• Molecules and Cells
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• v.26 no.6
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• pp.606-610
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• 2008

Phenoloxidase (PO), a melanin-forming enzyme around the foreign bodies, is an important component of the host defense system in invertebrates. Pro-PO is the enzymatically inactive zymogen form of PO. In the Drosophila genome, three Pro-PO isoforms have been identified to date. These include Pro-PO1 and 2, which are primarily expressed in crystal cells, and Pro-PO3, which is predominantly found in the lamellocytes. In this study, we demonstrated that Drosophila Pro-PO3, but not Pro-PO1 or 2, is enzymatically active in its zymogen form. These findings were evidenced by spectacular melanin forming capacities of various cells and tissues that overexpressed these pro-enzymes. Furthermore, the melanization phenotype observed in the lamellocyte-enriched $hop^{Tum-l}$ mutant was drastically reduced in the absence of PPO3, indicating that PPO3 plays a major role in the lamellocyte-mediated spontaneous melanization process. Taken together, these findings indicate that the biochemical properties, activation mode and in vivo role of Pro-PO3 are likely distinct from those of the other two Pro-PO enzymes involved in Drosophila physiology.

• BMB Reports
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• v.34 no.1
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• pp.15-20
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• 2001

One of the innate immune reactions in invertebrates is the pro-phenoloxidase (pro-PO) activation system that is involved in the generation of superoxide, melanin synthesis, and the subsequent sequestration of foreign matter entering the hemocoel of the invertebrates. However, the molecular mechanism of this biological reaction is still obscure. To expand our understanding of the biological roles of the pro-PO activation system in invertebrates, we performed a yeast two-hybrid screening by using three regions of pro-PO as bait and a yeast two-hybrid cDNA library from Tenebrio molitor larvae as prey We isolated a novel partial cDNA clone that encodes a glycine-rich protein that interacted with the active phenoloxidase (termed phenoloxidase interacting protein, POIP). POIP consists of two domains: One is an N-terminal unique domain and the other is a C-terminal glycine-rich domain. The C-terminal glycine-rich domain showed sequential homology with those of insect antifungal proteins. Also, the yeast two-hybrid screen in a reverse orientation (using POIP as bait) yielded PO, suggesting that the PO-POIP interaction is specific. By using a 315 bP PCR fragment of the N-terminal unique region of POIP, we cloned the full-length cDNA of POIP from the Tenebruo cDNA library constructed by using E. coli injected larvae. The interaction analysis between PO, and a truncated fragment lacking the N-terminal unique region of POIP, indicated that the N-terminal unique region is necessary for interaction between PO and POIP. The expression level of the POIP mRNA is increased by bacterial injection into T. molitor larvae. This suggests that POIP might be engaged in the humoral defense reaction.

• The Korea Journal of Herbology
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• v.30 no.6
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• pp.33-38
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• 2015

Objectives : Portulaca oleracea (PO) have been used as a traditional medicine to treat inflammatory diseases in Korea. However, the anti-inflammatory effect of PO ethanol extract on lipopolysaccharide (LPS)-induced inflammation is not well-known. Therefore, this study was performed to identify the anti-inflammatory effect of PO on LPS induced inflammatory.Methods : Identification of PO was conducted by comparison with purified standards by HPLC. To measure out the cytotoxicity of PO, author performed the MTT assay. To evaluate the anti-inflammatory effects of PO, author examined the inflammatory mediators such as nitric oxide (NO) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin, (IL)-1β and IL-6) on RAW 264.7 cells. Author also examined molecular mechanisms such as mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-κB) activation by western blot.Results : Three major components (peaks 1, 2, 3) were detected in both varieties and peak 1 was characterized as caffeic acid, peak 2 as p-coumaric acid, and peak 3 as ferulic acid by comparison of chromatographic properties with authentic standards. Extract from PO itself did not have any cytotoxic effect in RAW 264.7 cells. PO inhibited LPS-induced productions of inflammatory mediators such as NO and pro-inflammatory cytokines in RAW 264.7cells. In addition, PO inhibited the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun NH₂-terminal kinase (JNK) and NF-κB activation in RAW 264.7 cells.Conclusions : Above experiment data can be an important indicator for the identification of PO and this study suggest that treatment of PO could reduce the LPS-induced inflammation. Thereby, PO could be used as a protective agent against inflammation.

• BMB Reports
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• v.46 no.5
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• pp.264-269
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• 2013

Pattern recognition receptors are known to participate in the activation of Prophenoloxidase system. In this study, a 1,3-${\beta}$-D-glucan recognition protein was detected for the first time in Antheraea pernyi larvae (Ap-${\beta}GRP$). Ap-${\beta}GRP$ was purified to 99.9% homogeneity from the hemolymph using traditional chromatographic methods. Ap-${\beta}GRP$ specifically bind 1,3-${\beta}$-D-glucan and yeast, but not E. coli or M. luteus. The 1,3-${\beta}$-D-glucan dependent phenoloxidase (PO) activity of the hemolymph inhibited by anti-Ap-${\beta}GRP$ antibody could be recovered by addition of purified Ap-${\beta}GRP$. These results demonstrate that Ap-${\beta}GRP$ acts as a biosensor of 1,3-${\beta}$-Dglucan to trigger the Prophenoloxidase system. A trace mount of 1,3-${\beta}$-D-glucan or Ap-${\beta}GRP$ alone was unable to trigger the proPO system, but they both did. Ap-${\beta}GRP$ was specifically degraded following the activation of proPO with 1,3-${\beta}$-Dglucan. These results indicate the variation in the amount of Ap-${\beta}GRP$ after specific immune challenge in A. pernyi hemolymph is an important regulation mechanism to immune response.

• BMB Reports
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• v.46 no.7
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• pp.358-363
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• 2013

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein.

• Korean journal of applied entomology
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• v.42 no.4
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• pp.353-360
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• 2003

Immunodepression can be required for entomopathogenic bacteria to induce their potent pathogenicities to the target insects. Here, we raise a hypothesis that the capacity of a pathogenic bacterium to induce the target insect immunodepression has positive relationship with the degree of pathogenicity. X. nematophilus had 1,200 times as potent as another entomopathogenic bacterium, Staphylococcus gallinarum against the fifth instar larvae of silkworm, Bombyx mori, when they were Injected into the hemocoel. Although both bacteria had significant cytotokic effect on the hemocytes of B. mori, X. nematophilus gave faster and greater cytotoxicity than did S. gallinarum. In cellular immune reactions, B. mori could form 20 hemocyte nodules against the bacterial injection with 5${\times}$10$\^$5/ cells. The number of the hemocyte nodules was significantly depressed when live X. nematophilus was inject-ed, but not in S. gallinarum. Activation of prophenoloxidase (proPO) was depressed in the bacterial injection. The depression of PO activation was significantly greater in X. nematophilus infection than in S. gallinarum injection. Lysozyme activity was induced by the injection of S. gallinarum at 4 h after the treatment, but not induced in X. nematophilus at all the time. These results showed that X. nemato-philus induced greater immunodepression against B. mori and resulted in higher pathogenicity than did S. gallinarum. Therefore, this study suggests that the immunodepression induced by entomopathogenic bacteria has positive relationship with their pathogenicity.