Comparison of skin mucus lectins among longtooth grouper Epinephelus bruneus and giant grouper E. lanceolatus as well as the hybrid, Kue-Tama, and their binding abilities to the skin fluke Benedeniaepinepheli.

Interspecific hybrids of farm-raised fish are becoming popular in aquaculture owing to their advantages over pure species, including improved growth and higher resistance to infectious diseases. Kue-Tama is a recently established hybrid grouper derived from the longtooth grouper Epinephelus bruneus (♀) × giant grouper E. lanceolatus (♂). In our previous study, this hybrid showed significantly higher resistance against the skin fluke Benedenia epinepheli, a problematic parasite in grouper farming, than the longtooth grouper. In the present study, we explored lectins in the skin mucus of hybrids and their parent species. While C-type lectins of approximately 15 kDa were obtained from longtooth groupers, additional C-type lectins with molecular masses of approximately 20 and 30 kDa, as well as 45-kDa F-type lectin, were also detected in Kue-Tama and giant groupers. Semi-quantitative reverse transcript-polymerase chain reaction (RT-PCR) demonstrated that the gene expression levels of both C-type and F-type lectins were significantly higher in the skin of the hybrid and giant groupers than that of the longtooth grouper. In addition, some skin mucus lectins of the hybrid and giant groupers were bound to the fluke, suggesting that these lectins conferred resistance to parasitic infections.

l-fucoside localization in the gills of the genus Takifugu and its possible implication in the parasitism of Heterobothrium okamotoi (Monogenea: Diclidophoridae).

BACKGROUND: The monogenean parasite Heterobothrium okamotoi only parasitizes the gills of Takifugu rubripes. In this study, we hypothesized that the carbohydrates contribute to high host specificity of H. okamotoi. METHODS: T. rubripes, T. niphobles, T. snyderi, and T. pardalis were used for UEA I staining of the gills and an in vivo challenge test against H. okamotoi. To examine the effect of l-fucose, an in vitro detachment test was conducted using the host's gills. Additionally, fucosylated proteins were isolated from the membrane proteins of T. niphobles gills. RESULTS: The location of l-fucoside and the infection dynamics in four species were correlated to some extent; H. okamotoi detached relatively quickly from T. niphobles possessing l-fucoside both on the surface of the gills and in certain types of cells, including mucus cells, but detached slowly from T. snyderi possessing l-fucoside in only certain types of cells, including mucus cells. Under the conditions examined, H. okamotoi exhibited minimal detachment from T. rubripes and T. pardalis, and l-fucoside was not detected. The significantly higher detachment rate of H. okamotoi from the host's gills incubated in l-fucose-containing medium compared with the controls suggests that l-fucose in the non-host gills induced detachment of H. okamotoi. Four fucosylated proteins, including mucin5AC-like, were identified as potential factors for the detachment of H. okamotoi. CONCLUSIONS: Fucosylated proteins covering the surface of non-host gills might contribute to H. okamotoi detachment. GENERAL SIGNIFICANCE: This research shows the possible involvement of oligosaccharides in the host specificity of monogenean parasites.

Discovery of Teleost Plasma Kallikrein/Coagulation Factor XI-Like Gene from Channel Catfish (Ictalurus punctatus) and the Evidence that the Protein Encoded by it Acts as a Lectin.

Mammalian plasma kallikrein (PK) and coagulation factor XI (fXI) are serine proteases that play in the kinin-kallikrein cascade and in the blood clotting pathway. These proteases share sequence homology and have four apple domains (APDs) and a serine protease domain (SPD) from their N-terminus to C-terminus. No homologs of these proteases are believed to be present in fish species, except for lobe-finned fish. Fish, however, have a unique lectin, named kalliklectin (KL), which is composed of APDs only. In the present study, we found genomic sequences encoding a protein with both APDs and SPD in a few cartilaginous and bony fishes, including the channel catfish Ictalurus punctatus, using bioinformatic analysis. Furthermore, we purified two ~ 70 kDa proteins from the blood plasma of the catfish using mannose-affinity and gel filtration chromatography sequentially. Using de novo sequencing with quadrupole time-of-flight tandem mass spectrometry, several internal amino acid sequences in these proteins were mapped onto possible PK/fXI-like sequences that are thought to be splicing variants. Exploration of APD-containing proteins in the hagfish genome database and phylogenetic analysis suggested that the PK/fXI-like gene originated from hepatocyte growth factor, and that the gene was acquired in a common ancestor of jawed fish. Synteny analysis provided evidence for chromosomal translocation around the PK/fXI-like locus that occurred in the common ancestor of holosteans and teleosts after separation from the lobe-finned fish lineage, or gene duplication into two chromosomes, followed by independent gene losses. This is the first identification of PK/fXI-like proteins in teleosts.

