Micropropagation and Shoot Tip Cryopreservation of 'Sunny Gold' Freesia.

Cryopreservation is a promising method for the long-term preservation of plant germplasm, especially for vegetatively propagated species like freesias. In this study, we investigate streamlining the cryopreservation process for 'Sunny Gold' Freesia, starting from effective in vitro initiation and proliferation using various plant growth regulator combinations. We also assess the impact of subculture on regrowth rates after cryopreservation. The shoot tips were successfully initiated in vitro after sterilization. The shoots were multiplied an average of three times in media containing N6-benzyladenine and kinetin. The regrowth rates of non-cryopreserved shoot tips excised from different subculture cycles did not differ significantly, with rates of 44% observed for plants from more than five subcultures and 47% for those from three subcultures. However, only the shoot tips excised from cultures subjected to three subculture cycles were able to recover after cryopreservation, with a regrowth rate of 31%. Our findings lay the groundwork for the development of an efficient cryopreservation protocol for freesias in the future.

Enantiomeric CopA3 dimer peptide suppresses cell viability and tumor xenograft growth of human gastric cancer cells.

The CopA3 dimer peptide is a coprisin analog that has an anticancer effect against human cancer cells in vitro. In this study, we investigated the anticancer activity of the enantiomeric CopA3 dimer peptide in human gastric cancer cell lines as well as in an in vivo tumor xenograft model. Enantiomeric CopA3 reduced gastric cancer cell viability and exhibited cytotoxicity against cancer cells. Enantiomeric CopA3-induced cell death was mediated by specific interactions with phosphatidylserine and phosphatidylcholine, membrane components that are enriched in cancer cells, in a calcein leakage assay. Moreover, acridine orange/ethidium bromide staining, flow cytometric analysis, and Western blot analysis showed that enantiomeric CopA3 induced apoptotic and necrotic gastric cancer cell death. The antitumor effect was also observed in a mouse tumor xenograft model in which intratumoral inoculation of the peptide resulted in a significant decrease in the SNU-668 gastric cancer tumor volume. In addition, periodic acid-Schiff and hematoxylin staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed apoptotic and necrotic cell death in tumor masses treated with greater than 150 µg CopA3. Collectively, these results indicate that the enantiomeric CopA3 dimer peptide induces apoptosis and necrosis of gastric cancer cells in vitro and in vivo, indicating that the peptide is a potential candidate for the treatment of gastric cancer, which is a common cause of cancer and cancer deaths worldwide.

Anticancer Activity of the Antimicrobial Peptide Scolopendrasin VII Derived from the Centipede, Scolopendra subspinipes mutilans.

Previously, we performed de novo RNA sequencing of Scolopendra subspinipes mutilans using high-throughput sequencing technology and identified several antimicrobial peptide candidates. Among them, a cationic antimicrobial peptide, scolopendrasin VII, was selected based on its physicochemical properties, such as length, charge, and isoelectric point. Here, we assessed the anticancer activities of scolopendrasin VII against U937 and Jurkat leukemia cell lines. The results showed that scolopendrasin VII decreased the viability of the leukemia cells in MTS assays. Furthermore, flow cytometric analysis and acridine orange/ethidium bromide staining revealed that scolopendrasin VII induced necrosis in the leukemia cells. Scolopendrasin VII-induced necrosis was mediated by specific interaction with phosphatidylserine, which is enriched in the membrane of cancer cells. Taken together, these data indicated that scolopendrasin VII induced necrotic cell death in leukemia cells, probably through interaction with phosphatidylserine. The results provide a useful anticancer peptide candidate and an efficient strategy for new anticancer peptide development.

Nutritional Composition and Bioactive Constituents of Artificial Culture of Ophiocordyceps longissima (Ascomycetes).

