| 1. |
- Agianian, Bogos, et al.
(författare)
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Preliminary characterization of hemolymph coagulation in Anopheles gambiae larvae
- 2007
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 31:9, s. 879-888
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Tidskriftsartikel (refereegranskat)abstract
- Hemolymph coagulation is a first response to injury, impeding infection, and ending bleeding. Little is known about its molecular basis in insects, but clotting factors have been identified in the fruit fly Drosophila melanogaster. Here, we have begun to study coagulation in the aquatic larvae of the malaria vector mosquito Anopheles gambiae using methods developed for Drosophila. A delicate clot was seen by light microscopy, and pullout and proteomic analysis identified phenoloxidase and apolipophorin-I as major candidate clotting factors. Electron microscopic analysis confirmed clot formation and revealed it contains fine molecular sheets, most likely a result of lipophorin assembly. Phenoloxidase appears to be more critical in clot formation in Anopheles than in Drosophila. The Anopheles larval clot thus differs in formation, structure, and composition from the clot in Drosophila, confirming the need to study coagulation in different insect species to learn more about its evolution and adaptation to different lifestyles.
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| 2. |
- Bidla, Gawa, et al.
(författare)
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Activation of Insect Phenoloxidase after Injury : Endogenous versus Foreign Elicitors
- 2009
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 1:4, s. 301-308
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme phenoloxidase (PO) is one of the first immune molecules that was identified in invertebrates. Recently, the immune function of PO has been challenged. We tested how PO is activated following injury in 2 insects, i.e. the fruit fly Drosophila melanogaster and the wax moth Galleria mellonella. Rapid PO activation in Drosophila was limited to discrete areas of the hemolymph clot which forms after injury. Surprisingly, unlike systemic PO activation during bacterial sepsis, clot melanization was not sensitive to microbial elicitors in our assay. Instead, Drosophila clot melanization was activated by endogenous signals such as apoptotic cells and was superinduced by phosphatidylserine, a negatively charged phospholipid normally found on the inner surface of the plasma membrane and exposed during apoptosis. In contrast, melanization in G. mellonella hemolymph was stronger and more uniform and was sensitive to peptidoglycan. This shows that both exogenous and endogenous signals can trigger the same immune mechanism in species and context-dependent ways. Our findings have implications for the evolutionary dynamics of immune mechanisms and are in agreement with recent comparisons of insect immune transcriptomes.
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| 3. |
- Bidla, Gawa, et al.
(författare)
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Crystal cell rupture after injury in Drosophila requires the JNK pathway, small GTPases and the TNF homolog eiger
- 2007
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 120:7, s. 1209-1215
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Tidskriftsartikel (refereegranskat)abstract
- The prophenoloxidase-activating cascade is a key component of arthropod immunity. Drosophila prophenoloxidase is stored in crystal cells, a specialized class of blood cells from which it is released through cell rupture. Within minutes after bleeding, prophenoloxidase is activated leading to visible melanization of the clot matrix. Using crystal cell rupture and melanization as readouts to screen mutants in signal transduction pathways, we show that prophenoloxidase release requires Jun N-terminal kinase, small Rho GTPases and Eiger, the Drosophila homolog of tumor necrosis factor. We also provide evidence that in addition to microbial products, endogenous signals from dying hemocytes contribute to triggering and/or assembly of the prophenoloxidase-activating cascade, and that this process can be inhibited in vitro and in vivo using the viral apoptotic inhibitor p35. Our results provide a more comprehensive view of immune signal transduction pathways, with implications for immune reactions where cell death is used as a terminal mode of cell activation.
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| 4. |
- Bidla, Gawa, et al.
(författare)
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Hemolymph coagulation and phenoloxidase in Drosophila larvae
- 2005
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 29:8, s. 669-679
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Tidskriftsartikel (refereegranskat)abstract
- Hemolymph coagulation is a first response to wounding in insects. Although studies have been performed in large-bodied insects such as the moth Galleria mellonella, less is known about clotting in Drosophila melanogaster, the insect most useful for genetic and molecular analyses of innate immunity. Here we show the similarities between clots in Drosophila and Galleria by light- and electron microscopy. Phenoloxidase changes the Drosophila clot's physical properties through cross-linking and melanization, but it is not necessary for preliminary soft clot formation. Bacteria associate with the clot, but this alone does not necessarily kill them. The stage is now set for rapid advances in our understanding of insect hemolymph coagulation, its roles in immune defense and wound healing, and for a more comprehensive grasp of the insect immune system in general.
