Won Duk Yoon

Cytotoxicity and hemolytic activity of jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae) venom

The recent bloom of a giant jellyfish Nemopilema nomurai has caused a danger to sea bathers and fishery damages in the waters of China, Korea, and Japan. The present study investigated the cytotoxic and hemolytic activities of crude venom extract of N. nomurai using a number of in vitro assays. The jellyfish venom showed a much higher cytotoxic activity in H9C2 heart myoblast than in C2C12 skeletal myoblast (LC(50)=2 microg/mL vs. 12 microg/mL, respectively), suggesting its possible in vivo selective toxicity on cardiac tissue. This result is consistent with our previous finding that cardiovascular function is a target of the venom. In order to determine the stability of N. nomurai venom, its cytotoxicity was examined under the various temperature and pH conditions. The activity was relatively well retained at low environmental temperature (<or=20 degrees C) and dramatically lost at high temperature (>or=60 degrees C). In pH stability test, the venom has abruptly lost its activity at low pH environment (pH<or=4). Interestingly enough, however, its activity was not significantly affected even at the highest pH environment tested (pH<or=12) in the present study. Additionally, hemolytic activity of the venom was examined using the erythrocytes of cat, dog, human, rabbit and rat. Venom concentration-dependent hemolysis could be observed from 10 microg/mL of protein equivalents or higher with variable potencies in different species, among which dog erythrocyte was the most susceptible to the venom (EC(50)=151 microg/mL). SDS-PAGE analysis of N. nomurai venom showed the molecules of 20-40 kDa and 10-15 kDa appeared to be the major protein components of the venom.

Cytotoxic cytochalasins from the endozoic fungus Phoma sp. of the giant jellyfish Nemopilema nomurai

Four new cytochalasin derivatives (1-4), together with cytochalasin B (5), were isolated from the fungus Phoma sp. obtained from the giant jellyfish Nemopilema nomurai. The planar structure and relative stereochemistry were established by analysis of 1D and 2D NMR data. The absolute configuration was defined by the modified Moshers method. The compounds showed significant cytotoxicity against a small panel of human solid tumor cell lines (A549, SK-OV-3, SK-MEL-2, XF 498, and HCT15) with IC(50) values in the range of 0.5-30 μM. The cytochalasin B (5) showed obvious cytotoxicity with IC(50) of 7.9 μM against HeLa human cervical carcinoma cells.

protective effect of tetracycline against dermal toxicity induced by Jellyfish venom.

Background Previously, we have reported that most, if not all, of the Scyphozoan jellyfish venoms contain multiple components of metalloproteinases, which apparently linked to the venom toxicity. Further, it is also well known that there is a positive correlation between the inflammatory reaction of dermal tissues and their tissue metalloproteinase activity. Based on these, the use of metalloproteinase inhibitors appears to be a promising therapeutic alternative for the treatment of jellyfish envenomation. Methodology and Principal Findings Tetracycline (a metalloproteinase inhibitor) has been examined for its activity to reduce or prevent the dermal toxicity induced by Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom (NnV) using in vitro and in vivo models. HaCaT (human keratinocyte) and NIH3T3 (mouse fibroblast) incubated with NnV showed decreases in cell viability, which is associated with the inductions of metalloproteinase-2 and -9. This result suggests that the use of metalloproteinase inhibitors, such as tetracycline, may prevent the jellyfish venom-mediated local tissue damage. In vivo experiments showed that comparing with NnV-alone treatment, tetracycline pre-mixed NnV demonstrated a significantly reduced progression of dermal toxicity upon the inoculation onto rabbit skin. Conclusions/Significance It is believed that there has been no previous report on the therapeutic agent of synthetic chemical origin for the treatment of jellyfish venom-induced dermonecrosis based on understanding its mechanism of action except the use of antivenom treatment. Furthermore, the current study, for the first time, has proposed a novel mechanism-based therapeutic intervention for skin damages caused by jellyfish stings.

