Michiko Kono

Purification and some properties of a tetrodotoxin binding protein from the blood plasma of kusafugu, Takifugu niphobles

A tetrodotoxin binding protein has been purified from the plasma of the puffer fish kusafugu, Takifugu niphobles, through DEAE-cellulose treatment, ammonium sulfate fractionation, Sephadex gelfiltrations and Sephacryl S-200 and Cellulofine A-500 column chromatography. Final purification by HPLC on a TSK G-3000 SL column yielded a protein which showed only a single protein peak. The molecular weight of the protein was estimated to be 116,000 and 91,000 by SDS-PAGE and mass spectrometry, respectively. A blast search on the amino-terminal amino acid sequence of the purified protein revealed that the protein had no homology to any other protein on data base.

Accumulation of tetrodotoxin and 4,9-anhydrotetrodotoxin in cultured juvenile kusafugu Fugu niphobles by dietary administration of natural toxic komonfugu Fugu poecilonotus liver

Non-toxic cultured juvenile kusafugu Fugu niphobles were fed with a diet containing highly toxic natural komonfugu Fugu poecilonotus liver until the 30th day (8.0 microg of TTX and 3.7 microg of 4,9-anhydroTTX/fish/day), and then fed with a non-toxic diet until the 240th day. During the 30-240th day, five or six fish were periodically sampled six times, and the contents of TTX and 4,9-anhydroTTX in each tissue were determined. The total TTX and 4,9-anhydroTTX accumulated in all tissues tested was not significantly changed during the experimental period, both being kept at 70% of administrated doses. However, in the liver, the TTX content accounted to be 120 microg (50% of administrated) on the 30th day, and then it gradually decreased to 50 microg until the 240th day, while 4,9-anhydroTTX content was kept at approximately 40 microg (40% of administrated) during all the experimental periods. In contrast to the liver, in the skin, TTX and 4,9-anhydroTTX were 40 and 5 microg, respectively, on the 30th day, and then gradually increased to 80 and 24 microg, respectively, until the 240th day. In the intestine, TTX and 4,9-anhydroTTX contents were kept at 25 and 12 microg, respectively, during all the experimental periods. According to these results, we assumed that a part of TTX accumulated in the liver was slowly transferred to the skin.

Purification and molecular cloning of a chitinase expressed in the hepatopancreas of the penaeid prawn Penaeus japonicus

A protein with chitinase activity was purified from the hepatopancreas of the penaeid prawn Penaeus japonicus, and the amino acid sequences of several amino acid fragments were determined. A cDNA clone was isolated and sequenced which contained the coding sequence of the enzyme. The conceptually translated protein (named Pjchi-3) was a member of the chitinase family based on sequence similarities to chitinases of non-crustacean species, as was the case with Pjchi-1 and 2, two chitinase homologues previously isolated from P. japonicus.

Molecular analysis of two genes, DD9A and B, which are expressed during the postmolt stage in the decapod crustacean Penaeus japonicus

In decapod crustaceans, deposition of calcium carbonate crystals (calcification) in the exoskeleton takes place during the postmolt phase of the molt cycle. In an attempt to identify proteins which regulate the calcification process, the differential display technique was used to identify genes which were specifically expressed in the integument during the postmolt stage in the penaeid prawn Penaeus japonicus. One of the genes thus identified, named DD9A, was expressed in the epithelial cells of the tail fan. DD9A encoded a putative precursor of a secreted protein of 113 amino acids which exhibited sequence similarities to a group of crustacean and insect cuticular proteins, suggesting that DD9A was a protein component of the exoskeleton. Another gene, DD9B, which was also transcribed specifically during the postmolt period was identified based on its sequence similarity to DD9A. Potential roles of the DD9A protein in the calcification of the exoskeleton will be discussed.

The DD5 gene of the decapod crustacean Penaeus japonicus encodes a putative exoskeletal protein with a novel tandem repeat structure

A gene, named DD5, was identified in the penaeid prawn Penaeus japonicus and its cDNA cloned and sequenced. DD5 is expressed in the epidermal cells underlying the exoskeleton and the transcripts are detected specifically during the postmolt stage of the molt cycle. Sequence analysis of the conceptual protein product suggests that the DD5 protein is a component of the exoskeleton. The bulk of the protein consists of tandem repeats of a unit sequence of approximately 100 amino acids. The repeated sequences are highly homologous to one another and each of them includes a variant of the Rebers--Riddiford consensus sequence.

