Biomedical subjects

Yuji Nagashima

Publications and source records attributed to Yuji Nagashima.

26 records · Page 2Linked to original sources

PubMed2003

An epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea.

Three peptide toxins (gigantoxins I-III) with crab toxicity were isolated from the sea anemone Stichodactyla gigantea by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T and their complete amino acid sequences were determined. Gigantoxins II (44 residues) and III (48 residues) have LD(50) (against crabs) of 70 and 120 microg/kg, respectively, and are analogous to the known type 1 and 2 sea anemone sodium channel toxins, respectively. On the other hand, gigantoxin I (48 residues) is potently paralytic to crabs (ED(50) 215 microg/kg), although its lethality is very weak (LD(50)>1000 microg/kg). Interestingly, gigantoxin I has 31-33% homologies with mammalian epidermal growth factors (EGFs), with the same location of six cysteine residues. In accordance with the sequence similarity, gigantoxin I exhibits EGF activity as evidenced by rounding of A431 cells and tyrosine phosphorylation of the EGF receptor in the cells, although much less potently than human EGF. Gigantoxin I is the first example of EGF-like toxins of natural origin.

Amino Acid Sequence↗

PubMed2003

Occurrence of type 3 sodium channel peptide toxins in two species of sea anemones (Dofleinia armata and Entacmaea ramsayi).

From two species of sea anemones, Dofleinia armata and Entacmaea ramsayi, three peptide toxins (two from the former and one from the latter) with crab toxicity were purified and completely sequenced. The three toxins (30-32 residues) are highly homologous to each other and also to PaTX from Entacmaea actinostoloides, a type 3 sea anemone sodium channel toxin. This study reveals that there is a family of PaTX-like toxins in sea anemones.

Amino Acid Sequence↗

PubMed2003

In vitro accumulation of tetrodotoxin in pufferfish liver tissue slices.

The liver tissue slices of pufferfish accumulate in vitro tetrodotoxin (TTX), when incubated with minimum essential medium containing TTX. In the case of Takifugu rubripes liver slices incubated at a concentration of 25 microg TTT/ml, TTX of 3.9 microg/g was first detected at 2h and increased to 15 microg/g at 48h. The TTX content accumulated was not decreased, even when the slices were further incubated without TTX for additional 48h. Another species of pufferfish T. paradalis also showed similar trend in TTX accumulation, except they accumulated higher concentration of TTX (36.4 microg/g at 48h) than T. rubripes. On the contrary, in the cases of the liver slices from parrot-bass Oplegnathus fasciatus, green ling Hexagrammos otakii and filefish Thamnaconus modestus incubated at a concentration of 25 microgTTX/ml, TTX of 3-4 microg/g was detected even at 0.5h. However, no significant change in TTX contents was recognized during the incubation for 48h. Further incubation of the filefish liver slices without TTX for additional 48h did not decrease the TTX content. It is unlikely that the liver slices of filefish as well as pufferfish rapidly excrete TTX. These results suggest that the difference in the accumulation of TTX between pufferfish and filefish livers is ascribable to the difference not in the TTX excreting ability but in the ability to take up TTX.

Animals↗

PubMed2003

Primary structure of echotoxin 2, an actinoporin-like hemolytic toxin from the salivary gland of the marine gastropod Monoplex echo.

Echotoxins are 25 kDa proteins with both hemolytic and lethal activities, previously purified from the salivary gland of the marine gastropod Monoplex echo. In this study, a cDNA encoding echotoxin 2 was cloned by RT-PCR, 3'-RACE and 5'-RACE, based on its partial amino acid sequence. The full-length echotoxin 2 cDNA (1000 bp) obtained contains an open reading frame (825 bp) coding for a precursor protein of 274 amino acid residues. Mature echotoxin 2 composed of 226 amino acid residues is assumed to be produced by post-translational removal of N-terminal 23 residues (predicted as a signal peptide) and C-terminal 25 residues from the precursor protein. Very interestingly, a homology search revealed that echotoxin 2 is analogous to actinoporins, 20 kDa pore-forming hemolysins reported from various sea anemones. In addition to the similarities in biological activity, molecular size and basicity between echotoxin 2 and actinoporins, two prominent structural features, an N-terminal amphiphilic alpha-helix and an aromatic patch comprising Trp and Tyr residues, both of which are important for the pore-forming activity of actinoporins, are also recognized in echotoxin 2. However, echotoxin 2 is distinguishable from actinoporins in having Cys residues and lacking an RGD motif.

Amino Acid Sequence↗

PubMed2003

[In vitro evaluation of allergenicity of dried food powders manufactured for food provocation test].