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| 5. |
- Dushay, Mitchell S.
(författare)
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Insect hemolymph clotting
- 2009
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 66:16, s. 2643-2650
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Forskningsöversikt (refereegranskat)abstract
- The clot's appearance in different large-bodied insects has been described, but until recently, little was known about any insect clot's molecular makeup, and few experiments could directly test its function. Techniques have been developed in Drosophila (fruit fly) larvae to identify clotting factors that can then be tested for effects on hemostasis, healing, and immunity. This has revealed unanticipated complexity in the hemostatic mechanisms in these larvae. While the clot's molecular structure is not yet fully understood, progress is being made, and the loss of clotting factors has been shown to cause subtle immune defects. The few similarities between coagulation in different insect species and life stages, and the current state of knowledge about coagulation in insects are discussed.
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| 6. |
- Ekengren, Sophia, et al.
(författare)
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A humoral stress response in Drosophila.
- 2001
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Ingår i: Curr Biol. - 0960-9822. ; 11:9, s. 714-8
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Tidskriftsartikel (refereegranskat)abstract
- The ability to react to unfavorable environmental changes is crucial for survival and reproduction, and several adaptive responses to stress have been conserved during evolution [1-3]. Specific immune and heat shock responses mediate the elimination of invading pathogens and of damaged proteins or cells [4-6]. Furthermore, MAP kinases and other signaling factors mediate cellular responses to a very broad range of environmental insults [7-9]. Here we describe a novel systemic response to stress in Drosophila. The Turandot A (TotA) gene encodes a humoral factor, which is secreted from the fat body and accumulates in the body fluids. TotA is strongly induced upon bacterial challenge, as well as by other types of stress such as high temperature, mechanical pressure, dehydration, UV irradiation, and oxidative agents. It is also upregulated during metamorphosis and at high age. Strikingly, flies that overexpress TotA show prolonged survival and retain normal activity at otherwise lethal temperatures. Although TotA is only induced by severe stress, it responds to a much wider range of stimuli than heat shock genes such as hsp70 or immune genes such as Cecropin A1.
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| 7. |
- Hedengren, Marika, et al.
(författare)
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Relish, a central factor in the control of humoral but not cellular immunity in Drosophila.
- 1999
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Ingår i: Mol Cell. - : Elsevier. - 1097-2765. ; 4:5, s. 827-37
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Tidskriftsartikel (refereegranskat)abstract
- The NF-kappa B-like Relish gene is complex, with four transcripts that are all located within an intron of the Nmdmc gene. Using deletion mutants, we show that Relish is specifically required for the induction of the humoral immune response, including both antibacterial and antifungal peptides. As a result, the Relish mutants are very sensitive to infection. A single cell of E. cloacae is sufficient to kill a mutant fly, and the mutants show increased susceptibility to fungal infection. In contrast, the blood cell population, the hematopoietic organs, and the phagocytic, encapsulation, and melanization responses are normal. Our results illustrate the importance of the humoral response in Drosophila immunity and demonstrate that Relish plays a key role in this response.
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| 8. |
- Karlsson, C, et al.
(författare)
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Proteomic analysis of the Drosophila larval hemolymph clot
- 2004
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:50, s. 52033-52041
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Tidskriftsartikel (refereegranskat)abstract
- Components of the insect clot, an extremely rapid forming and critical part of insect immunity, are just beginning to be identified (1). Here we present a proteomic comparison of larval hemolymph before and after clotting to learn more about this process. This approach was supplemented by the identification of substrates for the enzyme transglutaminase, which plays a role in both vertebrate blood clotting (as factor XIIIa) and hemolymph coagulation in arthropods. Hemolymph proteins present in lower amounts after clotting include CG8502 (a protein with a mucin-type domain and a domain with similarity to cuticular components), CG11313 (a protein with similarity to prophenoloxidase-activating proteases), and two phenoloxidases, lipophorin, a secreted gelsolin, and CG15825, which had previously been isolated from clots (2). Proteins whose levels increase after clotting include a ferritin-subunit and two members of the immunoglobulin family with a high similarity to the small immunoglobulin-like molecules involved in mammalian innate immunity. Our results correlate with findings from another study of coagulation (2) that involved a different experimental approach. Proteomics allows the isolation of novel candidate clotting factors, leading to a more complete picture of clotting. In addition, our two-dimensional protein map of cell-free Drosophila hemolymph includes many additional proteins that were not found in studies performed on whole hemolymph.