In this study, the compositions of Ophiocordyceps longissima mycelia, synnemata, and fruiting bodies were first analyzed in order to clarify its chemical basis for development as a health food or medicine. We found that the contents of crude protein, polysaccharides, and macroelements were highest in mycelia, whereas effective components, including mannitol, ergosterol, adenosine, inosine, Zn, and Se, were lowest in mycelia. Polysaccharide, mannitol, and ergosterol levels in synnemata (2.33, 4.54, and 0.66 g/100 g, respectively) were similar to those in fruiting bodies, but was significantly different from those of mycelia (4.79, 1.77, and 0.43 g/100 g). Trehalose content in fruiting bodies (2.15 g/100 g) was >4 times higher than that in synnemata (0.5 g/100 g). Adenosine content in fruiting bodies (0.024 g/100 g) was 3-4 times higher than that of synnemata, whereas inosine, cytosine, guanosine, and uridine (0.093, 0.145, 0.053, and 0.073 g/100 g) were highest in synnemata. Cu and Se were lower in mycelia (3.30 × 10⁻4 and 3.1 × 10⁻5 g/100 g) than in synnemata (1.85 × 10⁻³ and 7.2 × 10⁻5 g/100 g) and fruiting bodies (1.67 × 10⁻³ and 4.3 × 10⁻5 g/100 g). As, Hg, Cd, and Pb in mycelia were under the limit of edible fungus health standard; Pb was not found, but Cd, Hg, and As were detected in fruiting bodies. These findings suggest that O. longissima could be utilized in different culture methods according to market demand and might be a possible health food or medicinal resource.

First report of Oryctes rhinoceros nudivirus (Coleoptera: Scarabaeidae) causing severe disease in Allomyrina dichotoma in Korea.

Oryctes rhinoceros nudivirus (OrNV) has been known to cause severe disease in coconut palm rhinoceros beetle, Oryctes rhinoceros, in Southeastern Asia and is used as a biological control to reduce the pest population. Here, we report for the first time that the OrNV may have landed on Korea and may be the major pathogen for diseased larvae of Korean horn beetle, Allomyrina dichotoma. After peroral inoculation, over 60% of infected larvae perished in 6 wk. This viral disease spreads very fast in several locations throughout Korea. This threat not only makes economic loss of local farms rearing A. dichotoma larvae but also may disturb the ecosystem by transmitting to wild A. dichotoma.

Anticancer activity of CopA3 dimer peptide in human gastric cancer cells.

CopA3 is a homodimeric α-helical peptide derived from coprisin which is a defensin-like antimicrobial peptide that was identified from the dung beetle, Copris tripartitus. CopA3 has been reported to have anticancer activity against leukemia cancer cells. In the present study, we investigated the anticancer activity of CopA3 in human gastric cancer cells. CopA3 reduced cell viability and it was cytotoxic to gastric cancer cells in the MTS and LDH release assay, respectively. CopA3 was shown to induce necrotic cell death of the gastric cancer cells by flow cytometric analysis and acridine orange/ethidium bromide staining. CopA3-induced cell death was mediated by specific interactions with phosphatidylserine, a membrane component of cancer cells. Taken together, these data indicated that CopA3 mainly caused necrosis of gastric cancer cells, probably through interactions with phosphatidylserine, which suggests the potential utility of CopA3 as a cancer therapeutic.

Comparison of in vitro and in vivo phototoxicity tests with S-(-)-10,11-dihydroxyfarnesic acid methyl ester produced by Beauveria bassiana KACC46831.

Beauveria bassiana is a fungi that is well-known for demonstrating a resistance to environmental change. To confirm whether S-(-)-10,11-dihydroxyfarnesic acid methyl ester (DHFAME) produced by Beauveria bassiana KACC46831 causes phototoxicity when used for cosmetic purposes due to its anti-tyrosinase activity, we conducted in vitro and in vivo phototoxicity tests. There were no significant changes or damage observed in the compound-treated group with regards to skin phototoxicity, while 8-methoxypsoralen, which served as a positive control, induced toxic effects. The in vitro 3T3 neutral red uptake assay, an alternative assessment, was used for further confirmation of the phototoxicity. The results showed that DHFAME did not exhibit phototoxicity at the designated concentrations, with or without UV irradiation in the 3T3 cells. These results indicated that the methyl ester produced by Beauveria bassiana KACC46831 does not induce phototoxicity in the skin. Therefore, the results of the present study indicate that DHFAME shows potential for use as a cosmetic ingredient that does not cause skin phototoxicity.

S-(-)-10,11-dihydroxyfarnesoic acid methyl ester inhibits melanin synthesis in murine melanocyte cells.

