Biomedical subjects

Yuji Nagashima

Publications and source records attributed to Yuji Nagashima.

At least 19 recordsLinked to original sources

PubMed2006

Molecular cloning and expression of two new allergens from Anisakis simplex.

The nematode Anisakis simplex is a marine parasite that causes allergy as well as anisakiasis. Although five Anisakis allergens have already been identified, immunoblotting studies suggested that unidentified allergens still exist. In this study, an expression cDNA library constructed from A. simplex was subjected to immunoscreening using an Anisakis-allergic patient serum, and two positive clones coding for allergens (named Ani s 5 and 6) were obtained. Ani s 5 (152 amino acid residues) is homologous with nematode proteins belonging to the SXP/RAL-2 protein family and Ani s 6 (84 amino acid residues) with serine protease inhibitors from various animals. Of the 28 patient sera examined, seven and five reacted to recombinant Ani s 5 and 6 expressed in Escherichia coli, respectively. By inhibition immunoblotting experiments using the recombinant allergens as inhibitors, natural Ani s 5 could be identified as a 15-kDa protein in the crude extract of A. simplex but natural Ani s 6 could not be identified probably due to its low expression. In conclusion, Ani s 5 and 6 are new allergens of A. simplex that are specific to some Anisakis-allergic patients.

Allergens↗

PubMed2006

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli.

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.

Amino Acid Sequence↗

PubMed2006

Molecular cloning of grammistins, peptide toxins from the soapfish Pogonoperca punctata, by hemolytic screening of a cDNA library.

A novel method, based on the hemolytic screening of a cDNA phage library, was developed to isolate cDNAs encoding grammistins (antibacterial peptide toxins) of the soapfish Pogonoperca punctata. As a result, cDNAs encoding six grammistins were isolated and elucidated for their nucleotide sequences. In common with the grammistins, the precursor protein is composed of a highly conserved signal peptide, a considerably conserved propeptide that is characterized to contain a pair of basic residues (Lys-Arg) at plural positions including the C-terminus and one copy of a mature peptide. This precursor organization is similar to those of dermaseptins, antibacterial peptides from the frog skin.

Amino Acid Sequence↗

PubMed2006

Purification, properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom.

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of alpha-subunit (702 amino acid residues) and beta-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX alpha- and beta-subunits share as high as 87 and 95% sequence identity with the SNTX alpha- and beta-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX beta-subunit with the reported VTX beta-subunit is 90%, being lower than that with the SNTX beta-subunit.

Amino Acid Sequence↗

PubMed2006

Caspase-independent apoptosis induced in rat liver cells by plancitoxin I, the major lethal factor from the crown-of-thorns starfish Acanthaster planci venom.

Plancitoxin I, the major lethal factor from the crown-of-thorns starfish Acanthaster planci venom, is quite unique not only in exhibiting potent hepatotoxicity but also in sharing high sequence homology with mammalian deoxyribonulease II. In this study, morphological and biochemical changes in rat liver epithelial cells (TRL 1215 cells) treated with the toxin were examined to understand the mechanism by which plancitoxin I displays hepatotoxicity. AlamarBlue assay established that plancitoxin I is cytolethal to TRL 1215 cells. This cytolethalithy was ascribable to apoptotic cell death. Nuclear fragmentation evidenced by either Diff-Quick or Hoechst 33258 staining, DNA fragmentation by TUNEL assay and electrophoretic analysis on agarose gel and phosphatidylserine externalization by flow cytometric analysis of annexin V-FITC stained cells were all characteristics of apoptosis. The observed apoptosis was shown to be independent of the caspase 3 cascade that is generally accepted as the effector of the apoptotic process. Very interestingly, experiments using FITC-labeled plancitoxin I proved that the toxin can enter the nucleus of TRL 1215 cells. Our results suggested that plancitoxin I induces apoptosis of TRL 1215 cells through the following procedure: binding to a specific receptor in the cytoplasmic membrane, entering the cell, entering the nucleus and degrading DNA.