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| 9. |
- Korayem, Ahmed, et al.
(författare)
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Evidence for an immune function of lepidopteran silk proteins
- 2007
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 352:2, s. 317-322
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Tidskriftsartikel (refereegranskat)abstract
- Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution.
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| 10. |
- Korayem, A M, et al.
(författare)
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A Drosophila salivary gland mucin is also expressed in immune tissues : evidence for a function in coagulation and the entrapment of bacteria
- 2004
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Ingår i: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 0965-1748 .- 1879-0240. ; 34:12, s. 1297-1304
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Tidskriftsartikel (refereegranskat)abstract
- Our studies on the developmental regulation of glycosylation in Drosophila melanogaster led us to identify and characterize gp 150, an ecdysone-regulated mucin that is found in hemocytes, the gut (peritrophic membrane) and in the salivary glands. We are particularly interested in mucin immune functions and found that gp 150 is released from larval hemocytes, becomes part of the clot and participates in the entrapment of bacteria. By RT-PCR and RNAi experiments, we identified gp 150 as the previously described 171-7, an ecdysone-induced salivary glue protein. We discuss the evolutionary and biochemical implications of the dual use of salivary proteins for immune functions in insects. Further molecular characterization of such shared proteins may enable a better understanding of the properties of proteins involved in containment and elimination of microbes, as well as hemostasis and wound repair.
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| 11. |
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| 12. |
- Lesch, Christine, et al.
(författare)
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A role for Hemolectin in coagulation and immunity in Drosophila melanogaster
- 2007
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 31:12, s. 1255-1263
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Tidskriftsartikel (refereegranskat)abstract
- Hemolectin has been identified as a candidate clotting factor in Drosophila. We reassessed the domain structure of Hemolectin (Hml) and propose that instead of C-type lectin domains, the two discoidin domains are most likely responsible for the protein's lectin activity. We also tested Hml's role in coagulation and immunity in Drosophila. Here we describe the isolation of a new hml allele in a forward screen for coagulation mutants, and our characterization of this and two other hml alleles, one of which is a functional null. While loss of Hml had strong effects on larval hemolymph coagulation ex vivo, mutant larvae survived wounding. Drosophila thus possesses redundant hemostatic mechanisms. We also found that loss of Hml in immune-handicapped adults rendered them more sensitive to Gram(-) bacteria infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.
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| 13. |
- Lindgren, Malin, et al.
(författare)
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Fondue and transglutaminase in the Drosophila larval clot
- 2008
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Ingår i: Journal of insect physiology. - : Elsevier BV. - 0022-1910 .- 1879-1611. ; 54:3, s. 586-592
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Tidskriftsartikel (refereegranskat)abstract
- Hemolymph coagulation is vital for larval hemostasis and important in immunity, yet the molecular basis of coagulation is not well understood in insects. Of the larval clotting factors identified in Drosophila, Fondue is not conserved in other insects, but is notable for its effects on the clot's physical properties, a possible function in the cuticle, and for being a substrate of transglutaminase. Transglutaminase is the only mammalian clotting factor found in Drosophila, and as it acts in coagulation in other invertebrates, it is also likely to be important in clotting in Drosophila. Here we describe a Fondue-GFP fusion construct that labels the cuticle and clot, and show that chemical inhibition and RNAi knockdown of the Drosophila transglutaminase gene affect clot properties and composition in ways similar to knockdown of the fon gene. Thus, Fondue appears to be incorporated into the cuticle and is a key transglutaminase substrate in the clot. This is also the first direct functional confirmation that transglutaminase acts in coagulation in Drosophila.
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| 14. |
- Markovic, Maja Pavlovic, et al.
(författare)
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Drosophila lamin mutations cause melanotic mass formation and lamellocyte differentiation
- 2009
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Ingår i: Molecular Immunology. - : Elsevier BV. - 0161-5890 .- 1872-9142. ; 46:16, s. 3245-3250
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Tidskriftsartikel (refereegranskat)abstract
- The fruit fly immune system is a valuable model for invertebrate and innate immunity. Cellular immune reactions in Drosophila are of great interest, especially the molecular genetic mechanisms of hemocyte differentiation and the encapsulation of foreign bodies. Here we report that changes in the lamin gene cause melanotic masses. These darkened clusters of cells result from autoimmune-like encapsulation of self-tissue, as shown by the presence in lam larvae of lamellocytes, effector hemocytes that appear in larvae following wounding or parasitization. Lamins thus affect immunity in Drosophila, and lam mutations can serve as genetic tools to dissect cellular immune signaling and effector pathways.