Unique properties of prothrombin in the bullhead shark Heterodontus japonicus: the first report of purification and characterization of a blood coagulation factor in Chondrichthyes.

Prothrombin is a serine protease precursor of the blood coagulation system. In this study, the primary structure of prothrombin of a cartilaginous fish, bullhead shark (Heterodontus japonicus), was determined using RNA-Seq and the protein was purified from the blood plasma. Bullhead shark prothrombin was found to be comprised of four domains, as in the case of reported mammalian homologues. Two arginine residues that should be cleaved by activated factor X were found in the amino acid sequence of the shark prothrombin, but only one of the two cleavage sites for thrombin or meizothrombin was conserved. The apparent molecular mass of the shark prothrombin on SDS-PAGE was 110 kDa, whereas that of its amino acid sequence was 65 kDa. Potential N-glycosylation sites were found at 79th, 108th, 121st, 179th, 199th, 507th, and 527th asparagine residues in the shark prothrombin, and treatment with N-glycosidase reduced the molecular mass to 65 kDa. This indicates that, in contrast to human prothrombin, which has only 7-kDa N-glycans, the prothrombin of the shark is highly N-glycosylated. This study is the first to report on the purification and characterization of blood coagulation factors in a cartilaginous fish.

Addition of a Vascular Bundle Accelerates Bone Union in Femoral Bone Defects.

BACKGROUND: The Masquelet method has become increasingly popular for the treatment of bone defects in recent years. In this method, an induced membrane (IM) with abundant blood circulation, stem cells, and osteogenesis-promoting factors is formed by implanting bone cement during the first surgery. This IM stimulates bone formation in the bone defect after implantation of the bone graft during the second surgery. However, the Masquelet method requires two surgeries and thus a longer treatment period. In the present study, we investigated whether bone defects could be reconstructed in a single surgery by introducing a vascular bundle into the bone defect as an alternative to the IM, in addition to bone grafting. METHODS: Thirty-six 12-week-old female Sprague-Dawley rats were used. After creating a 5-mm long bone defect in the femur, a mixture of autologous and artificial bone was grafted into the defect, and a saphenous arteriovenous vascular bundle was introduced. The animals were divided into three groups: the control group (bone defect only), the BG group (bone grafting only), and the BG + V group (bone grafting + vascular bundle introduction). After surgery, radiological and histological evaluations were performed to assess osteogenesis and angiogenesis in bone defects. RESULTS: In the BG + V group, significant bone formation was observed in the bone defect on radiological and histological evaluations, and the amount of bone formation was significantly higher than that in the other two groups. Furthermore, cortical bone continuity was observed in many specimens in the BG + V group. On histological evaluation, the number of blood vessels was also significantly higher in the BG + V group than in the other two groups. CONCLUSION: Our results suggest that the introduction of a vascular bundle in addition to bone grafting can promote bone formation in bone defects and allow for complete bone defect reconstruction in a single surgery.

Skin extension with a digito-lateral flap and early active finger extension training for Dupuytren contracture: A retrospective study.