Animals↗

PubMed2006

Nitrogen:Phosphorus supply ratio may control the protein and total toxin of dinoflagellate Alexandrium tamarense.

The protein and total toxin of dinoflagellate Alexandrium tamarense at the exponential growth phase were studied at four N:P supply ratios=8, 16, 24 and 48 by maintaining the N concentration at 880 microM with variable P concentrations without nutrient limitation. Because A. tamarense grew well at all N:P supply ratios, they might synthesize RNA which contains high phosphorus and consequently low N:P atomic ratio of cells might be retained during exponential growth phase. Cellular protein:C ratio and toxin:C ratio depended on N:P supply ratio, suggesting that intracellular biochemical composition of A. tamarense might vary due to N:P supply conditions. These biochemical changes could not be detected by only investigations of cellular N:C atomic ratio which was independent on N:P supply ratio. Total cellular toxin contents of A. tamarense increased with increasing N:P supply ratio, indicated that total cellular toxin contents of A. tamarense might be stimulated by relative P-deficiency. In situ P concentration of the Seto Inland Sea of Japan has been reduced since 1980s the environmental regulation issued by Japanese Government, and therefore N:P supply ratio of input water from adjacent rivers has became higher than the Redfield ratio. The present study may suggest that the reduction of P supply into ambient sea water might cause A. tamarense to accumulate more toxin within the cell, in result, noxious A. tamarense would be more influential to marine organisms in coastal ecosystem.

Animals↗

PubMed2006

Isolation and cDNA cloning of a potassium channel peptide toxin from the sea anemone Anemonia erythraea.

A potassium channel peptide toxin (AETX K) was isolated from the sea anemone Anemonia erythraea by gel filtration on Sephadex G-50, reverse-phase HPLC on TSKgel ODS-120T and anion-exchange HPLC on Mono Q. AETX K inhibited the binding of (125)I-alpha-dendrotoxin to rat synaptosomal membranes, although much less potently than alpha-dendrotoxin. Based on the determined N-terminal amino acid sequence, the nucleotide sequence of the full-length cDNA (609bp) encoding AETX K was elucidated by a combination of degenerate RT-PCR, 3'RACE and 5'RACE. The precursor protein of AETX K is composed of a signal peptide (22 residues), a propart (27 residues) ended with a pair of basic residues (Lys-Arg) and a mature peptide (34 residues). AETX K is the sixth member of the type 1 potassium channel toxins from sea anemones, showing especially high sequence identities with HmK from Heteractis magnifica and ShK from Stichodactyla helianthus. It has six Cys residues at the same position as the known type 1 toxins. In addition, the dyad comprising Lys and Tyr, which is considered to be essential for the binding of the known type 1 toxins to potassium channels, is also conserved in AETX K.

Amino Acid Sequence↗

PubMed2005

Molecular cloning of two toxic phospholipases A2 from the crown-of-thorns starfish Acanthaster planci venom.

The full-length cDNAs encoding two toxic phospholipases A2 (AP-PLA2-I and -II) from the crown-of-thorns starfish Acanthaster planci venom were individually cloned by RT-PCR, 3'RACE and 5'RACE. In common with both AP-PLA2s, the precursor protein is composed of a signal peptide, a propeptide and a mature protein (136 and 135 residues for AP-PLA2-I and -II, respectively). The four motifs (Ca2+-binding loop, Ca2+-binding site, active site and catalytic network) characteristic of groups I and II PLA2s are well conserved in both AP-PLA2s. In addition to this, the presence of the elapid and pancreatic loops and the involvement of a propeptide in the precursors suggested that AP-PLA2s are highly analogous to the group IB PLA2s. However, when compared to the amino acid sequence of bovine pancreatic PLA2, the representative group IB PLA2, AP-PLA2s require some amino acid insertions and deletions in the region 76-100, as previously observed for the starfish Asterina pectinifera PLA2s. Furthermore, the phylogenetic tree made clearly demonstrated that AP-PLA2s and A. pectinifera PLA2s are distinguishable from the group IB PLA2s as well as other PLA2s, being classified into a new group.