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| 15. |
- Munoz-Alarcon, Andres, et al.
(författare)
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Characterization of lamin mutation phenotypes in Drosophila and comparison to human laminopathies
- 2007
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 2:6, s. e532-
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Tidskriftsartikel (refereegranskat)abstract
- Lamins are intermediate filament proteins that make up the nuclear lamina, a matrix underlying the nuclear membrane in all metazoan cells that is important for nuclear form and function. Vertebrate A-type lamins are expressed in differentiating cells, while B-type lamins are expressed ubiquitously. Drosophila has two lamin genes that are expressed in A- and B-type patterns, and it is assumed that similarly expressed lamins perform similar functions. However, Drosophila and vertebrate lamins are not orthologous, and their expression patterns evolved independently. It is therefore of interest to examine the effects of mutations in lamin genes. Mutations in the mammalian lamin A/C gene cause a range of diseases, collectively called laminopathies, that include muscular dystrophies and premature aging disorders. We compared the sequences of lamin genes from different species, and we have characterized larval and adult phenotypes in Drosophila bearing mutations in the lam gene that is expressed in the B-type pattern. Larvae move less and show subtle muscle defects, and surviving lam adults are flightless and walk like aged wild-type flies, suggesting that lam phenotypes might result from neuromuscular defects, premature aging, or both. The resemblance of Drosophila lam phenotypes to human laminopathies suggests that some lamin functions may be performed by differently expressed genes in flies and mammals. Such still-unknown functions thus would not be dependent on lamin gene expression pattern, suggesting the presence of other lamin functions that are expression dependent. Our results illustrate a complex interplay between lamin gene expression and function through evolution.
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| 16. |
- Scherfer, C, et al.
(författare)
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Isolation and characterization of hemolymph clotting factors in Drosophila melanogaster by a pullout method
- 2004
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Ingår i: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 14:7, s. 625-629
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Tidskriftsartikel (refereegranskat)abstract
- Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates [1]. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly [2, 3]. Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads. The validity of the assay was demonstrated by characterization of mutants. We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria. We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris. Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins. Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) [4,5] that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin [6], and we confirm that hemolectin mutant larvae show clotting defects.
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| 17. |
- Scherfer, Christoph, et al.
(författare)
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The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation
- 2006
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Ingår i: Developmental Biology. - : Elsevier BV. - 0012-1606 .- 1095-564X. ; 295, s. 156-163
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Tidskriftsartikel (refereegranskat)abstract
- Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. Jon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.
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| 18. |
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| 19. |
- Theopold, Ulrich, et al.
(författare)
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Mechanisms of Drosophila Immunity : An Innate Immune System at Work
- 2008
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Ingår i: Current Immunology Rewiews. - 1573-3955. ; 3:4, s. 276-288
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Forskningsöversikt (refereegranskat)abstract
- Insect immune systems which lack the type of adaptive immunity known in vertebrates rely on several mechanisms including solid barriers against the environment, rapid coagulation of hemolymph after wounding, the formation of aggregates that immobilize and kill foreign invaders, phagocytosis, and the production of antimicrobial peptides. The mode of action and the regulation of the expression of antimicrobial peptides have been studied intensively for more than three decades and are now increasingly well understood. In addition, the characterization of several key molecules involved in other branches of insect immunity has led to a deeper and much more comprehensive understanding of innate immunity in insects. Here we focus on the current status of our view of immunity in the vinegar fly Drosophila melanogaster, the best characterized insect model. We also discuss how evolutionary and ecological forces may have shaped immune responses in Drosophila as compared to other insect species. Finally, several infection models reveal finely-tuned and pathogen-dependent interactions between Drosophila immunity and fly physiology.
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| 20. |
- Theopold, Ulrich, et al.
(författare)
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The Drosophila clotting system and its messages for mammals
- 2014
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 42:1, s. 42-46
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Tidskriftsartikel (refereegranskat)abstract
- Drosophila has been increasingly used as a model to study hemolymph clotting. Proteomics and bioinformatics identified candidate clotting-factors, several of which were tested using genetics. Mutants and lines with reduced expression of clotting-factors show subtle effects after wounding, indicating that sealing wounds may rely on redundant mechanisms. More striking effects are observed after infection, in particular when a natural infection model involving entomopathogenic nematodes is used. When translated into mammalian models these results reveal that mammalian blood clots serve a similar immune function, thus providing a new example of the usefulness of studying invertebrate models.
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