The development of antimelanogenic agents is important for the prevention of serious aesthetic problems such as melasmas, freckles, age spots, and chloasmas. In the course of screening for melanin synthesis inhibitors, we found that the culture broth from an insect morphopathogenic fungus, Beauveria bassiana CS1029, exhibits potent antimelanogenic activity. We isolated and purified an active metabolite and identified it as S-(-)-10,11-dihydroxyfarnesoic acid methyl ester (dhFAME), an insect juvenile hormone. To address whether dhFAME inhibits melanin synthesis, we first measured the size of the melanin biosynthesis inhibition zone caused by dhFAME. dhFAME also showed inhibitory activity against mushroom tyrosinase in Melan-a cells. Intracellular, dose-dependent tyrosinase inhibition activity was also confirmed by zymography. In addition, we showed that dhFAME strongly inhibits melanin synthesis in Melan-a cells. Furthermore, we compared levels of TYR, TRP-1, TRP-2, MITF, and MC1R mRNA expression by reverse-transcription polymerase chain reaction and showed that treatment of Melan-a cells with 35 µM dhFAME led to an 11-fold decrease in TYR expression, a 6-fold decrease in TRP-2 expression, and a 5-fold decrease in MITF expression. Together, these results indicate that dhFAME is a potent inhibitor of melanin synthesis that can potentially be used for cosmetic biomaterial(s).

Anticancer activity of a synthetic peptide derived from harmoniasin, an antibacterial peptide from the ladybug Harmonia axyridis.

Harmoniasin is a defensin-like antimicrobial peptide identified from the ladybug Harmonia axyridis. Among the synthetic homodimer peptide analogues derived from harmoniasin, HaA4 has been found to have antibacterial activity without hemolytic activity. In this study, we investigated whether HaA4 has anticancer activity against human leukemia cell lines such as U937 and Jurkat cells. HaA4 manifested cytotoxicity and decreased the cell viability of U937 and Jurkat cells in MTS assay and LDH release assay. We found that HaA4 induced apoptotic and necrotic cell death of the leukemia cells using flow cytometric analysis, acridine orange/ethidium bromide staining and nucleosomal fragmentation of genomic DNA. Activation of caspase-7 and -9 and fragmentation of poly (ADP-ribose) polymerase was detected in the HaA4-treated leukemia cells, suggesting induction of a caspase-dependent apoptosis pathway by HaA4. Caspase-dependent apoptosis was further confirmed by reversal of the HaA4-induced viability reduction by treatment of Z-VAD-FMK, a pan-caspase inhibitor. In conclusion, HaA4 caused necrosis and caspase-dependent apoptosis in both U937 and Jurkat leukemia cells, which suggests potential utility of HaA4 as a cancer therapeutic agent.

Antimicrobial activity of the synthetic peptide scolopendrasin ii from the centipede Scolopendra subspinipes mutilans.

The centipede Scolopendra subpinipes mutilans is a medicinally important arthropod species. However, its transcriptome is not currently available and transcriptome analysis would be useful in providing insight into a molecular level approach. Hence, we performed de novo RNA sequencing of S. subpinipes mutilans using next-generation sequencing. We generated a novel peptide (scolopendrasin II) based on a SVM algorithm, and biochemically evaluated the in vitro antimicrobial activity of scolopendrasin II against various microbes. Scolopendrasin II showed antibacterial activities against gram-positive and -negative bacterial strains, including the yeast Candida albicans and antibiotic-resistant gram-negative bacteria, as determined by a radial diffusion assay and colony count assay without hemolytic activity. In addition, we confirmed that scolopendrasin II bound to the surface of bacteria through a specific interaction with lipoteichoic acid and a lipopolysaccharide, which was one of the bacterial cell-wall components. In conclusion, our results suggest that scolopendrasin II may be useful for developing peptide antibiotics.

Characterization and cDNA cloning of a defensin-like peptide, harmoniasin, from Harmonia axyridis.

We compared the mRNA expression profile of the Harmonia axyridis larvae that were either untreated or treated with LPS. The extracted mRNAs were subjected to ACP RTPCR analysis using a combination of arbitrary primers and oligo (dT) primer. Among the 47 DEGs differentially expressed, we identified a cDNA showing homology with defensin-like antibacterial peptide. The cDNA showed a putative 32-residue signal sequence and a 50-residue mature peptide named harmoniasin. We also investigated the antibacterial activity of the harmoniasin analog, which exhibited potent antibacterial activities against Gramnegative and -positive bacteria strains and it also evidenced no hemolytic activity.