Amino Acid Sequence↗

PubMed2005

Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina.

Two peptide toxins, acrorhagin I (50 residues) and II (44 residues), were isolated from special aggressive organs (acrorhagi) of the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. The LD50 against crabs of acrorhagin I and II were estimated to be 520 and 80 microg/kg, respectively. 3'- and 5'-RACE established the amino acid sequences of the acrorhagin precursors. The precursor of acrorhagin I is composed of both signal and mature peptides and that of acrorhagin II has an additional sequence (propart) between signal and mature peptides. Acrorhagin I has no sequence homologies with any toxins, while acrorhagin II is somewhat similar to spider neurotoxins (hainantoxin-I from Selenocosmia hainana and Tx 3-2 from Phoneutria nigriventer) and cone snail neurotoxin (omega-conotoxin MVIIB from Conus magus). In addition, analogous peptides (acrorhagin Ia and IIa) were also cloned during RT-PCR experiments performed to confirm the nucleotide sequences of acrorhagins. This is the first to demonstrate the existence of novel peptide toxins in the sea anemone acrorhagi.

Amino Acid Sequence↗

PubMed2005

Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata.

Three peptide toxins (Am I-III) with crab toxicity were isolated from the sea anemone Anthopleura maculata by gel filtration and reverse-phase HPLC. Am I was weakly lethal to crabs (LD50 830 microg/kg) and Am III was potently lethal (LD50 70 microg/kg), while Am II was only paralytic (ED50 420 microg/kg). The complete amino acid sequences of the three toxins were determined by cDNA cloning based on 3'-Race and 5'-Race. Although Am III (47 residues) is an analogue of the well-known type 1 sea anemone sodium channel toxins, both Am I (27 residues) and II (46 residues) are structurally novel peptide toxins. Am I is a new toxin having no sequence homologies with any toxins. Am II shares 28-39% identity with the recently characterized sea anemone toxins inhibiting specialized ion channels, BDS-I and II from Anemonia sulcata and APETx1 and 2 from Anthopleura elegantissima. The precursor proteins of the three toxins are commonly composed of a signal peptide, a propart with a pair of basic residues (Lys-Arg) at the end and the remaining portion. Very interestingly, the Am I precursor protein contains as many as six copies of Am I.

Amino Acid Sequence↗

PubMed2005

Further isolation and characterization of grammistins from the skin secretion of the soapfish Grammistes sexlineatus.

Soapfishes contain peptide toxins (grammistins) in the skin secretion. Two grammistins (Gs 1 and Gs 2) and six grammistins (Pp 1, Pp 2a, Pp 2b, Pp 3, Pp 4a and Pp 4b) have already been isolated from Grammistes sexlineatus and Pogonoperca punctata, respectively. In this study, five grammistins (Gs A-E), together with grammistins Gs 1 and Gs 2, were further isolated from G. sexlineatus by gel filtration and reverse-phase HPLC. Sequence analyses revealed that grammistins Gs A (28 residues) and Gs C (26 residues) are analogous to grammistin Pp 3 and grammistin Gs B (12 residues) to grammistin Pp 1, while grammistins Gs D (13 residues) and Gs E (13 residues) are identical with grammistins Pp 1 and Pp 2b, respectively. Grammistins Gs A-C exhibited antibacterial activity with a broad spectrum against nine species of bacteria in common with the other grammistins but had no hemolytic activity differing from the other grammistins. Grammistins Gs A-E, Gs 1 and Gs 2 could release carboxyfluorescein entrapped within liposomes made of either phosphatidylcholine or phosphatidylglycerol/phosphatidylcholine (3:1), demonstrating their membrane-lytic activity. However, no clear relationship between the membrane-lytic activity and the biological activity of grammistins was recognized.

