Abstract 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 α subunits (αHr1 and αHr2) from the hemocyte mRNA of H. roretzi, by RT-PCR, and the entire coding sequence of αHr1 was determined from cDNA clones. αHr1 contains an I domain, the inserted domain characteristic of a subset of mammalian α subunits, including the leukocyte integrin family. A phylogenetic tree constructed for the α subunits also supports the ancestral position of αHr1 in the monophyletic cluster of I domain-containing α integrins. The αHr1 gene shows hemocyte-specific expression on Northern blot analysis. Western blot analysis and immunocytochemical staining of the hemocytes of H. roretzi using anti-αHr1 Ab showed that αHr1 subunits exist on the surface of a subpopulation of phagocytic hemocytes. Furthermore, anti-αHr1 Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, of yeast cells by ascidian hemocytes. These observations strongly suggest that αHr1 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.
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.
A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain ∼16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the “Ciona intestinalis Gene Collection Release I”. In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.
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.
The molecular mechanisms of the endogenous circadian clocks that allow most animals to adapt to environmental cycles have recently been uncovered. The draft genome of the ascidian, Ciona intestinalis, a model animal that is close to vertebrates, has been described. However, the C. intestinalis genome lacks the canonical clock genes such as Per, Bmal and Clock that are shared by vertebrates and insects. Here, we found the circadian rhythms at the physiological and molecular levels. The oxygen consumption rate was lower during the light phase and higher during the dark phase during a day, and the rhythm highly damped and continued under constant darkness. From the microarray analysis, the 396 spots (1.8% of the total; corresponding to 388 clones) were extracted as candidates for circadian expression. We confirmed the circadian expression of several candidate genes by northern blotting. Furthermore, three of four rhythmic expressed genes showed phase-shifts to prolonged light period. However, most of known clock genes did not oscillate. These data suggest that C. intestinalis have a unique molecular circadian clock and the daily environmental change is not such a strong effect for sea squirt in its evolution when compared to vertebrates and insects.
The aim of this study is to investigate the choice strategy under uncertainty. Female subjects were asked to choose their hypothetical marriage partners among 30 alternatives. Results were analyzed by comparing the positions of the accepted and the rejected alternatives on the preference ordinal scales. The preference scale may be divided into at least two regions; the positive and the negative regions. The subjects had a tendency to reject an alternative when more than one of its aspects were in the negative region, and to accept an alternative when all of its aspects were in the positive region.
The Effects of Perfluoroalkyl Acids (Pfaas) Exposure in Utero on IGF2/H19 DNA Methylation in Cord BloodAbstract Number:2253 Sachiko Kobayashi*, Kaoru Azumi, Seiko Sasaki, Mayumi Ishizuka, Hiroyuki Nakazawa, Emiko Okada, Sumitaka Kobayashi, Houman Goudarzi, Sachiko Itoh, Chihiro Miyashita, Tamiko Ikeno, Atsuko Araki, Reiko Kishi Sachiko Kobayashi* Hokkaido University Centre for Environmental and Health Sciences, Japan, E-mail Address: [email protected] , Kaoru Azumi Hokkaido University Centre for Environmental and Health Sciences, Japan, E-mail Address: [email protected] , Seiko Sasaki Hokkaido University Graduate School of Medicine, Department of Public Health Sciences, Japan , Mayumi Ishizuka Laboratory of Toxicology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Japan , Hiroyuki Nakazawa Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University, Japan , Emiko Okada Hokkaido University Graduate School of Medicine, Department of Public Health Sciences, Japan , Sumitaka Kobayashi Hokkaido University Graduate School of Medicine, Department of Public Health Sciences, Japan , Houman Goudarzi Hokkaido University Centre for Environmental and Health Sciences, Japan , Sachiko Itoh Hokkaido University Centre for Environmental and Health Sciences, Japan , Chihiro Miyashita Hokkaido University Centre for Environmental and Health Sciences, Japan , Tamiko Ikeno Hokkaido University Centre for Environmental and Health Sciences, Japan , Atsuko Araki Hokkaido University Centre for Environmental and Health Sciences, Japan , and Reiko Kishi Hokkaido University Centre for Environmental and Health Sciences, Japan, E-mail Address: [email protected] AbstractBackground: Prenatal exposure to perfluoroalkyl acids (PFAAs) negatively influences children's health including reduced birth weight. Insulin-like growth factor 2 (IGF2) is an important fetal growth factor, whose methylation levels at regulatory regions are associated with birth weight. We hypothesized that prenatal exposure to PFAAs may affect fetal growth epigenetically via DNA methylation at IGF2/H19 locus.Aims: To evaluate the effects of PFAA exposures in utero on infants' IGF2/H19 and LINE1 DNA methylation.Methods: Of 514 pregnant women who were enrolled in the Sapporo Toho hospital cohort between 2002- 2005, 235 mother- child dyads whose exposure and methylation data were available were subjected to this study. Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) concentrations in maternal serum were measured by LC–MS/MS. Bisulfite pyrosequencing was used to quantify the methylation levels of two differentially methylated regions (DMRs) within IGF2/H19 locus, as well as LINE1 in cord blood DNA. Multiple regression analyses were performed to investigate the correlations between log10-transformed PFAA concentrations and DNA methylation levels at each locus.Results: Median concentrations of PFOS and PFOA were 5.0 ng/mL and 1.4 ng/mL respectively. After adjustment for potential confounders, IGF2 methylation was significantly decreased by 1.61% with a log10-unit increase of PFOA concentrations (ß = -1.61, 95% CI: -3.00 to -0.22). Although not statistically significant, PFOS exposure showed similar tendency with PFOA on IGF2 methylation (ß = -1.16, 95% CI: -2.97 to 0.66). H19 and LINE1 methylation levels were not altered significantly by any of these PFAA exposures.Conclusions: Our results suggest that prenatal exposure to PFOA and PFOS influences IGF2 methylation levels. Further investigations are necessary to elucidate the effects of the methylation status of IGF2 on birth weight as well as postnatal growth and obesity.
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.