CopA3 peptide from Copris tripartitus induces apoptosis in human leukemia cells via a caspase-independent pathway.

Our previous study demonstrated that CopA3, a disulfide dimer of the coprisin peptide analogue (LLCIALRKK), has antibacterial activity. In this study, we assessed whether CopA3 caused cellular toxicity in various mammalian cell lines. CopA3 selectively caused a marked decrease in cell viability in Jurkat T, U937, and AML-2 cells (human leukemia cells), but was not cytotoxic to Caki or Hela cells. Fragmentation of DNA, a marker of apoptosis, was also confirmed in the leukemia cell lines, but not in the other cells. CopA3-induced apoptosis in leukemia cells was mediated by apoptosis inducing factor (AIF), indicating induction of a caspase-independent signaling pathway.

Ovalicin ameliorates compound 48/80-induced atopic dermatitis-related symptoms.

Ovalicin, a secondary metabolite produced by an entomopathogenic fungus Metarhizium anisopliae var. anisopliae, is currently used as an efficient biological control agent against various agricultural insect pests, but not so many biological activities have been investigated. To assess whether ovalicin has potential in ameliorating atopic dermatitis-related symptoms in mice, we first sensitized skin in the dorsal neck of Balb/c mice using compound 48/80, and scrutinized whether the compound affected the atopic dermatitis-related symptoms. The results revealed that ovalicin significantly reduces scratching behavior in a concentration-dependent fashion. Moreover, the treatment inhibits the levels of the degranulation of mast cells by 65%, and levels of histamine release by 51% at a concentration of 10 µg/ml. Together, the present data strongly suggest that ovalicin elicits potential anti-atopic activities in mice.

Long-term preservation, regeneration, and cultivation of Paecilomyces tenuipes (Peck) Samson (Ascomycetes), an entomopathogenic fungus inoculated into the silkworm larva of Bombyx mori.

Paecilomyces tenuipes reportedly have anticancer and immune activities, along with various other medicinal uses. Cultured products with P. tenuipes are certified for use in food in South Korea, and processed goods containing this fungus have been developed in many countries, particularly South Korea, Japan, and China. Research on mass production technology-procured raw materials for the manufacture of P. tenuipes is very important; however, cultures of the fungus have been unstable. This study identified stable cultivation conditions, focusing on growth inhibition and revitalization. Moisture regulation and preservation of pupae inoculated with P. tenuipes were used to control growth inhibition and revitalization. When inoculated silkworm pupae were dehydrated to 4% moisture and preserved freeze-dried or at -70 degrees C, -20 degrees C, or 4 degrees C, the mycelia in their bodies were able to survive for 14 d. Inoculated silkworm pupae were rehydrated for 3 h and the mycelia within their bodies were recovered at 94.3-96.3%. Silkworm pupae at 4% moisture were able to survive for 135 d at temperatures < 4 degrees C and for 1 y after freeze-drying. Optimal conditions for synnemata induction were 25 degrees C and 100-300 1x.

Reduction of matrix metalloproteinase-9 expression by culture filtrate of Paecilomyces farinosus J3.

The aim of the present study was to investigate the anti-tumor effects of a culture filtrate of Paecilomyces farinosus J3. Various anti-tumor assays using B16 melanoma cells were carried out. Paecilomyces farinosus J3 significantly decreased the wound healing capability, invasiveness and angiogenic activity, which was confirmed by wound healing, human umbilical vein endothelial cell and invasion assays. Paecilomyces farinosus J3 strongly inhibited cell migration, tube formation and the angiogenic process in a concentration-dependent manner. Zymographic analysis also indicated a reduced expression of matrix metalloproteinase-9 (MMP-9), a 92-kDa gelatinase. Taken together, the results indicate that the anti-tumor activities of Paecilomyces farinosus J3 originate from the reduction of MMP-9 expression in B16F10 cells.

Anti-asthmatic activities in mycelial extract and culture filtrate of Cordyceps sphecocephala J201.