Abundance and distribution of Nemopilema nomurai (Scyphozoa, Rhizostomeae), in Korean waters in 2005–2013

Nemopilema nomurai have appeared massively since 2003 in Korean waters, and regular offshore monitoring has been performed since 2005 in the eastern Yellow Sea (YS), northwestern East China Sea (ECS), Korean southern waters (Ksw), and western East Sea (ES). Korea Jellyfish Monitoring Network (KoJEM) was established in 2006 for coastal jellyfish monitoring. From these, sight survey monitoring data were gathered and analyzed to extract the yearly and seasonal fluctuation of N. nomurai’s abundance and distribution. The first massive annual appearance of N. nomurai occurred in the northwestern ECS, off Shanghai, China, in mid May of 2005–2008 and of 2012–2013. In other years or concurrently with the appearance in the northwestern ECS in the same year, several individuals were found in the mid-eastern YS and Ksw, and southwestern ES. The population of N. nomurai at the northwestern ECS moved to the southeastern YS and Jeju Strait in June, and from the July to August it extended its distribution all around the Korean peninsula and persisted till October. Since October, the distribution was retracted into the southern YS and Ksw, and disappeared slowly. N. nomurai’s yearly abundance varied a lot, from nearly 0 inds 10−4m−2 in 2010 to 62.4 inds 10−4m−2 in 2005. The highest abundance was recorded in 2005, followed by 2007 and 2009, and the least abundance was in 2008 and 2010–2013. The results were compared with those of the Liaoning Bay, Bohai Sea, which led to speculation about a new seeding place of N. nomurai, and mortality during the early planktonic phase of N. nomurai was proposed as the determinant of the yearly variation in abundance.

Proteomics approach to examine the cardiotoxic effects of Nemopilema nomurai Jellyfish venom

UNLABELLED Nemopilema nomurai is one of the largest species of jellyfish in the world. It blooms mainly offshore of Korea, China, and Japan. Increasing population numbers of N. nomurai is increasing the risk of sea bathers to the jellyfish stings and accompanying envenomations. Cardiovascular effects, and cytotoxicity and hemolytic activities have been previously reported in rodent models. To understand the mechanism of cardiac toxicity, we examined the effect of N. nomurai jellyfish venom (NnV) at the proteome level on rat cardiomyocytes cell line H9c2 using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Cells treated with NnV displayed dose-dependent inhibition of viability. Cellular changes at proteome level were investigated after 6h and 12h of venom treatment. Electrophoretic examination revealed 72 protein spots displaying significant quantitative changes. These proteins were analyzed by MALDI-TOF/MS. Thirty four differentially expressed proteins were successfully identified; 24 proteins increased in quantity and 10 proteins decreased, compared to the respective controls. Proteins altered in content in Western blot analyses included myosin VII, annexin A2, aldose reductase, suppressor of cytokine signaling 1 (SOCS1), and calumenin, which are well-known marker proteins of cardiac dysfunctions. BIOLOGICAL SIGNIFICANCE This is the first report revealing the cardiac toxicity of NnV at the proteome level. NnV directly targeted proteins involved in cardiac dysfunction or maintenance. Suppressor of cytokine signaling 1 (SOCS1), which inhibits the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, was upregulated by NnV. Other proteins related to cardiac arrest that were over-expressed included aldose reductase and calumenin. These results clarify the underlying mechanism of cardiomyocyte damage caused by NnV. By inhibiting these particular targets and more precisely identifying the components of NnV-mediated cardiac toxicity, jellyfish venom-associated poisoning could be reduced or prevented.