Examination of transformation among tetrodotoxin and its analogs in the living cultured juvenile puffer fish, kusafugu, Fugu niphobles by intramuscular administration

In puffer fish, tetrodotoxin (TTX) exists as the major toxin with chemically equilibrium analogs (4-epiTTX, 4,9-anhydroTTX) and chemically non-equilibrium analogs (deoxy analogs, 11-oxoTTX, 4-S-cysteinylTTX). There are two purposes to this study: 1) to search for the reason why TTX is the most major analog in puffer fish, even 4,9-anhydroTTX is chemically more stable, 2) to investigate whether or not chemically non-equilibrium analogs are transformed in puffer fish, because these were predicted to be biosynthetic intermediates. Pure TTX, 4-epiTTX, 4,9-anhydroTTX, and 11-oxoTTX were separately administrated to the cultured non-toxic juvenile puffer fish kusafugu, Fugu niphobles by intramuscular injection. Sixteen days after administration, TTX analogs in the whole fish were analyzed by LC-fluorescent detection and LC/MS. By the administration of TTX, 4-epiTTX, and 4,9-anhydroTTX, 34-40% of the administrated doses of the toxins were accumulated, and 4,9-anhydroTTX has become the major toxin after inter-conversion. This result indicates discrepancy from the previous ones wherein TTX was predominantly accumulated when TTXs were administrated through diets; this suggests that dietary administration might be necessary to accumulate TTX as the major toxin, and not 4,9-anhydroTTX. Transformations from TTX to deoxy analogs or 11-oxoTTX, or from 11-oxoTTX to TTX were not detected in this study.

Pigmentation of cultured red sea bream, Chrysophrys major, using astaxanthin from Antarctic krill, Euphausia superba, and a mysid, Neomysis sp.

Abstract Pigmentation enhancement in cultured red sea bream, Chrysophrys major , was investigated using Antarctic krill, Euphausia superba , and a mysid, Neomysis sp., as a source of astaxanthin. Diets fortified with processed Antarctic krill (krill meal) and its acetone extract, containing 0.82–4.92 mg carotenoids/100 g dry weight, and raw krill and raw mysid supplemented diets, containing about 2.00 mg carotenoids/100 g wet weight, were formulated and tested for carotenoid deposition. The rate of carotenoid deposition in fish fed with raw krill and raw mysid was significantly higher and resulted in distinct pigmentation. The groups fed with the krill meal and acetone extract diets showed varied concentrations of skin carotenoids and resulted in faint pigmentation. Pigmented fish then fed on a carotenoid-free diet for the same length of time showed no apparent differences in the skin pigmentation although the detectable amounts of carotenoids varied. The bream converted some of the dietary astaxanthin to skin tunaxanthin.

Kinetic Analysis of a Chitinase from Red Sea Bream, Pagrus major

Kinetic analysis was done on the 46-kDa chitinase (EC 3.2.1.14) purified from the stomach of red sea bream, Pagrus major, using glycolchitin and N-acetylchitooligosaccharides (GlcNAcn, n=2–6) as substrates. High activity was observed at two pHs, such as 2.5 and 9.0, toward glycolchitin as seen in other insect chitinases, and also at both pH 2.5 and 5.0 even toward a short substrate, N-acetylchitopentasaccharide. Allosamidin competitively inhibited chitinase with Ki value of 0.0214 μM at pH 2.5 and 0.0024 μM at pH 9.0 in the reaction of glycolchitin. Substrate inhibition was observed in the reaction of N-acetylchitopentasaccharide. The anomeric forms of the products from N-acetylchitooligosaccharides were analyzed to be β anomer by the high pressure liquid chromatography (HPLC) method. The data for both β-anomer formation and allosamidin inhibition suggest that red sea bream chitinase belongs to family 18 of glycosyl hydrolases. This suggestion is also supported by the results for the N-terminal amino acid sequence.

Kinetics of a Chitinase from a Prawn, Penaeus japonicus

Kinetic analysis was done on a chitinase (EC 3.2.1.14) purified from the liver of a prawn, Penaeus japonicus, using N-acetylchitooligosaccharides (GlcNAcn, n = 2 to 6), p-nitrophenyl N-acetylchitooligosaccharides (pNp-GlcNAcn, n = 1 to 5), and colloidal chitin as the substrates. The enzyme hydrolyzed GlcNAc4 to two molecules of GlcNAc2, GlcNAc5 to GlcNAc2 plus GlcNAc3, and GlcNAc6 by two ways to GlcNAc2 plus GlcNAc4 (87%), and two molecules of GlcNAc3 (13%). Neither GlcNAc2 nor GlcNAc3 was hydrolyzed. The Km and kcat were 0.249 mm and 3.38 sec−1 for GlcNAc4,0.018 mm and 2.67 sec−1 for GlcNAc5, and 0.005 mm and 2.72 sec−1 for GlcNAc6, respectively. The cleavage patterns of p-nitrophenyl N-acetylchitooligosaccharides were different from those of the corresponding N-acetylchitooligosaccharides. The enzyme hydrolyzed colloidal chitin to produce mainly GlcNAc2 and a trace of GlcNAc3. Allosamidin inhibited prawn chitinase in a competitive way with a Ki of 0.1 μm and IC50 of 0.14 μm. These results suggest that p...