In the surgical management of Dupuytren contracture (DC), Y-V plasty (YV) and Z-plasty (ZP) are techniques often used for skin extension. However, achieving sufficient skin extension with these procedures alone is often difficult. Therefore, we addressed this issue with an adjunctive digito-lateral flap (DLF) and report the clinical results of the surgery using a DLF in addition to YV and ZP. Fifteen patients with DC (15 affected fingers) underwent partial fasciectomy using a DLF in addition to YV or ZP, and early active finger extension training was performed immediately after the operation. The flap survival rate, preoperative and postoperative extension angle, Tonkin contracture improvement (TCI) rate, and Tubiana staging grades were evaluated. The contracture sites were at 4 proximal interphalangeal (PIP) and 3 metacarpophalangeal (MP) joints of the little finger and 4 PIP and MP joints each of the ring and little fingers. All the flaps survived, and the extension angle improved at the final observation from a preoperative mean of -45° to -3° and -55° to 5° for the PIP and MP joints, respectively. One patient with PIP joint contracture treated in the early stage of the study experienced a persistent 5° limitation of extension, even though the TCI rate was satisfactory (91.9%) and the outcome was "good." Full extension of the joints was achieved in 15 patients, in whom the TCI rate was 100% and the outcome was "very good." This technique was able to solve 3 important steps to achieve full extension: intraoperatively, wound closure, and rehabilitation. We attained and maintained long-term full extension intraoperatively and immediately after surgery and obtained very good treatment results, as shown in this study. In conclusion, highly favorable clinical outcomes were achieved through the combination of a DLF with YV and ZP. Skin extension with a DLF is a useful surgical technique for DC.

Repeated translocation of a supergene underlying rapid sex chromosome turnover in Takifugu pufferfish.

Recent studies have revealed a surprising diversity of sex chromosomes in vertebrates. However, the detailed mechanism of their turnover is still elusive. To understand this process, it is necessary to compare closely related species in terms of sex-determining genes and the chromosomes harboring them. Here, we explored the genus Takifugu, in which one strong candidate sex-determining gene, Amhr2, has been identified. To trace the processes involved in transitions in the sex-determination system in this genus, we studied 12 species and found that while the Amhr2 locus likely determines sex in the majority of Takifugu species, three species have acquired sex-determining loci at different chromosomal locations. Nevertheless, the generation of genome assemblies for the three species revealed that they share a portion of the male-specific supergene that contains a candidate sex-determining gene, GsdfY, along with genes that potentially play a role in male fitness. The shared supergene spans ∼100 kb and is flanked by two duplicated regions characterized by CACTA transposable elements. These results suggest that the shared supergene has taken over the role of sex-determining locus from Amhr2 in lineages leading to the three species, and repeated translocations of the supergene underlie the turnover of sex chromosomes in these lineages. These findings highlight the underestimated role of a mobile supergene in the turnover of sex chromosomes in vertebrates.

Postoperative early and proactive grip strength training program for distal radius fractures promotes earlier recovery of grip strength: A retrospective study.

ABSTRACT: The use of volar locking plates (VLPs) for distal radius fractures has remarkably improved clinical outcomes; however, there are some reports of delayed recovery of grip strength. Since January 2019, we have been conducting an early and proactive grip strength training program (EGTP). In this program, 20 minutes of grip strength training-using a gripper with a load of 0.7 kg-was initiated from 2 weeks after surgery; the load was then gradually increased. From 6 weeks postsurgery, daily home grip strength training was performed using a gripper with a load of 5 kg, provided to the patient.We investigated whether the introduction of the EGTP could lead to earlier recovery of grip strength. We also examined whether the EGTP caused postoperative correction loss at the fractured site, or contributed to the early improvement of wrist function.Thirty-nine patients who underwent surgery using VLPs for distal radius fractures were included in this study; 20 followed the EGTP (EGTP group) and 19 patients did not (NGTP group). For these patients, grip strength and range of motion of the wrist joint were evaluated both 3 and 6 months postoperatively. The Quick Disabilities of the Arm, Shoulder, and Hand (qDASH) scores were also evaluated 6 months postoperatively. Additionally, corrective losses of radial inclination (RI), palmar tilt (PT), and ulnar variance (UV)-occurring from immediately postsurgery to 6 months after surgery-were evaluated.At both 3 and 6 months postoperatively, the grip strength of the EGTP group was significantly higher than that of the NGTP group. Regarding range of motion, only palmar flexion was significantly improved in the EGTP group at 3 months postoperatively. Conversely, no differences in corrective losses of RI, PT, and UV, or in qDASH scores, were observed between the two groups.The results of this study suggest that the EGTP can provide early recovery of grip strength and palmar flexion of the wrist without causing corrective loss at the fracture site.

