• Molecules and Cells
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• 제26권6호
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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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• 제34권1호
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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.

• 대한본초학회지
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• 제30권6호
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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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• 제46권5호
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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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• 제46권7호
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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.

• 한국응용곤충학회지
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• 제42권4호
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• pp.353-360
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• 2003

곤충병원세균이 효과적으로 병원력을 발휘하는 데 대상 곤충의 면역저하가 요구될 수 있다. 본 연구는 대상곤충의 면역저하가 병원세균의 살충능력에 직접 연관된다는 가설을 세웠다. 곤충병원세균을 5령 누에(Bombyx mori)의 혈강에 접종하였을 때, X. nematophilus는 다른 곤충병원세균인 Staphyiococcus gallinarum 보다 1,200배 높은 병원력을 보였다. 비록, 두 곤충병원세균은 누에의 혈구세포에 대해서 독성효과를 가지고 있지만, X. nematophiius는 S. gallinarum과 비교해보다 빠르고 높은 세포독성 영향을 보였다. 세포성 면역반응에서 5${\times}$$10^{5}$농도로 세균이 접종되었을 때, 누에는 약 20개 정도의 소낭을 형성하였다. 특히, 살아있는 X. nematophilus가 접종된 누에에서 소낭 형성은 크게 감소하였지만, S. gullinarum은 소낭 형성 감소에 영향을 주지 않았다. 두 세균 모두는 prophenoloxidase (proPO)의 활성화를 억제시켰다. 그러나 X. nematophilus.가 S. gallinarum보다 높게 PO 활성화를 억제시켰다. 라이소자임의 활성유도는 S. gallinarum접종 후 4시간에서 관찰되었으나, X. nematophilus를 접종하였을 때는 라이소자임의 활성이 전혀 관찰되지 않았다 이러한 결과들은 누에에서 X. nematophilus가 S. gallinarum보다 더 큰 면역저하를 유발하여 병원성을 높였다는 것을 보여준다. 따라서 본 연구는 곤충병원세균에 의해서 유발된 면역저하는 곤충병원세균의 병원성과 연관되어 있다는 것을 제시한다.다.