• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.315-319
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• 2003

A novel method is developed to yield virus titers in 10 h, is easy to .perform using 96-well plates, and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immune-staining. The titer is determined by counting foci produced due to infection of virus under a fluorescent microscopy. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post infection time of 4 h. Therefore, 10 hours were enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer.

• YAKHAK HOEJI
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• v.45 no.5
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• pp.484-490
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• 2001

Mylabris phalerata(MP) is an insect that has been used for the treatment of cancer in oriental medicine. To evaluate the anticancer activity of Mylabris phalerata, We measured the cytotoxicity of Mylabris phalerata solvent fractions such as MC, EA, BuOH and residual layers on U937, human monocytic leukemia cells. Of those fractions BuOH layer of Mylabris phalerata was the most effective with ID$_{50}$ of 140$\mu\textrm{g}$/ml. It effectively caused DNA fragmentation from the concentration of 50$\mu\textrm{g}$/ml, showed apoptotic nucleus by tenets assay and expressed apototic portion stained by Annexin-V. It also induced the activation of caspase-3 and cleavage of the substrate poly (ADP-ribose) polymerase (PARP). These results suggest BuOH layer of Mylabris phalerata exerts anticancer activity by induction of apoptosis via activation of caspase-3 protease.e.

• The Korean Journal of Pesticide Science
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• v.12 no.2
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• pp.184-191
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• 2008

Entomopathogenic bacteria (Xenorhabdus nematophila, X. sp. and Photorhabdus temperata subsp. temperata) isolated from entomopathogenic nematodes express potent insecticidal activity in insect hemocoel. They are also known to suppress insect immune mediation by inhibiting phospholipase $A_2$, leading to host immunosuppression. This study analyzed effects of their cultured broths on inhibiting insect immunosuppression. For this, we removed all bacterial cells using $0.2\;{\mu}m$ pore sized membrane from the bacteria-cultured broth. All three sterilized cultured media, in dose-dependent manners, significantly inhibited hemocyte-spreading behavior of 5th instar larvae of Spodoptera exigua. However, they showed differential inhibitory activities among different bacterial species, in which X. nematophila showed the most potent inhibitory activity. This immunosuppressive effect was applied to increase the pathogenicity of Bacillus thuringiensis (Bt). All three bacterial cultured broths including bacterial cells significantly potentiated Bt pathogenicity against young S. exigua larvae when each of them was orally administered in a mixture of low dose of Bt. Finally, we tested the effect of oral administration of the cultured media containing the immunosuppressive compound(s) secreted by the bacteria. The membrane-sterilized cultured broths were mixed with the low dose of Bt and then orally administered to the young S. exigua. Only the cultured medium of X. nematophila showed increase of Bt pathogenicity. These results indicated that the; cultured media of the three bacteria possessed immunosuppressive factor(s), which may act to potentiate Bt toxicity to young S. exigua larvae.

• Applied Microscopy
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• v.18 no.2
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• pp.102-118
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• 1988

This study was carried out to examine in vitro first whether the storage proteins, which the fat bodies of last larvae from Hyphantria cunea secrete into haemolymph, can be uptaked by the fat body cells of prepupa and then how the uptaked storage proteins can be accumulated in the fat body cells, if uptaken. The fat bodies which had been isolated from last instar larvae were cultured in 1 ml of Grace's insect medium containing $50{\mu}l$ of $^{3}H$-leucine (5.0 mCi/mol, Dupont) at $28{\pm}2^{\circ}C$ for 6 hrs. After the homogenates of the cultured fat bodies were centrifuged at 10,000 rpm for 10 minutes, the proteins included in the supernatant were separated by polyacrylamide gel electrophoreses (non-SDS, 6%). The next treatment of the electrophoresed gel was followed by rinsing. A storage protein band of several bands in the rinsed gel was sliced off. With elution of sliced storage protein bands in Tris-glycine buffer, the purification of radioactive storage proteins from fat bodies was finished. After the purified radioactive storage proteins were added in Grace's insect midis containing fat bodies of the prepupae, they were cultured for the randomly following minutes given as 3, 5, 7, 10, 15, 20 and 30 and for the randomly following hours given as 1, 2, 3 and 4 respectively. The double fixations of the cultured fat bodies in aldehyde and $OsO_4$, were followed by preparation of ultrathin sections from Epon-Araldite blocks through dehydration and embedding. The electron microscope autoradiographic treatment of all prepared sections were performed by the dipping method (Kim et al., 1987). The finally prepared specimens were examined with electron microscope. The fat body cells of the prepupa could be found to uptake the storage preteins of the last instar larvae, which were included in the culture medium, mostly by formation of coated vesicles. The in vitro uptake of the storage proteins actively occurred by 30 minutes after the addition of purified storage proteins in the culture medium. After culture for 7 minutes with the storage proteins, the uptaked radioactive storage proteins labelled a number of lysosomal granules. After culture for 20 minutes with the storage proteins, the radioactive storage proteins were finally incorporated and accumulated in lipid droplets and protein granules. The frequency in the fat body cell of radiolabelled lipid droplets occurs approximately 60%, while the frequency, in which the radiolabelled protein granules occurs in a fat body cell, is approximately 40%.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.65-70
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• 2002

A TGF-${\beta}1$/GFP monomeric fusion protein was cloned from pPK9A and pGFP-Cl plasmid by PCR amplification. The fusion protein was expressed in a $Bac-To-Bac^{TM}$ baculovirus expression system. A 45 kDa fusion protein was purified using an Ni-NTA column with 300 mM imidazol from a cell lysate infected with recombinant viruses for 72 h post-infection. The fusion protein cross-reacted with the commercial $TGF-{\beta}1$ polyclonal Ab as well as Ab raised against a precursor, monomeric $TGF-{\beta}1$, and GFP. The binding activity of the fusion protein with a $TGF-{\beta}1$ receptor was examined. Fluorescence was observed in Mv1Lu cells, yet not in insect cells treated with the fusion protein. No fluorescence was detected in Mv1Lu cells incubated with the fusion protein treated with Ab prior to the binding reaction, or with GFP alone, thereby indicating that the binding of the fusion protein was specific to $TGF-{\beta}1$ with a receptor.