Vitellogenin-derived fragment in embryos of Japanese flounder Paralichthys olivaceus with binding and bactericidal activities against an infectious bacterium via an interaction with saccharides.

Thirty- and 90-kDa proteins with binding ability to Edwardsiella tarda, a causative bacterium of Edwardsiellosis in fish, were purified from the embryo of Japanese flounder Paralichthys olivaceus. The proteins were isolated with affinity chromatography, in which the bacterium was used as a ligand and galactose, mannose, and ethylenediaminetetraacetic acid (EDTA) were used as elution agents, followed by gel filtration chromatography. N-terminal amino acid sequencing and liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis revealed that the 90-kDa protein was lipovitellin heavy-chain (LvH), which is one of the proteolytically cleaved products of maternal vitellogenin (Vg) and represents the main precursor of the egg yolk in teleosts, and the 30-kDa protein was an N-terminal bit of LvH. On the other hand, Vg in the serum of the mother fish did not bind to E. tarda. While the 90-kDa protein did not show anti-bacterial activity, the 30-kDa protein strongly exhibited activity toward E. tarda, with a minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) below 0.06 µM, suggesting that the latter protein plays an important role during embryogenesis in the flounder. This is the first report showing that Vg-derived products have monosaccharides-binding activity and a fragment derived from LvH exhibits bactericidal activity.

Galactose-Binding C-Type Lectin Promotes Cellular Aggregation of Coelomocytes in Sea Cucumber.

Echinoderms have a large coelomic cavity containing coelomocytes. When the coelomic fluid is removed from the cavity, the cells aggregate immediately. We found that a fraction or an extract of the intestine of the sea cucumber, Apostichopus japonicus, markedly accelerated cellular movement and aggregation on a glass slide, and this effect was clearly inhibited by galactose. We successfully purified the aggregation-promoting factor, a 16 kDa protein, from the intestine. TOF-MS analysis followed by de novo sequencing revealed that the protein is a C-type lectin. RNA-seq data and cDNA cloning demonstrated the factor to be a novel lectin, named AjGBCL, consisting of 158 aa residues in the mature form. Microscopic observation revealed that most of the aggregating cells moved toward aggregates and not to an intestinal fragment, suggesting that AjGBCL is not a chemoattractant but a cellular aggregation-inducing factor that may induce aggregates to release chemoattractant. We report, for the first time, an endogenous molecule that promotes coelomocyte aggregation in echinoderms.

Rapalink-1 and Hydroxychloroquine Exhibit an Additive Effect in Undifferentiated Pleomorphic Sarcoma by Inducing Apoptosis.

BACKGROUND/AIM: Advanced undifferentiated pleomorphic sarcoma (UPS) has a poor prognosis and there are few treatments that can improve overall survival. Recently, Rapalink-1, a third-generation mammalian target of rapamycin (mTOR) kinase inhibitor, has been developed and shown to be effective against other tumours. However, mTOR inhibitors have been shown to induce autophagy and resistance to anti-cancer drugs. This study aimed to investigate the antitumor effects of Rapalink-1 with an autophagy inhibitor. MATERIALS AND METHODS: The antitumor effect of Rapalink-1 and/or hydroxychloroquine in three UPS cell lines was examined via cell viability analysis, western blotting, flow cytometry and immunofluorescence. RESULTS: Rapalink-1 decreased cell proliferation and inhibited the PI3K/mTOR pathway. Combined treatment with Rapalink-1 and hydroxychloroquine enhanced the antitumor effect compared to treatment with Rapalink-1 alone by blocking the autophagy-inducing effect of mTOR inhibitors. CONCLUSION: Combined treatment with Rapalink-1 and hydroxychloroquine may be used as a potential therapeutic agent against UPS.

