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1.
Am J Pathol ; 187(8): 1867-1878, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28606795

RESUMO

Optic gliomas are brain tumors characterized by slow growth, progressive loss of vision, and limited therapeutic options. Optic gliomas contain various amounts of myxoid matrix, which can represent most of the tumor mass. We sought to investigate biological function and protein structure of the myxoid matrix in optic gliomas to identify novel therapeutic targets. We reviewed histological features and clinical imaging properties, analyzed vasculature by immunohistochemistry and electron microscopy, and performed liquid chromatography-mass spectrometry on optic gliomas, which varied in the amount of myxoid matrix. We found that although subtypes of optic gliomas are indistinguishable on imaging, the microvascular network of pilomyxoid astrocytoma, a subtype of optic glioma with abundant myxoid matrix, is characterized by the presence of endothelium-free channels in the myxoid matrix. These tumors show normal perfusion by clinical imaging and lack histological evidence of hemorrhage organization or thrombosis. The myxoid matrix is composed predominantly of the proteoglycan versican and its linking protein, a vertebrate hyaluronan and proteoglycan link protein 1. We propose that pediatric optic gliomas can maintain blood supply without endothelial cells by using invertebrate-like channels, which we termed primitive myxoid vascularization. Enzymatic targeting of the proteoglycan versican/hyaluronan and proteoglycan link protein 1 rich myxoid matrix, which is in direct contact with circulating blood, can provide novel therapeutic avenues for optic gliomas of childhood.


Assuntos
Vasos Sanguíneos/patologia , Células Endoteliais/patologia , Neovascularização Patológica/patologia , Glioma do Nervo Óptico/patologia , Endotélio/patologia , Humanos , Glioma do Nervo Óptico/irrigação sanguínea , Estudos Retrospectivos
2.
Genetics ; 204(3): 1075-1087, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27585844

RESUMO

The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. We have made the surprising discovery that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes Thrombospondin (Tsp) and Tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. We further demonstrate that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, we propose that shared extracellular protein-protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment.


Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Músculo Esquelético/metabolismo , Tendões/metabolismo , Animais , Fatores de Coagulação Sanguínea/genética , Proteínas Sanguíneas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Hemolinfa/metabolismo , Músculo Esquelético/fisiologia , Ligação Proteica , Tendões/fisiologia , Trombospondinas/genética , Trombospondinas/metabolismo
3.
Dev Comp Immunol ; 42(1): 42-6, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23545286

RESUMO

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.


Assuntos
Transtornos da Coagulação Sanguínea/imunologia , Proteínas Sanguíneas/imunologia , Proteínas de Drosophila/imunologia , Drosophila melanogaster/imunologia , Hemolinfa/imunologia , Animais , Coagulação Sanguínea/genética , Coagulação Sanguínea/imunologia , Transtornos da Coagulação Sanguínea/genética , Biologia Computacional/métodos , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Humanos , Mamíferos , Proteômica/métodos
4.
J Insect Physiol ; 58(10): 1376-81, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22885376

RESUMO

Drosophila larval coagulation factors have been identified in vitro. Better understanding of insect hemolymph coagulation calls for experiments in vivo. We have characterized a fondue (fon) mutation and null alleles isolated by imprecise excision of a transposable element. Loss of fon was pupal lethal, but adults could be recovered by expressing the UAS::fonGFP construct of Lindgren et al. (2008). Despite their lethality, fon mutations did not affect larval survival after wounding either when tested alone or in combination with a mutation in the hemolectin clotting factor gene. This reinforces the idea of redundant hemostatic mechanisms in Drosophila larvae, and independent pleiotropic functions of the fondue protein in coagulation and a vital process in metamorphosis.