• Journal of Microbiology and Biotechnology
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• v.29 no.11
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• pp.1790-1798
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• 2019

Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'-end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28℃ but weak at 30℃. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.

• The Korean Journal of Mycology
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• v.49 no.4
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• pp.477-486
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• 2021

A survey of insect-associated fungi in Korea revealed a novel fungal strain isolated from the bean bug Riptortus clavatus (Heteroptera: Alydidae). Culturally and morphologically, the fungal strain designated KNUF-20-INY03, shares features with members of the genus Acaulium. Phylogenetic analyses based on the concatenated nucleotide sequences of the internal transcribed spacer regions (ITS) regions and partial sequences of the translation elongation factor 1-alpha (TEF1-α), and β-tubulin (β-TUB), and large subunit of the nuclear ribosomal RNA (LSU) genes showed that the isolate is part of a clade that includes other Acaulium species, but it occupies a distinct phylogenetic position. Based on the shape, size, and color of its conidia and conidiogenous cells, strain KNUF-20-INY03 is readily distinguishable from the closely related A. acremonium, A. albonigrescens, A. caviariformis, A. pannemaniae, and A. retardatum. The conidial length-to-width ratio (1.6) of the novel isolate is significantly lower than that of A. acremonium (1.9), A. albonigrescens (2.4), and A. pannemaniae (2.4), and KNUF-20-INY03 produces hyaline conidia and elliptical conidiogenous cells while A. caviariformis forms brown conidia and A. retardatum produces flask-shaped conidiogenous cells. Thus, both phylogenetic and morphological analyses indicate that this strain is a novel species in the genus Acaulium, and we propose the name Acaulium microspora sp. nov.

• Biomedical Science Letters
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• v.26 no.4
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• pp.267-274
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• 2020

The larva of Protaetia brevitarsis Lewis (P. brevitarsis), edible insect, is traditionally consumed as alternative source of nutrients and has various health benefits. However, the exact pharmaceutical effects of P. brevitarsis on inflammatory response are still not well understood. Thus, we investigated the anti-inflammatory effects and mechanisms of P. brevitarsis in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. We investigated the effects of P. brevitarsis on the expression levels of inflammatory-related genes, including inflammatory cytokines, prostaglandin E2 (PGE2), cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in LPS-stimulated RAW264.7 cells. To understand the anti-inflammatory mechanism of P. brevitarsis, we explored the regulatory effect of P. brevitarsis on nuclear factor (NF)-κB and caspase-1 activation. The findings of this study demonstrated that P. brevitarsis inhibits the LPS-induced inflammatory cytokine and PGE2 levels, as well as COX-2 and iNOS expression. Moreover, we confirmed that the anti-inflammatory effect of P. brevitarsis occurs via suppression of the activation of NF-κB and caspase-1. Conclusively, these findings provide experimental evidence that P. brevitarsis may be useful candidate for the treatment of inflammatory-related diseases.

• International Journal of Industrial Entomology and Biomaterials
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• v.47 no.2
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• pp.90-98
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• 2023

The human eye, constantly exposed to solar radiation, can be damaged by UV radiation. In particular, ultraviolet B (UVB)-induced damage plays an important role in retinal degeneration and cell aging. In this study, we investigated the protective effects of the methanol extract of Oxya chinensis sinuosa (OCM), an edible insect known for its high protein content (64.2%), and various pharmacological effects, on human retinal pigment epithelial cells. ARPE-19 cells were treated with OCM and subsequently UVB irradiated. Our results showed that OCM effectively attenuates UVB-induced cell damage by reducing MAPK phosphorylation (JNK and p38 MAPK). Additionally, OCM increased the phosphorylation of Akt, and cell cycle regulators, including p21 and p27, in a dose-dependent manner. Moreover, OCM treatment increased ARPE-19 cell proliferation by activating the S6K1/S6 pathway. This study suggests that OCM prevents UVB-induced retinal cell damage by increasing cell proliferation via ROS reduction, suggesting its potential as a functional therapeutic superfood against retinal cell damage.

• Applied Microscopy
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• v.33 no.2
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• pp.131-143
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• 2003

A Differentiating pathway of hemocytes in vitro and in vivo of grasshopper, Euprepocnemis shirakii was described using light and electron microscopes. In the interior of body, the stem cells of the hemopoietic organ differentiated into six types of cells respectively which are prohemoyte, plasmatocyte, granulocyte I, granulocyte II, spherulocyte and oenocytoid. The formation of these hemocytes was derived from the stem cells surrounded by a reticular cell. Hemopoietic tissue cultured in the insect media differentiated different hemocytes, but none of them underwent any mitotic division. Morphological features of the cultured cells in media were essentially the same as those of the hemocytes differentiated from the stem cells in vivo. These results were shown that each stem cell could differentiate into different types of hemocytes. It was confirmed that the stem cells possessed the pluripotent differentiation ability to directly each hemocyte, and that the once formed hemocytes in vivo and in vitro didn t undergo further transformation to other hemocytes. The maintenance of circulating hemocytes in grasshopper had been depended on the widely spreading hemopoietic organ situated in the upper surface of the dorsal alary muscle and located on the first to eighth segments.