Amino Acid Sequence↗

PubMed2004

Variability in toxicity of the dinoflagellate Alexandrium tamarense in response to different nitrogen sources and concentrations.

Nitrogen (N) supply in pulses was simulated by exposing Alexandrium tamarense which was acclimatized at low N concentration (6 microM-N) to sudden increase in concentrations of nitrate, ammonium and urea, and the variability in toxicity due to nutrient status of A. tamarense was examined. The toxin composition did not vary dramatically among the three N sources, however, ammonium induced the highest concentration of intracellular toxin, followed by urea and then nitrate. Therefore, populations utilizing high ammonium concentration could be more toxic than those growing on nitrate or urea. The toxin content was dependent on the cellular N status of nitrate grown cells only, suggesting that the competition for N in toxin production with other metabolic pathways such as growth may be different among N sources. The relationship between toxin and nutrient status is a complex interaction and it involves the redistribution of cellular N within the cells. Understanding the toxin dynamics of natural populations in relation to nutrient is essential for the mitigation of harmful dinoflagellates in a given coastal ecosystem.

Animals↗

PubMed2004

Purification and properties of proteinaceous trypsin inhibitors in the skin mucus of pufferfish Takifugu pardalis.

A screening assay for inhibitory activity against trypsin in skin mucus from 29 species of fishes reveals a wide distribution of trypsin inhibitors in skin mucus and relatively high antitryptic activity in pufferfish of the family Tetraodontidae. Two trypsin inhibitors termed TPTI 1 and 2 were purified to homogeneity from the skin mucus of Takifugu pardalis by salting out, lectin affinity, anion exchange FPLC and gel filtration HPLC. Both inhibitors are acidic glycoproteins, with an apparent molecular mass of 57 kDa in SDS-PAGE, pI below 4 and 1.9% reducing sugar for TPTI 1 and with an apparent molecular mass of 47 kDa in SDS-PAGE, pI 5.2 and 0.8% reducing sugar for TPTI 2. The inhibitors effectively repress the catalytic activity of trypsin and alpha-chymotrypsin, and therefore can be classified as serine protease inhibitors. The inhibitory constants against trypsin were 4.9x10(-8) M for TPTI 1 and 3.9x10(-8) M for TPTI 2. Both inhibitors react with trypsin at a molar ratio of 1:1, although TPTI 1 reversibly inactivates the proteolytic activity of trypsin non-competitively and TPTI 2, competitively. The trypsin inhibitors in the skin mucus of T. pardalis may function as defense substances to neutralize serine proteases released by invasive pathogens.

Amino Acids↗

PubMed2004

Purification and molecular cloning of a major allergen from Anisakis simplex.

A heat-stable allergen with a molecular weight of 21 k was purified from larvae of the nematode Anisakis simplex by gel filtration, anion-exchange FPLC and reverse-phase HPLC. When analyzed by immunoblotting and ELISA, seven of eight patient sera reacted to the 21 k allergen, demonstrating that this protein is a major allergen of A. simplex. A full-length cDNA encoding the 21 k allergen was cloned by a combination of 3'RACE and screening of an expression library with DIG-labeled DNA probes. The precursor of the 21 k allergen was judged to be composed of a signal peptide (23 residues) and a mature protein (171 residues). As compared to the N-terminal amino acid sequence (up to the 17th residue) of Ani s 1 previously identified as the major allergen, the 21 k allergen has only one replacement, suggesting that the 21 k allergen belongs to the same protein family of Ani s 1. Although the 21 k allergen was found to have 30-40% sequence identity with Kunitz-type trypsin inhibitor domain containing hypothetical proteins of Caenorhabditis elegans, it lacked inhibitory activity against trypsin. The 21 k allergen was successfully expressed in Escherichia coli as a GST-fusion protein showing reactivity with IgE in patient sera.

Allergens↗

PubMed2004

Determination of tetramine in marine gastropods by liquid chromatography/electrospray ionization-mass spectrometry.

