Masatoshi Nozaki

Apoptotic effect in the glioma cells induced by specific protein extracted from Okinawa Habu (Trimeresurus flavoviridis) venom in relation to oxidative stress

Okinawa Habu (Trimeresurus flavoviridis) venom is well known for its toxic efficacy, from which one kind of specific protein, Okinawa Habu apoxin protein-1 (OHAP-1) has been extracted. The purpose of this study was to investigate whether OHAP-1 could induce apoptosis in some glioma cells, and if so, to elucidate the possible mechanism involved. Three malignant glioma cell lines were tested. The malignant glioma cell lines were rat C6 and human RBR 17T, U251. OHAP-1 inhibited growth of all cell lines. Whether or not the apoptosis had been induced was determined by using DNA gel electrophoresis, DNA flow cytometry and TUNEL assay. After OHAP-1 treatment, DNA fragmentation, an increase in the percentage of subdiploid DNA content, and TUNEL positive cells were found in the C6, RBR17T, and U251 cells. Furthermore, OHAP-1 showed L-amino acid oxidase (LAAO) activity. In order to study the mechanism of apoptosis induced by OHAP-1, the changes of intracellular reactive oxygen species (ROS) were measured using flow cytometry, and the expression of p53 protein was examined using immunohistochemistry. OHAP-1 was found to generate ROS and increase the expression of p53 protein in glioma cells. The inhibiting effect of OHAP-1 on three tested cells was reversed when an antioxidant of either catalase or reduced glutathione (GSH) was added; its apoptotic effect correspondingly became weaker. In this study, the apoptotic effect of OHAP-1 on some malignant glioma cells was confirmed, and it could be that this effect might be mediated through promoting the generation of intracellular ROS and p53 protein expression in glioma cells. It was suggested that OHAP-1 is promising as a potential candidate for clinical tumor therapy.

Novel proteinaceous toxins from the nematocyst venom of the Okinawan sea anemone Phyllodiscus semoni Kwietniewski

The Okinawan sea anemone Phyllodiscus semoni is known to cause cases of severe stinging. We isolated P. semoni toxins 60A and 60B (PsTX-60A and PsTX-60B; ca. 60 kDa) as the major toxins from the isolated nematocysts of this species for the first time. PsTX-60A and PsTX-60B showed lethal toxicity to the shrimp Palaemon paucidence when administered via intraperitoneal injection (LD(50) values: 800-900 and 800 microg/kg, respectively) and hemolytic activity toward a 0.8% suspension of sheep red blood cells (ED(50) values: 600 and 300 ng/ml, respectively). Furthermore, we sequenced the cDNA encoding PsTX-60A. The deduced amino acid sequence of PsTX-60A did not show any similarity to previously reported proteins. The N-terminal amino acid sequence of PsTX-60B showed homology with that of PsTX-60A. These toxins represent a novel class of cytolytic proteinaceous toxins.

A New Polypeptide Toxin from the Nematocyst Venom of an Okinawan Sea Anemone Phyllodiscus semoni (Japanese name “unbachi-isoginchaku”)

The venomous sea anemone Phyllodiscus semoni causes cases of severe stinging. We isolated Phyllodiscus semoni toxin 20A (PsTX-20A), a hemolytic and lethal polypeptide (20 kDa), from the nematocyst venom of this species for the first time. Furthermore, we sequenced the cDNA encoding PsTX-20A. The deduced amino acid sequence of PsTX-20A showed that this toxin was a new member of the actinoporin family, which consists of several cytolytic polypeptides originating from sea anemones. PsTX-20A showed lethal toxicity to the shrimp Palaemon paucidens when administered via intraperitoneal injection (LD50, 50 μg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50, 80 ng/ml).

Purification of three antihemorrhagic factors from the serum of a mongoose (Herpestes edwardsii)

Three antihemorrhagic factors (AHF-1, AHF-2 and AHF-3) were purified from the serum of H. edwardsii, a mongoose, by a combination of gel filtration on a Sephadex G-200 column and high performance liquid chromatography with a TSK gel DEAE-5PW column. Each of the purified antihemorrhagic factors showed a single band on polyacrylamide gel disc electrophoresis. The three antihemorrhagic factors inhibited the hemorrhagic activity of HR 1 and HR 2, the hemorrhagic principles from the snake venom of Trimeresurus flavoviridis Okinawa. AHF-1, AHF-2 and AHF-3 were stable at temperatures from 0 degrees to 60 degrees C and at pH values between 2.0 and 11.0. The same molecular weight (65,000) was obtained for the three antihemorrhagic factors. No precipitin lines were found for the purified antihemorrhagic factors with the venom of T. flavoviridis Okinawa and its hemorrhagic principles, HR 1 and HR 2.