Food provocation test (FPT) is one of important diagnostic methods for food allergy, but no standard antigens for FPT have yet been developed. In this study, dried powders were manufactured from five kinds of foods (cow's milk, hen egg, chicken, soybean and wheat) by spray-drying or freeze-drying and examined in vitro for their usefulness as antigens for FPT. In SDS-PAGE, the migration pattern of the extract from each powder was the same as or closely similar to that of the extract from its material. When analyzed by ELISA, a good correlation (r=0.853-0.978) in the reactivity with sera from food-allergic patients was observed between the extracts from each powder and its material. Moreover, in cow's milk, hen egg and soybean, almost the same ELISA inhibition curves were drawn, regardless of whether the extracts from each powder and its material were used as immobilized antigens or inhibitors. These results demonstrated that each powder contains the same allergens as its material at almost the same levels, being useful as an antigen for FPT. Favorably, the powders were found to be stored without significant changes in IgE reactivity at -20 degrees C or 5 degrees C for more than 18 months, although their storage at room temperature was suggested to be avoided.

Allergens↗

PubMed2002

Properties of proteinaceous toxins in the salivary gland of the marine gastropod (Monoplex echo).

Three proteinaceous toxins (named echotoxins 1, 2, and 3) with both lethal and hemolytic activities were purified from the salivary gland of the marine gastropod (Monoplex echo) by gel filtration on Sephadex G-75 and cation-exchange FPLC on Mono S, although marked reduction in toxicity was observed in the latter chromatography. When subjected to reverse-phase HPLC on TSKgel Phenyl-5PW RP, echotoxin 2 afforded a single peak, while both echotoxins 1 and 3 were further separated into two components, respectively. All echotoxins are monomeric simple proteins with a molecular mass of about 25kDa and their N-termini are blocked. Amino acid compositions of echotoxins 1a, 1b, and 2 are closely similar to one another, being characterized by the abundance of Gly and Ala and the absence of half-Cys, Met, and Trp. Of the sugars, lipids and glycolipids tested, gangliosides potently inhibited the hemolytic activity of echotoxins. Furthermore, ganglioside G(T1b) inhibited both in vivo hemolysis and lethality in mice displayed by echotoxins. It is thus concluded that echotoxins injected into mice induce rapid hemolysis by binding to receptors (possibly gangliosides) gangliosides in the erythrocyte membrane, eventually leading to death of mice.

Amino Acids↗

PubMed2002

A tetrodotoxin-binding protein in the hemolymph of shore crab Hemigrapsus sanguineus: purification and properties.

The shore crab Hemigrapsus sanguineus hemolymph contains soluble proteins that bind tetrodotoxin (TTX) and are responsible for high resistance of the crab to TTX. The TTX-binding protein was purified from the hemolymph by ultrafiltration, lectin affinity chromatography and gel filtration HPLC. The purified protein gave only one band in native-polyacrylamide gel electrophoresis (PAGE), confirming its homogeneity. Its molecular weight was estimated to be about 400k by gel filtration HPLC, while it was estimated to be about 82k under non-reducing conditions and about 72 and 82k under reducing conditions by SDS-PAGE, indicating that the TTX-binding protein was composed of at least two distinct subunits. The TTX-binding protein was an acidic glycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% amino sugar. The protein selectively bound to TTX, with a neutralizing ability of 6.7 mouse unit TTX/mg protein, but not to paralytic shellfish poisoning toxins. However, its neutralizing activity was almost lost by treatments with enzymes (protease XIV, thermolysin, trypsin, amyloglucosidase and alpha-amylase) and denaturing agents (1% SDS, 1% dithiothreitol, 8 M urea and 6 M guanidine hydrochloride), suggesting the involvement of both proteinaceous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein.

Amino Acids↗

PubMed2002

[Toxicity and tetramine contents of salivary glands from carnivorous gastropods].

Salivary glands from 29 species of marine carnivorous gastropods in nine families were examined for lethal activity against mice and tetramine content. Mouse lethality was assayed by intravenous injection of buffer extracts into mice, and was detected in 14 species. Heat-stability tests confirmed that toxins in four species were thermolabile, while those in eight species were thermostable. Based on the tetramine contents determined by the colorimetric method using methanolic extracts, the thermostable toxins in seven species (Neptunea eulimatalamellosa, N. vinosa, N. arthritica, N. bulbacea, N. intersculpta f. pribiloffensis, N. intersculpta f. frater pilsbry and Hemifusus tuba) were considered to be tetramine contained at high levels (more than 900 micrograms/g salivary gland), but that in one species (Buccinum opisthoplectum) appeared to be a low-molecular-weight compound differing from tetramine. It is interesting that one (Hemifusus tuba) of the seven species containing high amounts of tetramine belongs to the family Melongenidae, although the other six Neptunea species are members of the family Buccinidae, as expected from previous studies.

Animals↗