| 1. |
- Diao, Yupu, et al.
(författare)
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Existence of Prophenoloxidase in Wing Discs : A Source of Plasma Prophenoloxidase in the Silkworm, Bombyx mori
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:7, s. e41416-
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Tidskriftsartikel (refereegranskat)abstract
- In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.
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| 2. |
- Kong, Lulu, et al.
(författare)
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Thermolysin Damages Animal Life Through Degradation of Plasma Proteins Enhanced by Rapid Cleavage of Serpins and Activation of Proteases
- 2015
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Ingår i: Archives of Insect Biochemistry and Physiology. - : Wiley. - 0739-4462 .- 1520-6327. ; 88:1, s. 64-84
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Tidskriftsartikel (refereegranskat)abstract
- Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.
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| 3. |
- Lu, Anrui, et al.
(författare)
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Recombinant Drosophila prophenoloxidase 1 is sequentially cleaved by alpha-chymotrypsin during in vitro activation
- 2014
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Ingår i: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 102C, s. 154-165
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Tidskriftsartikel (refereegranskat)abstract
- Insect prophenoloxidase (PPO) is an essential innate immunity protein to induce pathogen into melanization. In Bombyx mori, pro-phenoloxidase-activating enzyme (PPAE) can directly cleave and activate PPO. However, PPO in Manduca sexta cannot be cleaved into active phenoloxidase (PO) by serine proteases unless cofactors are involved, which indicates that PPO activation is complicated. Here we use recombinant Drosophila melanogaster prophenoloxidase 1 (rPPO1) to study the mechanism of PPO activation induced by a typical serine protease, alpha-chymotrypsin. Small amounts of alpha-chymotrypsin cleave rPPO1 at the N- and C-terminus to produce a large fragment rPPO1(N1/C1) that needs further cleavage by alpha-chymotrypsin to produce a smaller fragment rPO1(60-kD) with PO activity. rPO1(60-kD) oxidizes dopamine without being affected by high temperature, or by having salt and Ethylene diamine tetraacetic acid (EDTA) in the solution. After incubation with dopamine, rPO1(60-kD) cannot be detected using reducing SDS-PAGE due to formation of a large complex. Trypsin, another typical serine protease, cleaves rPPO1 at the N- and C-terminus to produce a small fragment rPPO1(N'/C') without PO activity. Several rPPO1 mutants were created through over-expressing active fragments that have direct PO activity. They are easily cleaved by low amounts of alpha-chymotrypsin without increasing PO activity. Therefore, rPPO1 can be sequentially cleaved in at least three places by alpha-chymotrypsin to produce activated rPO1(60-kD).
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| 4. |
- Shao, Qimiao, et al.
(författare)
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Hindgut Innate Immunity and Regulation of Fecal Microbiota through Melanization in Insects
- 2012
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:17, s. 14270-14279
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Tidskriftsartikel (refereegranskat)abstract
- Many insects eat the green leaves of plants but excrete black feces in an as yet unknown mechanism. Insects cannot avoid ingesting pathogens with food that will be specifically detected by the midgut immune system. However, just as in mammals, many pathogens can still escape the insect midgut immune system and arrive in the hindgut, where they are excreted out with the feces. Here we show that the melanization of hindgut content induced by prophenoloxidase, a key enzyme that induces the production of melanin around invaders and at wound sites, is the last line of immune defense to clear bacteria before feces excretion. We used the silkworm Bombyx mori as a model and found that prophenoloxidase produced by hindgut cells is secreted into the hindgut contents. Several experiments were done to clearly demonstrate that the blackening of the insect feces was due to activated phenoloxidase, which served to regulate the number of bacteria in the hindgut. Our analysis of the silkworm hindgut prophenoloxidase discloses the natural secret of why the phytophagous insect feces is black and provides insight into hindgut innate immunity, which is still rather unclear in mammals.
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