Edwardsiella tarda の赤血球凝集能に関与する線毛遺伝子群の同定

We determined the nucleotide sequence of a 8.6 kb DNA region containing etfA encoding a putative fimbrial major subunit from chromosomal DNA of Edwardsiella tarda KG8401 which expresses mannose-resistant hemagglutination (MRHA). This region contained three novel genes, etfBCD, at the downstream region of etfA. The deduced amino acid sequences of EtfABCD contained conserved fimbrial domains; fimbrial protein, fimbrial chaperone, fimbrial usher and fimbrial protein, respectively. Escherichia coli transformed with the cloned etf operon expressed MRHA and fimbriae that reacted with rabbit antiserum against the fimbrial major subunit of E. tarda, showing the implication of the fimbriae in the hemagglutination of E. tarda

Identification and Characterization of the Larval Settlement Pheromone Protein Components in Adult Shells of Crassostrea gigas: A Novel Function of Shell Matrix Proteins

The global decline of natural oyster populations emphasizes the need to improve our understanding of their biology. Understanding the role of chemical cues from conspecifics on how oysters occupy appropriate substrata is crucial to learning about their evolution, population dynamics, and chemical communication. Here, a novel role of a macromolecular assembly of shell matrix proteins which act as Crassostrea gigas Settlement Pheromone Protein Components in adult shells is demonstrated as the biological cue responsible for gregarious settlement on conspecifics. A bioassay-guided fractionation approach aided by biochemical and molecular analyses reveals that Gigasin-6 isoform X1 and/or X2 isolated from adult shells is the major inducing cue for larval settlement and may also play a role in postlarva–larva settlement interactions. Other isolated Stainsall-stainable acidic proteins may function as a co-factor and a scaffold/structural framework for other matrix proteins to anchor within this assembly and provide protection. Notably, conspecific cue-mediated larval settlement induction in C. gigas presents a complex system that requires an interplay of different glycans, disulfide bonds, amino acid groups, and phosphorylation crosstalk for recognition. These results may find application in the development of oyster aquacultures which could help recover declining marine species and as targets of anti-fouling agents

Comparative analysis of the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) from macrophages exposed to high virulent and low virulent strains of Edwardsiella tarda.

We previously reported that high virulent strain (NUF251) of Edwardsiella tarda has an ability to prevent the production of reactive oxygen species by macrophages, and is even capable of surviving and multiplying within Japanese flounder (Paralichthys olivaceus) peritoneal macrophages, whereas the low virulent strain (NUF194) has no such ability. In this study, we found that NUF251 and NUF194 induced NO and TNF-alpha production from Japanese flounder peritoneal macrophages, and NUF251 caused faster induction of NO release and much higher level of TNF-alpha production than NUF194. In addition, similar differences between two strains in terms of the induction of NO and TNF-alpha production were also observed in mouse macrophage cell line RAW264.7 cells. Our results suggest that the potent ability to induce the production of NO and TNF-alpha from macrophages may be one of the factors responsible for the virulence of E. tarda

Larval settlement and metamorphosis of Mytilus coruscus in response to varying bacterial capsular polysaccharide

Marine invertebrates are the backbone of marine biodiversity and play a pivotal role in the marine ecosystem. The life cycle of most marine invertebrates includes the settlement and metamorphosis stage, which is induced by marine biofilms, but the mechanism is still enigmatic. In the present study, we constructed the capsular polysaccharide (CPS) synthesis gene capC-deleted mutant of Pseudoalteromonas marina by gene knockout and then compared the phenotype, the biofilm-forming ability, the effect on settlement and metamorphosis of Mytilus coruscus, and the exopolysaccharide and CPS levels between the mutant and the wild-type strains to explicate the relationship between bacteria and mussels. The study presented that the phenotype and biofilm-forming ability between the wild-type and ΔcapC strains had no significant difference, but the inducing activity of ΔcapC biofilms on larval settlement and metamorphosis decreased significantly (p < 0.05). Compared with the wild-type, the CPS content of ΔcapC strain significantly decreased by 38.07%, accompanied by the increase of c-di-GMP. Meanwhile, the biomass of α-polysaccharides and β-polysaccharides on ΔcapC biofilms decreased significantly (p < 0.05). Thus, the CPS synthesis gene could modulate c-di-GMP, which regulates bacterial polysaccharide secretion, and then impact larval settlement and metamorphosis of mussels. This work brings an entry point to deeply understand the interaction between bacterial polysaccharide and larval recruitment

Purification, molecular cloning, and some properties of a manganese-containing superoxide dismutase from Japanese flounder (Paralichthys olivaceus).