Apoptosis induced by box jellyfish (Chiropsalmus quadrigatus) toxin in glioma and vascular endothelial cell lines.

This study was made to investigate whether Chiropsalmus Quadrigatus toxins (CqTX), which isolated from box jellyfish C. Quadrigatus venom, could induce apoptosis in human U251 and rat C6 malignant glioma cells and transformed vascular endothelial ECV 304 cell lines. Cell viability was estimated by MTT assay. Apoptosis was evaluated using TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labeling (TUNEL) method and DNA gel electrophoresis. Furthermore, the expression of p53 protein was examined immunohistochemically in the U251 cells. After the CqTX treatment, the growth of all cell lines was inhibited, the fragmented DNA was observed and some cells became TUNEL positive. The expression of p53 protein was increased in the tested U251 cells. The results suggested that CqTX induced apoptosis in these cell lines. The promotion of the p53 expression might be one mechanism of apoptosis induced by CqTX in the glioma cells.

Purification of an antihemorrhagic factor from the serum of the non-venomous snake Dinodon semicarinatus

An antihemorrhagic factor was purified from the serum of Dinodon semicarinatus, a non-venomous snake (Akamata), by a series of high performance liquid chromatographies with a TSK gel DEAE-5PW column. The purified antihemorrhagic factor showed a single band on polyacrylamide gel disc electrophoresis and inhibited the hemorrhagic activity of HR1 and HR2, the hemorrhagic factors of Trimeresurus flavoviridis Okinawa. The antihemorrhagic factor was stable from 0 degrees to 60 degrees and at pH values 2.0-11.0. The molecular weight of the factor was estimated to be 59,000 and 52,000 by a gel filtration and SDS-disc electrophoresis, respectively, suggesting that it consists of a single subunit, as we also found for the antihemorrhagic factors of the mongoose Herpestes edwardsii.

Neutralization of hemorrhagic snake venoms by sera of Trimeresurus flavoviridis (Habu), Herpestes edwardsii (mongoose) and Dinodon semicarinatus (Akamata)

The sera of T. flavoviridis (Habu), H. edwardsii (mongoose) and D. semicarinatus (Akamata, non-venomous snake) were tested for their capacity to neutralize 28 species of hemorrhagic snake venoms in vitro. The sera of these animals neutralized a variety of hemorrhagic venoms, suggesting a common structure for a hemorrhagic factor and a similar mechanism of neutralization for the antihemorrhagic factor in the sera. The serum of T. flavoviridis neutralized the lethal toxicity of the T. flavoviridis venom but could not neutralize those of the other hemorrhagic venoms at all. The sera of H. edwardsii and D. semicarinatus did not inhibit the activity of all hemorrhagic venoms.

Characterization of three hemorrhagic factors from the venom of Okinawa habu (Trimeresurus flavoviridis)

Three hemorrhagic factors, HR1, HR2a and HR2b, of Okinawa habu venom were characterized in terms of their subunit structure, amino acid composition, metal content and immunological properties. HR1 is a dimer (mol. wt 90,000) consisting of two identical subunits at 25 degrees C, but polymerizes to form a tetramer at 4 degrees C. Two peaks corresponding to the dimer and the tetramer were observed upon ultracentrifugation analysis at 20 degrees C. HR2a and HR2b are monomers (mol. wt 24,000 and 19,000, respectively). HR1, HR2a and HR2b contain 407, 203 and 161 amino acids, respectively and the respective mol. wt based on the amino acid composition are 45,988, 23,075 and 18,457. The hemorrhagic factors contain Zn2+, Ca2+ and Mg2+, and were irreversibly inhibited by incubation with chelating reagents. The three hemorrhagic factors were immunologically distinguished from each other, and the hemorrhagic activities were inhibited by the respective antiserum. The activity of HR2a was also inhibited by the antiserum against HR2b.

Purification and crystallization of hemorrhagic factor, HR2b, from the venom of Trimeresurus flavoviridis (habu)

The hemorrhagic factor, HR2b, was purified from the venom of Trimeresurus flavoviridis (habu) by a combination of gel filtration, cation exchange column chromatography and high performance liquid chromatography. The purified HR2b was homogeneous by the criteria of ultracentrifugation and SDS-disc electrophoresis. The mol. wt of HR2b was 18,000 and 18,500 by gel filtration on Sephadex G-50 and by SDS-disc electrophoresis, respectively, indicating a monomer structure for the hemorrhagic factor. Crystals of HR2b, taking the form of thin plates, were obtained in the presence of ammonium sulfate.