Cloning of Metalloproteinase 17 Genes from Oriental Giant Jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae).

We previously demonstrated that Nemopilema nomurai jellyfish venom metalloproteinases (JVMPs) play a key role in the toxicities induced by N. nomurai venom (NnV), including dermotoxicity, cytotoxicity, and lethality. In this study, we identified two full-length JVMP cDNA and genomic DNA sequences: JVMP17-1 and JVMP17-2. The full-length cDNA of JVMP17-1 and 17-2 contains 1614 and 1578 nucleotides (nt) that encode 536 and 525 amino acids, respectively. Putative peptidoglycan (PG) binding, zinc-dependent metalloproteinase, and hemopexin domains were identified. BLAST analysis of JVMP17-1 showed 42, 41, 37, and 37% identity with Hydra vulgaris, Acropora digitifera, Megachile rotundata, and Apis mellifera venom metalloproteinases, respectively. JVMP17-2 shared 38 and 36% identity with H. vulgaris and A. digitifera, respectively. Alignment results of JVMP17-1 and 17-2 with other metalloproteinases suggest that the PG domain, the tissue inhibitor of metalloproteinase (TIMP)-binding surfaces, active sites, and metal (ion)-binding sites are highly conserved. The present study reports the gene cloning of metalloproteinase enzymes from jellyfish species for the first time. We hope these results can expand our knowledge of metalloproteinase components and their roles in the pathogenesis of jellyfish envenomation.

Proteomic Investigation to Identify Anticancer Targets of Nemopilema nomurai Jellyfish Venom in Human Hepatocarcinoma HepG2 Cells.

Nemopilema nomurai is a giant jellyfish that blooms in East Asian seas. Recently, N. nomurai venom (NnV) was characterized from a toxicological and pharmacological point of view. A mild dose of NnV inhibits the growth of various kinds of cancer cells, mainly hepatic cancer cells. The present study aims to identify the potential therapeutic targets and mechanism of NnV in the growth inhibition of cancer cells. Human hepatocellular carcinoma (HepG2) cells were treated with NnV, and its proteome was analyzed using two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF/MS). The quantity of twenty four proteins in NnV-treated HepG2 cells varied compared to non-treated control cells. Among them, the amounts of fourteen proteins decreased and ten proteins showed elevated levels. We also found that the amounts of several cancer biomarkers and oncoproteins, which usually increase in various types of cancer cells, decreased after NnV treatment. The representative proteins included proliferating cell nuclear antigen (PCNA), glucose-regulated protein 78 (GRP78), glucose-6-phosphate dehydrogenase (G6PD), elongation factor 1γ (EF1γ), nucleolar and spindle-associated protein (NuSAP), and activator of 90 kDa heat shock protein ATPase homolog 1 (AHSA1). Western blotting also confirmed altered levels of PCNA, GRP78, and G6PD in NnV-treated HepG2 cells. In summary, the proteomic approach explains the mode of action of NnV as an anticancer agent. Further characterization of NnV may help to unveil novel therapeutic agents in cancer treatment.

The Hair Growth-Promoting Effect of Rumex japonicus Houtt. Extract.

Rumex japonicus Houtt. is traditionally used as a medicinal plant to treat patients suffering from skin disease in Korea. However, the beneficial effect of Rumex japonicus Houtt. on hair growth has not been thoroughly examined. Therefore, the present study aims to investigate the hair growth-promoting effect of Rumex japonicus (RJ) Houtt. root extract using human dermal papilla cells (DPCs), HaCaT cells, and C57BL/6 mice model. RJ induced antiapoptotic and proliferative effects on DPCs and HaCaT cells by increasing Bcl-2/Bax ratio and activating cellular proliferation-related proteins, ERK and Akt. RJ also increased ß-catenin via the inhibition of GSK-3ß. In C57BL/6 mice model, RJ promoted the anagen induction and maintained its period. Immunohistochemistry analysis demonstrated that RJ upregulated Ki-67 and ß-catenin expressions, suggesting that the hair growth effect of RJ may be mediated through the reinforcement of hair cell proliferation. These results provided important insights for the possible mechanism of action of RJ and its potential as therapeutic agent to promote hair growth.

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 His(69), Asp(117), and Ser(216). 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.

Evaluation of Phototoxic and Skin Sensitization Potentials of PLA 2 -Free Bee Venom.

Bee venom (BV) from honey bee (Apis mellifera L.) has been used in oriental medicine and cosmetic ingredients because of its diverse pharmacological activities. In many studies, among BV components, phospholipase A2 (PLA2) is known as a major player in BV-induced allergic reaction. Therefore, we removed PLA2 from BV using ultrafiltration and then investigated in vitro phototoxicity and in vivo skin sensitization of PLA2-free BV (PBV) in comparison with regular BV. The 3T3 neutral red uptake phototoxicity assay can be appropriated to identify the phototoxic effect of a test substance upon the exposure of ultraviolet A. Chlorpromazine, a positive control, showed high levels of photoirritation factor and mean photo effect values, while BV and PBV had less of these values. Local lymph node assay is an alternative method to evaluate skin sensitization potential of chemicals. BALB/c mice were treated with p-phenylenediamine (PPD, positive control), BV, or PBV. In all of PPD concentrations, stimulation indexes (SI) as sensitizing potential of chemicals were ≥1.6, determined to be sensitizer, while SI levels of BV and PBV were below 1.6. Thus, based on these findings, we propose that both BV and PBV are nonphototoxic compounds and nonsensitizers.

Improved Therapeutic Profiles of PLA2-Free Bee Venom Prepared by Ultrafiltration Method.

Bee venom (BV) has long been used in traditional Eastern and Western medicine for chronic inflammation, pain and skin therapy. Human exposure to BV, however, often causes unwanted adverse effects and is even fatal in some cases. Phospholipase A2 (PLA2) of BV is now suspected to play a key role in these adverse effects. We investigated the potential use of PLA2-free bee venom (PBV) as a replacement for BV in cosmetic products. PBV prepared by molecular weight cut-off ultrafiltration exhibits a superior profile in comparison with regular BV, by inhibiting elastase activity and suppressing the induction of nitric oxide (NO) and metalloproteinase-9 (MMP-9), while retaining the effects of cell proliferation and protection against ultraviolet B (UVB)-induced damage in human dermal fibroblast cells. PBV thus appears to be more promising than BV as a cosmetic ingredient with a reduced potential for adverse reactions in the recipient.