Assuntos
Proteínas Sanguíneas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Hemolinfa/fisiologia , Hemostasia , Animais , Drosophila melanogaster/genética , Feminino , Larva/fisiologia , Masculino , Mutação
5.
Dev Comp Immunol ; 36(2): 274-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21641926

RESUMO

Coagulation involving both hemocytes and humoral factors is important for insect survival and immune defense. Hemolectin is a major larval clotting factor in Drosophila, and hemolymph from hml mutants does not clot ex vivo. Yet surprisingly third instar hml larvae survived injury as well as controls. The number of hemocytes in circulation changes during larval development. Reasoning that this could affect coagulation, we studied larval survival after injury at different stages. We found that hml larvae survived less than controls when injured during the feeding stage with fewer hemocytes. This important in vivo result reinforces the role of Hemolectin in larval hemostasis. A subtle effect of hml on immunity was found in adults. Similar experiments on hml mutant larvae gave different results, but feeding stage hml larvae were differentially sensitive to infections with different strains of Serratia marcescens.


Assuntos
Proteínas de Drosophila/imunologia , Drosophila/imunologia , Lectinas/imunologia , Animais , Coagulação Sanguínea , Hemostasia , Larva/imunologia , Análise de Sobrevida
6.
Mol Immunol ; 46(16): 3245-50, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19716177

RESUMO

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.


Assuntos
Autoimunidade/genética , Diferenciação Celular/genética , Proteínas de Drosophila/genética , Reação a Corpo Estranho/genética , Hemócitos/imunologia , Laminas/genética , Mutação/imunologia , Animais , Diferenciação Celular/imunologia , Proteínas de Drosophila/imunologia , Drosophila melanogaster , Reação a Corpo Estranho/imunologia , Laminas/imunologia
7.
Cell Mol Life Sci ; 66(16): 2643-50, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19418022

RESUMO

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.


Assuntos
Coagulação Sanguínea/fisiologia , Drosophila melanogaster/fisiologia , Hemolinfa/fisiologia , Animais , Coagulação Sanguínea/genética , Coagulação Sanguínea/imunologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Hemócitos/metabolismo , Hemócitos/fisiologia , Hemolinfa/química , Hemolinfa/microbiologia , Larva/genética , Larva/fisiologia , Modelos Biológicos
8.
J Innate Immun ; 1(4): 301-8, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-20375588

RESUMO

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.


Assuntos
Drosophila melanogaster/imunologia , Hemolinfa/imunologia , Monofenol Mono-Oxigenase/imunologia , Mariposas/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Coagulação Sanguínea/imunologia , Drosophila melanogaster/enzimologia , Hemolinfa/enzimologia , Imunidade Inata/genética , Larva/enzimologia , Larva/imunologia , Melaninas/imunologia , Melaninas/metabolismo , Mariposas/enzimologia , Peptidoglicano/imunologia , Fosfatidilserinas/imunologia
9.
J Insect Physiol ; 54(3): 586-92, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18222466

RESUMO

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.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Hemolinfa/metabolismo , Transglutaminases/metabolismo , Animais , Drosophila/enzimologia , Larva/enzimologia , Larva/metabolismo
10.
PLoS One ; 2(6): e532, 2007 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-17565385

RESUMO

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.


Assuntos
Senilidade Prematura/fisiopatologia , Drosophila/genética , Lamina Tipo A/genética , Mutação/genética , Doenças Neuromusculares/fisiopatologia , Animais , Animais Geneticamente Modificados , Comportamento Animal , Sobrevivência Celular , Humanos , Lamina Tipo A/deficiência , Longevidade , Fenótipo
11.
Dev Comp Immunol ; 31(12): 1255-63, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17509683

RESUMO

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(-) bacterial infection. This demonstrates an immunological role of this clotting protein and reinforces the importance of the clot in insect immunity.


Assuntos
Proteínas de Drosophila/imunologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/imunologia , Hemolinfa/fisiologia , Lectinas/imunologia , Lectinas/metabolismo , Animais , Coagulação Sanguínea , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Genes de Insetos , Hemócitos/citologia , Hemócitos/metabolismo , Hemolinfa/imunologia , Hemostasia , Larva/genética , Larva/imunologia , Larva/fisiologia
12.
J Cell Sci ; 120(Pt 7): 1209-15, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17356067

RESUMO

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.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Hemócitos/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Animais , Apoptose , Drosophila/citologia , Drosophila/enzimologia , Drosophila/genética , Drosophila/imunologia , Ativação Enzimática , Hemolinfa/metabolismo , Imunidade Inata/genética , Modelos Biológicos , Monofenol Mono-Oxigenase/sangue
13.
Dev Comp Immunol ; 31(9): 879-88, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17287020

RESUMO

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.


