycle. Sleep stages were scored both visually and with a spindle and delta-wave, real-time, automatic analysing system. A cubic growth-curve model showed that the frequency of sleep spindles changed markedly over the menstrual cycle: spindle frequency was lowest about 18 days before onset of menses and highest 3 days before onset of menses. Slow waves did not change. The percentages of Stage 1 and REM sleep showed small changes during the menstrual cycle, and other parameters of visually scored sleep showed no tendency to change. Spindle frequency may reflect the effects of sex hormones on the reticular thalamic nucleus and may be a quantitative marker of premenstrual sleep disturbances.