Tetramine (tetramethylammonium ion) is found at high levels (several mg/g) in the salivary gland of buccinid gastropods and has been involved in numerous poisoning incidents after ingestion of those gastropods. A sensitive and selective determination method for tetramine, which is based on a combination of liquid chromatography (LC) and electrospray ionization-single quadrupole mass spectrometry (ESI-MS), was developed. Following separation by LC on a cation-exchange column, tetramine was easily detected by simultaneous monitoring of a molecular ion (m/z 74) at a cone voltage of 30 V and a fragment ion (m/z 58) at 70 V. A linear calibration curve was obtained in the range of 0.1-100 ng by plotting the peak areas of the molecular ion versus the amounts of tetramine. Spiking experiments demonstrated that tetramine in gastropod tissues can be determined by the LC/ESI-MS method, without being affected by sample matrices as well as the extration procedure. Applications of the new method to gastropod samples revealed that a small amount of tetramine is contained even in mid-gut gland and muscle and that tetramine in the salivary gland diffuses to other tissues during boiling and slow thawing.

Animals↗

PubMed2004

Plancitoxins, lethal factors from the crown-of-thorns starfish Acanthaster planci, are deoxyribonucleases II.

Two lethal factors (named plancitoxins I and II for major and minor toxins, respectively) with the same LD50 (i.v. injection into mice) of 140 microg/kg were purified from spines of the crown-of-thorns starfish Acanthaster planci. Injection of a sublethal dose of plancitoxin I or II into mice remarkably elevated serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, demonstrating that both toxins are potently hepatotoxic. Analysis by SDS-PAGE revealed that both plancitoxins are composed of two subunits (alpha-subunit of 10 kDa and beta-subunit of 27 kDa) bridged by a disulfide bond. Based on the determined N-terminal amino acid sequences of alpha- and beta-subunits, the full-length cDNA (1820 bp) encoding plancitoxin I was cloned by RT-PCR, 3'-RACE and 5'-RACE. alpha-Subunit (92 amino acid residues) and beta-subunit (240 residues) are coded in this order by the same cDNA. Interestingly, the deduced amino acid sequence of plancitoxin I showed 40-42% homologies with mammalian deoxyribonucleases II (DNases II). In addition, plancitoxin I exhibited DNA degrading activity with an optimum pH of 7.2. Plancitoxin I is the first example of toxic DNases II whose structures have been elucidated.

Alanine Transaminase↗

PubMed2004

Mechanism of the decrease of tetrodotoxin activity in modified seawater medium.

This study was designed to clarify the mechanism of the decrease of tetrodotoxin (TTX) toxicity during storage in a modified seawater medium (MSWM). When TTX was added to sterilized MSWM, the toxicity of TTX in the medium markedly decreased within 1 day, as determined by a mouse bioassay. HPLC (high-performance liquid chromatography) analysis showed that the peak of TTX was reduced and new unidentified peaks were observed. Omission of the P-1 metal solution from MSWM suppressed the decrease in TTX toxicity and the disappearance of TTX. Further studies indicated that boric acid in the P-1 metal solution triggers this toxicity decrease, indicating that TTX is chemically, not microbiologically, converted to unknown compounds in MSWM.

Animals↗

PubMed2003

Molecular cloning of an epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea.

An epidermal growth factor (EGF)-like toxin (gigantoxin I) and two sodium channel toxins (gigantoxins II and III), previously isolated from the sea anemone Stichodactyla gigantea, were cloned for their cDNAs. The precursor protein of gigantoxin I is composed of a signal peptide, propart and mature peptide, similar to those of gigantoxins II and III, and is much simpler in structure than those of mammalian EGFs. In addition, gigantoxin I as well as gigantoxins II and III was demonstrated to be contained in nematocysts, suggesting that gigantoxin I functions as a toxin in S. gigantea.

Amino Acid Sequence↗