Manganese-superoxide dismutase (Mn-SOD) from Japanese flounder (Paralichthys olivaceus) hepatopancreas has been purified with high purification (781-fold) and recovery (10.8%). The molecular mass of the purified enzyme was estimated to be 26kDa by SDS-PAGE under reducing conditions. In activity staining by native-PAGE, the Japanese flounder Mn-SOD gave three active bands and exhibited KCN-insensitive activity. In addition, the electrophoretic mobility of this enzyme was observed to be faster than that of Japanese flounder Cu,Zn-SOD. On the other hand, the N-terminal amino acid sequence of this Mn-SOD was determined to be 16 amino acid residues, and the sequence showed high homology to other Mn-SODs but not Japanese flounder Cu,Zn-SOD. Analysis of nucleotide and deduced amino acid sequences revealed that the Mn-SOD cDNA consisted of a 64bp 5\u27-non-coding region, a 675bp open reading frame encoding 225 amino acids, and a 465bp 3\u27-non-coding region. The first 27 amino acids containing a mitochondria-targeting signal were highly conserved among other Mn-SODs

Characteristics of a self-assembled fibrillar gel prepared from red stingray collagen

A translucent collagen gel was formed from a transparent acidic solution of red stingray collagen by adjusting to physiological ionic strength and pH in phosphate buffer and then incubating at 25?37°C. During fibril formation from red stingray collagen, the turbidity increased when the NaCl concentration was increased at constant pH and the rate of fibril formation was accelerated by higher pH or lower NaCl concentration. The T m of red stingray collagen fibrillar gel was estimated as 44.3 ± 3.5°C, which was higher than that of the collagen solution, 33.2°C. In addition, red stingray collagen gel maintained its shape without melting and was suitable for culture of mouse stromal cells at 37°C

A serine proteinase from the sarcoplasmic fraction of red sea bream Pagrus major is possibly derived from blood

Collagen degradation is known to be involved in the post mortem tenderization of fish muscle. A serine proteinase that is assumed to be related to collagen degradation after fish death was purified from the sarcoplasmic fraction of red sea bream Pagrus major by ammonium sulfate fractionation and column chromatography on Sephacryl S-300, Q Sepharose and Phenyl Sepharose CL-4B. The enzyme hydrolyzed gelatin and was obtained as a protein band of approximately 38 kDa upon sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. The N-terminal amino acid sequence of the enzyme was determined for 32 residues. A protein that had the same N-terminal amino acid sequence as the enzyme for ten residues was purified from the serum of red sea bream and showed the same characteristics as the enzyme. Therefore, it is suggested that the serine proteinase migrates from the blood to muscle and degrades muscle proteins after the death of the fish

養殖ブリやけ肉におけるⅠ型コラーゲンの変化

Yellowtails (Seriola quinqueradiata), which were cultured in summer at the water temperature of around 30-31℃, were used to make the model of burnt meat for the purpose of investigating the changes in type I collagen during the occurrence of burnt meat. “Burnt meat” (with lightness parameter, L *≧55) was observed just after slaughter in suffocate in air (SA) group and after 2 h storage in spinal corddestruction (SCD) group. Type I collagen decreased (33-37%) during the occurrence of burnt meat in SCD group. In addition, collagenase like protease activity was detected in muscle meat of yellowtail by usingsynthetic substrate, and was inhibited by EDTA. EDTA and pepstatin A suppressed the decrease of type I collagen, indicating that collagenase like protease and aspartic proteases may cause the changes in typeI collagen.海水温30－31℃で飼育されたブリを用いて，やけ肉モデル魚を作成し，筋肉Ⅰ型コラーゲンの変化を調べた。苦悶死ブリは致死直後に，即殺死ブリは保存２時間後にやけ肉 (L*≧55) が発生した。即殺死ブリでは，やけ肉発生に伴いⅠ型コラーゲンの減少(33－37%)が見られた。また，ブリ筋肉中に，合成基質を分解するコラゲナーゼ様酵素活性が認められ，その活性はEDTAにより阻害された。Ⅰ型コラーゲンの減少はEDTA及びペプスタチンAにより抑制されたことから，その減少にはコラゲナーゼ様酵素及びアスパルティックプロテアーゼが関与する可能性が示唆された