Target organ identification of jellyfish envenomation using systemic and integrative analyses in anesthetized dogs

Proper treatment of jellyfish envenomed patients can be successfully achieved only from an understanding of the overall functional changes and alterations in physiological parameters under its envenomation. The majority of previous investigations on jellyfish venoms have covered only a couple of parameters at a time. Unlike most other fragmentary jellyfish studies, we employed an integrative toxicological approach, including hemodynamics, clinical chemistry and hematology analyses, using N. nomurai jellyfish venom (NnV) in dogs. After the baseline measurements for mean arterial pressure (MAP), cardiac output (CO) and heart rate (HR), NnV was intravenously administered to the dogs at doses of 0.1 or 0.3mg/kg body weight. The dogs showed significant decreases in MAP (-27.4±3.7 and -48.1±9.9 mmHg), CO (-1.1±0.1 L/min and -1.0±0.2 L/min), and HR (-4.5±0.3 and -9.9±3.1 beats/min) comparing with the respective baseline controls. The onset of systemic hypotension and bradycardia occurred within 1 min of NnV injection and they lasted for 1-35 min, depending on the NnV doses. Interestingly, serum biochemical analyses of envenomed dogs exhibited dramatic increases of alkaline phosphatase (ALP), creatine phosphokinase (CPK), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), indicating its possible target organs. In conclusion, we have demonstrated simultaneously, for the first time, the multiple organ toxicities (cardiotoxic, myotoxic and hepatotoxic) of a scyphozoan jellyfish venom. Based on these results, an integrative toxinological approach using dogs appears to be effective in predicting jellyfish venom toxicities and designing their therapeutic strategies. We expect this method can be applied to other jellyfish venom research as well.

Description of feeding apparatus and mechanism innemopilema nomurai kishinouye (scyphozoa: rhizostomeae)

Feeding apparatus, mechanism and passage of ingested prey were described forNemopilema nomurai (Scyphozoa: Rhizostomeae).N. nomurai medusae without central mouths have developed complicated canal systems connecting the tip of the tentacle and oral arm to the gut cavity. The number of junctions in the canal system increases with the bell diameter. The prey is gathered by paralyzing nematocyst at the tentacles and by adhering cirri at the oral arms and scapulets. They are engulfed into the terminal pore located at the oral arms and scapulets, and entered into the gut cavity via the canal system. The estimated digestion time is 1 hour and 20 min. The diameter of terminal pore is always about 1 mm, implying that they could not eat prey larger than that pore size. On the other hand, ephyrae have central mouths and could swallow prey as large as adults could. Exploitation of the same size of food by adult and ephyra implies thatN. nomurai can affect seriously the whole food web, massively ingesting micro-and mesozoopl a n k t o n and cutting the energy transfer toward the higher level of carnivores.

Anticancer Effect of Nemopilema nomurai Jellyfish Venom on HepG2 Cells and a Tumor Xenograft Animal Model

Various kinds of animal venoms and their components have been widely studied for potential therapeutic applications. This study evaluated whether Nemopilema nomurai jellyfish venom (NnV) has anticancer activity. NnV strongly induced cytotoxicity of HepG2 cells through apoptotic cell death, as demonstrated by alterations of chromatic morphology, activation of procaspase-3, and an increase in the Bax/Bcl-2 ratio. Furthermore, NnV inhibited the phosphorylation of PI3K, PDK1, Akt, mTOR, p70S6K, and 4EBP1, whereas it enhanced the expression of p-PTEN. Interestingly, NnV also inactivated the negative feedback loops associated with Akt activation, as demonstrated by downregulation of Akt at Ser473 and mTOR at Ser2481. The anticancer effect of NnV was significant in a HepG2 xenograft mouse model, with no obvious toxicity. HepG2 cell death by NnV was inhibited by tetracycline, metalloprotease inhibitor, suggesting that metalloprotease component in NnV is closely related to the anticancer effects. This study demonstrates, for the first time, that NnV exerts highly selective cytotoxicity in HepG2 cells via dual inhibition of the Akt and mTOR signaling pathways, but not in normal cells.

Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai

An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT) and 3′ acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

Nemopilema nomurai Jellyfish venom treatment leads to alterations in rat cardiomyocytes proteome

This data article restrains data associated to the Choudhary et al. [1]. Nemopilema nomurai Jellyfish venom (NnV) can lead to cardiac toxicity. Here we analyzed the effect of NnV on rat cardiomyocytes cell line H9c2 at the proteome level using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). This analysis resulted in 34 proteins with differential expression. Here we provide the dataset for the proteins with amplified or reduced level as compare to control.