Molecular evolution of kalliklectin in teleost and identification of the novel type with eight apple domains in channel catfish, Ictalurus punctatus.

Kalliklectin is a unique fish-specific lectin that demonstrates sequence similarity to mammalian plasma kallikrein and coagulation factor XI, which are not lectins but proteases. Reported fish kalliklectins and these mammalian proteases comprise four characteristic "apple domains" (APDs). Bioinformatics analysis revealed that Siluriformes species possess anomalous kalliklectins comprising 6 to 16 APDs. Complementary DNA cloning showed that the full-length nucleotide sequence of Ictalurus punctatus consists of 2240 bp that encode 720 amino acid residues to produce a mature protein with a putative 18 amino acid N-terminus peptide sequence. This protein has a predicted molecular mass of 83,417.23 Da. Reverse transcription-polymerase chain reaction (RT-PCR) showed that this lectin gene expresses in the liver but not in any other tissues, including the mucosal tissues. This differential expression pattern makes this lectin unique compared to other lectins described in previous studies. We successfully detected an 85-kDa protein in the serum using western blotting analysis, suggesting that this lectin protein is produced by the liver and secreted into the bloodstream. We characterized a novel cDNA sequence encoding a new type of kalliklectin with eight APDs isolated from channel catfish, I. punctatus. Based on phylogenetic analysis, we speculated that there was a duplication of the third and fourth APD set in a common Siluriformes ancestor at some point after its separation from the common teleost ancestor and that these duplications then underwent independent repeats in different lineages resulting in the generation of the [APD1]-[APD2]-{[APD3]-[APD-4]} × n structure in modern catfishes.

Combined administration of rifampicin, ethambutol, and clarithromycin for the treatment of tenosynovitis of the hand caused by Mycobacterium avium complex: Case series and literature review.

ABSTRACT: We report the clinical results and problems of combined administration of rifampicin, ethambutol, and clarithromycin (REC) for the treatment of Mycobacterium avium complex (MAC) infection of the hand (hand MAC).Participants included 7 patients with hand MAC. After resection of the infected lesion, REC was prescribed for 12 months. For these patients, the site of infection, clinical course after initiation of REC, adverse drug effects (ADEs), and incidence of recurrence were evaluated.Sites of infection were the flexor tenosynovium in 5 patients, extensor tenosynovium in 1 patient, and both flexor and extensor tenosynovium in 1 patient. ADEs of REC occurred in 5 patients, and included visual disturbance caused by ethambutol in 2 patients, liver function abnormality caused by rifampicin in 2 patients, and fever with diarrhea caused by rifampicin in 1 patient. For 2 of these 5 patients, desensitization therapy was applied and REC was able to be reinstated. In the remaining 3 patients, the causative drugs were discontinued and levofloxacin, a new quinolone, was administered. Complete healing was achieved in 5 patients, and recurrence was observed in 2 patients. These 2 patients with recurrence included 1 patient in whom REC was completed and 1 patient in whom REC therapy was modified due to ADE.REC provided relatively good clinical results as a treatment for hand MAC. However, recurrences were observed even after the completion of REC and the use of an alternative drug. Optimal duration of REC and appropriate alternative drugs need to be identified in the future.

Effects of VEGF on Prefabricated Vascularized Bone Allografts in Rats.