This study investigated the effects of mycelial extract and culture filtrate of Cordyceps sphecocephala J201 on airway hyper-responsiveness, pulmonary immune cell infiltration, and Th2 cytokine expression in animal models of asthma. After Concanavalin A (Con A) activation of mouse primary spleen cells, the IL-4 and IL-13 cytokine expression were significantly decreased in the presence of the mycelial extract and culture filtrate of Cordyceps sphecocephala J201. The asthma model was induced by sensitization to ovalbumin by intraperitoneal (i.p.) injection treatment in mice. The Cordyceps sphecocephala J201 mycelial extract was injected in order to assess the effects of anti-asthmatic activity by comparing lung cell infiltration in ovalbumin-induced asthmatic mice. The results revealed that the increased IL-4, IL-13 and IL-25 expression were controlled by the mycelial extract and culture filtrate of Cordyceps sphecocephala J201, indicating that the extracts reduced the undesirable immune responses and/or cytokine expression exhibited in asthma.

Isolation and characterization of Psacotheasin, a novel Knottin-type antimicrobial peptide, from Psacothea hilaris.

We report the isolation and characterization of a novel knottin-type antimicrobial peptide from the yellow-spotted long-horned beetle Psacothea hilaris. A cDNA encoding a 56-mer knottin-type propeptide was identified and its predicted molecular mass and pI was 5.92 kDa and 8.28, respectively. A 34-mer mature peptide was also selected and named herein as psacotheasin. The antimicrobial activity of chemically synthesized psacotheasin against human bacterial pathogens was subsequently investigated. The results showed that psacotheasin exerted potent activities against both Gram-positive and Gram-negative bacterial strains. The present study suggests that psacotheasin can be applied to develop novel therapeutic agents.

Fruit Body Formation on Silkworm by Cordyceps militaris.

Injection inoculation protocols for fruit body formation of Cordyceps militaris (C. militaris) were investigated to improve the incidence of infection in the silkworm species Bombyx mori (B. mori). Injection, with suspensions of C. militaris hyphal bodies into living silkworm pupae, was used to test for fruit body production. Use of Daeseungjam rather than Baegokjam or Keumokjam varieties of B. mori is thought to be suitable for infection by C. militaris. From mounting, nine-day-old to 11-day-old pupae showed the best incidence of infection with a 100 µL injection volume. Silkworm pupae injected with a hyphal suspension concentration of more than 2 × 10(5) colony-forming unit (cfu) recorded a greater than 96% incidence of infection. Also, fruit bodies of C. militaris were induced and produced at a light intensity between 500 and 1,000 lx.

Isolation and Characterization of a Defensin-Like Peptide (Coprisin) from the Dung Beetle, Copris tripartitus.

The antibacterial activity of immune-related peptides, identified by a differential gene expression analysis, was investigated to suggest novel antibacterial peptides. A cDNA encoding a defensin-like peptide, Coprisin, was isolated from bacteria-immunized dung beetle, Copris tripartitus, by using differential dot blot hybridization. Northern blot analysis showed that Coprisin mRNA was up-regulated from 4 hours after bacteria injection and its expression level was reached a peak at 16 hours. The deduced amino acid sequence of Coprisin was composed of 80 amino acids with a predicted molecular weight of 8.6 kDa and a pI of 8.7. The amino acid sequence of mature Coprisin was found to be 79.1% and 67.4% identical to those of defensin-like peptides of Anomala cuprea and Allomyrina dichotoma, respectively. We also investigated active sequences of Coprisin by using amino acid modification. The result showed that the 9-mer peptide, LLCIALRKK-NH(2), exhibited potent antibacterial activities against Escherichia coli and Staphylococcus aureus.

Identification and distribution of two fungal species causing sclerotial disease on mulberry fruits in Korea.

A total of 520 overwintered sclerotia were collected from surface of soil under mulberry trees in six locations in Korea during February in 2006 and 2007. The collected sclerotia were tested for their germination in vitro and identified based on their morphological characteristics. Out of all sclerotia tested, 52.3% of the sclerotia germinated and produced two types of apothecia. The two types of fungi occurred from the sclerotia at the ratio of 49.8 vs. 50.2. The fungal type with cup-shaped apothecia was identified as Ciboria shiraiana and another type of fungus with club-shaped apothecia as Scleromitrula shiraiana. Taxonomy and distribution of the two sclerotial fungi were described and discussed.