Assuntos
Anopheles/crescimento & desenvolvimento , Coagulação Sanguínea , Hemolinfa , Animais , Anopheles/ultraestrutura , Drosophila melanogaster , Larva/crescimento & desenvolvimento , Larva/ultraestrutura , Microscopia Eletrônica de Transmissão
14.
Biochem Biophys Res Commun ; 352(2): 317-22, 2007 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-17126296

RESUMO

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.


Assuntos
Coagulação Sanguínea/imunologia , Hemolinfa/imunologia , Lepidópteros/imunologia , Seda/imunologia , Cicatrização/imunologia , Animais , Medicina Baseada em Evidências , Fibroínas/imunologia , Especificidade de Órgãos , Distribuição Tecidual
15.
Dev Biol ; 295(1): 156-63, 2006 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-16690050

RESUMO

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. fon 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.


Assuntos
Proteínas Sanguíneas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Hemolinfa/fisiologia , Receptores Toll-Like/metabolismo , Animais , Coagulação Sanguínea , Proteínas Sanguíneas/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Drosophila melanogaster/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Larva , Pupa , Interferência de RNA , Transdução de Sinais , Receptores Toll-Like/imunologia
16.
Dev Comp Immunol ; 29(8): 669-79, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-15854679

RESUMO

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.


Assuntos
Drosophila melanogaster/enzimologia , Drosophila melanogaster/imunologia , Hemolinfa/fisiologia , Larva/enzimologia , Larva/imunologia , Monofenol Mono-Oxigenase/metabolismo , Animais , Bactérias/imunologia , Coagulação Sanguínea , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/microbiologia , Hemolinfa/efeitos dos fármacos , Hemolinfa/imunologia , Hemolinfa/microbiologia , Imunidade Inata/imunologia , Larva/microbiologia , Microscopia Eletrônica de Varredura , Mariposas/imunologia , Mariposas/fisiologia , Neutrófilos/fisiologia
17.
Insect Biochem Mol Biol ; 34(12): 1297-304, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15544943

RESUMO

Our studies on the developmental regulation of glycosylation in Drosophila melanogaster led us to identify and characterize gp150, 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 gp150 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 gp150 as the previously described I71-7, 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.


Assuntos
Drosophila melanogaster/imunologia , Proteínas de Insetos/biossíntese , Proteínas de Insetos/fisiologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/fisiologia , Mucinas/biossíntese , Mucinas/fisiologia , Sequência de Aminoácidos , Animais , Bactérias/imunologia , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Ecdisona/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Glicosilação , Hemócitos , Hemolinfa , Imunidade Inata , Larva/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Salivares/metabolismo
18.
J Biol Chem ; 279(50): 52033-41, 2004 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-15466469

RESUMO

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.


Assuntos
Drosophila/fisiologia , Hemolinfa/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/fisiologia , Drosophila/genética , Drosophila/imunologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/fisiologia , Eletroforese em Gel Bidimensional , Genes de Insetos , Hemolinfa/imunologia , Larva/fisiologia , Dados de Sequência Molecular , Proteômica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transglutaminases/metabolismo
20.
Curr Biol ; 14(7): 625-9, 2004 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-15062105

RESUMO

Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly. 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) that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects.


Assuntos
Coagulação Sanguínea/fisiologia , Drosophila melanogaster/fisiologia , Hemolinfa/metabolismo , Animais , Fenômenos Fisiológicos Bacterianos , Western Blotting , Proteínas de Drosophila/sangue , Drosophila melanogaster/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas da Matriz Extracelular/sangue , Lectinas/sangue , Espectrometria de Massas , Microesferas , Monofenol Mono-Oxigenase
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