BACKGROUND: Massive bone defects after wide resection of malignant bone tumors or a serious injury require treatment using vascularized bone grafts. Although cadaveric bone allografts combined with vascularized bone autografts are currently thought to be ideal in terms of size and durability, this treatment requires the scarification of healthy bone tissue. In a previous study, we attempted to improve this situation by prefabricating a vascularized bone allograft in recipient rats. In this study, we added vascular endothelial growth factor (VEGF)-containing hydroxyapatite/collagen composite (HAp/Col) to a prefabricated vascularized bone allograft to stimulate angiogenesis, which is known to be important for bone formation. METHODS: Sprague Dawley rats (n = 50) were used as donors and Wistar rats (n = 50) as recipients. All rats were 9 weeks old. The recipient rats were divided into five groups according to the use of vascular bundles, HAp/Col, and an additive substance (VEGF). The bone allografts collected from the donors were transplanted into the thigh region of the recipients, and a saphenous vein and 10 µg HAp/Col with VEGF were inserted into the bone allografts through the slit. After 4 weeks, the transplanted bone allografts were harvested, and histologic and genetic evaluations were performed in relation to bone formation and resorption. RESULTS: The results showed that, compared with the control group, the implantation of the vascular bundles and VEGF-containing HAp/Col significantly stimulated angiogenesis and bone formation in the rats with the bone allografts. However, histological and genetic evaluations of bone resorption revealed that resorption was not observed in any group. CONCLUSION: These results suggest that VEGF-containing HAp/Col effectively stimulates angiogenesis and bone formation, but not bone resorption, in prefabricated vascularized bone allografts. This method could therefore become a useful tool for treating large bone defects.

Bone Union Enhancement by bFGF-Containing HAp/Col in Prefabricated Vascularized Allo-Bone Grafts.

BACKGROUND: We have developed a prefabricated vascularized allo-bone graft (PVAG) by implanting the saphenous vascular bundles of recipient rats into transplanted donor bones in a flow-through manner. We previously demonstrated that the angiogenetic and bone formative abilities of the PVAG are stimulated by the addition of a basic fibroblast growth factor (bFGF)-containing hydroxyapatite/collagen (HAp/Col). This study aimed to demonstrate that the bone union ability of the PVAG is similarly stimulated by the bFGF-containing HAp/Col composite. METHODS: Sprague-Dawley donor rats (n = 32) and Wistar recipient rats (n = 32) were used in this study. The PVAG was fixed to the femur of the recipient rat using K-wire (dimeter: 0.7 mm) pinning, followed by suturing with a 4-0 nylon suture. Recipients were divided into four groups: with or without vascular bundles, and with or without bFGF-containing HAp/Col. Rats were sacrificed 6 weeks after transplantation, and bone union, bone resorption, and angiogenesis were radiologically and histologically evaluated. RESULTS: Radiological analysis revealed a significant increase in callus formation and union rate, while histological analysis showed a significant increase in bone formation and angiogenesis in the group treated with both vascular bundles and bFGF. Bone resorption did not significantly increase in any of the evaluated groups. CONCLUSION: Osteogenic cells, osteoconductive scaffolds, growth factors, and mechanical environment are known to be important factors in the process of fracture healing. The PVAG developed herein contains osteogenic cells, osteoconductive scaffolds, and growth factors. In addition, the PVAG is rigidly fixed to the fracture site, providing a stable mechanical environment. Together, these four factors contributed to a good bone union. Furthermore, this method did not promote bone resorption. Thus, the addition of a vascular bundle and bFGF-containing HAp/Col makes it possible to create an ideal vascularized allo-bone graft for the reconstruction of massive bone defects.

Mannose-binding C-type lectins as defense molecules on the body surface of the sea urchin Pseudocentrotus depressus.

We found that the extract of the body wall of the sea urchin, Pseudocentrotus depressus, agglutinate Escherichia coli and is inhibited by mannose. A mannose-binding protein of 22 kDa was purified via affinity chromatography using mannose-agarose. Amino acid sequences obtained by Edman degradation and liquid chromatography quadrupole time-of-flight mass spectrometry followed by de novo sequencing suggested that the protein is a C-type lectin. Products of PCR with a degenerate primer pair and of RACE PCR for the cDNA of the 22 kDa protein were sequenced and produced two full-length cDNA sequences encoding C-type lectins. These two lectins, named P. depressus mannose-binding C-type lectin (PdMBCL) 1 and 2 are composed of 187 and 189 amino acid residues, including signal peptides, respectively, and share 86% identity in their mature form. PdMBCLs agglutinated Lactococcus garvieae, a Gram-positive fish pathogen. Reverse transcription PCR showed that both the genes for the PdMBCLs were expressed in the body wall and in other tissues. Furthermore, the lectins were detected from a rinse of the body surface. Taken together, the present study showed that PdMBCLs function as anti-microbial agents on the body surface of P. depressus.

Expression profile of kalliklectin, a soluble-type mannose receptor, during embryogenesis and early larval development in fugu (Takifugu rubripes).

Kalliklectin is a unique fish-specific lectin, whose sequence is similar to the heavy chain of mammalian plasma kallikrein and coagulation factor XI. In this study, we aimed to evaluate dynamic expression profiles of the lectin gene, during early developmental stages, in fugu, Takifugu rubripes. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the kalliklectin gene was not expressed until 14 h post-fertilization (hpf), while the mRNA was detected after 30 hpf. In real-time quantitative PCR (qPCR), the gene was first expressed at 10.5 hpf; then, the expression level increased with a peak at 30 hpf and then gradually decreased. On the other hand, western blotting with specific antibody detected the lectin protein at all tested stages, including the unfertilized egg, which suggests that the lectin detected in the early stages was a maternal factor. Immunohistochemistry demonstrated that kalliklectin was localized at the basement membranes of the newly hatched larvae, while the lectin was widely detected in epidermal cells in larva at 5 dph. A 40-kDa lectin was partially purified from unfertilized eggs using mannose-affinity chromatography, and the lectin was determined as kalliklectin by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis, which indicated that the lectin is functional in the eggs. The egg lectin can bind to Gram-positive bacterial pathogens of fish, such as Lactococcus garvieae and Streptococcus iniae. We conclude that fugu kalliklectin might be an important immunocomponent, transferred from mother to offspring.

Serum amyloid P-component/C-reactive proteins in fugu (Takifugu rubripes) egg with binding ability to disease-causing bacteria by carbohydrate-recognition.

Two galactose-binding proteins were purified from the eggs of Takifugu rubripes by affinity chromatography. These proteins were detected at 26 and 23 kDa under reducing and at 40 and 45 kDa under non-reducing conditions at SDS-PAGE. The peptide sequences from both proteins matched to short-type pentraxin. The 26-kDa lectin was glycosylated, while the other one was not, indicating that these could be glycoforms of pentraxin. Messenger RNA of pentraxin was detected in eggs and embryos at 1-cell stage, was undetectable till blastula, and finally detected again after gastrula, suggesting that the mRNAs in eggs and 1-cell embryos were maternal in origin, and autologous transcription of the gene occurred after blastula. Since they bind to pathogenic bacteria, egg pentraxins may have immunological functions during embryogenesis. This is the first study to show the presence of short-type pentraxin in fish eggs and the diversity of fish egg lectins.

D-mannose-specific Immunoglobulin M in Grass Puffer (Takifugu niphobles), a Nonhost Fish of a Monogenean Ectoparasite Heterobothrium okamotoi, Can Act as a Trigger for its Parasitism.

Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.

Transport of maternal transthyretin to the fetus in the viviparous teleost Neoditrema ransonnetii (Perciformes, Embiotocidae).

The molecular basis of viviparity in non-mammalian species has not been widely studied. Neoditrema ransonnetii, a surfperch, is a matrotrophic teleost whose fetuses grow by ovarian cavity fluid (OCF) ingestion and by nutrient absorption via their enlarged hindgut. We performed a proteomics analysis of N. ransonnetii plasma protein and found proteins specific to pregnant females; one of these was identified as transthyretin (TTR), a thyroid hormone distributor protein. We synthesized recombinant protein rNrTTR and raised an antibody, anti-rNrTTR, against it. Semi-quantitative analysis by western blotting using the antibody demonstrated that plasma TTR levels were significantly greater in pregnant fish than in non-pregnant fish. OCF and fetal plasma also contained high TTR levels. Immunohistochemical staining showed that large amounts of maternal TTR were taken up by fetal intestinal epithelial cells. These results indicate that maternal TTR is secreted into OCF and taken up by fetal enterocytes, presumably to deliver thyroid hormones to developing fetuses.