The main proteinases in Dermatobia hominis second and third instars larvae are serine-proteinases

Pires, F.A.; Moya-Borja, Gonzalo Efrain; Barreira, Jairo Dias; Pinho, Rosa Teixeira de; Alves, Carlos Roberto

doi:10.1016/j.vetpar.2007.01.001

Cited by 10 publications

(15 citation statements)

References 22 publications

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“…These results show that enzymatic activities detected in L1 are serine proteases of the chymotrypsin type. Similarly, the major proteases detected in larvae of other Diptera species also belong to the serine protease class (Tabouret et al 2003, Fazito do Vale et al 2007, Pires et al 2007). Regarding pH dependence, some serine protease enzymes were active at pH 5.5, while most presented an optimal activity between pH 7.5-9.5 (Fig.…”

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“…Such proteolytic mechanisms are highly regulated and are involved in a variety of physiologic processes, such as programmed cell death, stress responses (heat shock and anoxia), skeletal muscle atrophy, cellcell recognition, signal transduction and learning, morphogenesis, and photoreceptor light adaptation (Mykles 1998). Different classes of proteases such as cysteine proteases, metalloproteases and serino proteases have been described both in adults and larvae of several species of the Diptera (Han et al 1997, Rosenfeld & Vanderberg 1998, Cho et al 1999, Noriega et al 2002, Vierstraete et al 2003, Okuda et al 2005, Fazito do Vale et al 2007, Pires et al 2007, Rodrigues et al 2007). Despite the existence of information about morphology, taxonomy and ecology of this genus, as far as we know, no data has been published on the biochemical traits of O. thornax.…”

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Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

Cuervo¹,

Mesquita-Rodrigues²,

d’Avila-Levy³

et al. 2008

Mem. Inst. Oswaldo Cruz

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We report for the first time the expression of multiple protease activities in the first instar larva (L1) of the flesh fly Oxysarcodexia thornax (Walker). Zymographic analysis of homogenates from freshly obtained L1 revealed a complex proteolytic profile ranging from 21.5 to 136 kDa. Although some activities were detected at pH 3.5 and 5.5, the optimum pH for most of the proteolytic activities was between pH 7.5 and 9.5. Seven of 10 proteases were completely inactivated by phenyl-methyl sulfonyl-fluoride, suggesting that main proteases expressed by L1 belong to serine proteases class. Complete inactivation of all enzymatic activities was obtained using N-p-Tosyl-L-phenylalanine chloromethyl ketone (100 µM), a specific inhibitor of chymotrypsin-like serine proteases.Keywords: Oxysarcodexia thornax -Sarcophagidae -chymotrypsin-like serine proteasesThe genus Oxysarcodexia belongs to the Sarcophagidae family (flesh flies) whose major biological feature is the ovo-larvipary (Pape 1996). Females may deposit first instar larvae directly onto the vertebrate host at predisposing sites (causing secondary myiasis), carcasses, decomposing organic matter, and dung (Panu et al. 2000). Genus Oxysarcodexia is widely distributed in the Neotropical regions (Lopes 1946), and in Brazil, O. thornax (Walker) is found in both urban and rural areas of several states. This species is abundant in the state of Rio de Janeiro, occurring preferentially in the summer season, with the population peak between January and February (Oliveira et al. 2002).In biological systems, proteases may carry out (i) a regulatory function by activation or inactivation of specific proteins via selective proteolysis, and (ii) a nonspecific proteolytic function involved in general protein hydrolysis. Such proteolytic mechanisms are highly regulated and are involved in a variety of physiologic processes, such as programmed cell death, stress responses (heat shock and anoxia), skeletal muscle atrophy, cellcell recognition, signal transduction and learning, morphogenesis, and photoreceptor light adaptation (Mykles 1998). Different classes of proteases such as cysteine proteases, metalloproteases and serino proteases have been described both in adults and larvae of several species of the Diptera (Han et al. 1997, Rosenfeld & Vanderberg 1998, Cho et al. 1999, Noriega et al. 2002, Vierstraete et al. 2003, Okuda et al. 2005, Fazito do Vale et al. 2007, Pires et al. 2007, Rodrigues et al. 2007). Despite the existence of information about morphology, taxonomy and ecology of this genus, as far as we know, no data has been published on the biochemical traits of O. thornax. In the present study, the proteolytic profile of O. thornax first instar larvae was investigated using zymographic analysis. O. thornax adults were collected as previously described (Oliveira et al. 2002) and females were kept in a 50 ml plastic vial for 24 h at room temperature. Following death of the females, abundant live first instar larvae (L1) were released and immediately collected. Larvae were ...

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Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

Cuervo¹,

Mesquita-Rodrigues²,

d’Avila-Levy³

et al. 2008

Mem. Inst. Oswaldo Cruz

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“…Em estudos feitos com produtos E/S de D. Hominis, foram identificadas bandas protéicas que se situavam em uma faixa de distribuição entre 100 e 14 kDa (13) e, a partir de sobrenadante de macerado total de larvas de D. hominis em SDS-PAGE a 12% corado por prata, foram reveladas proteínas entre 94 a 14 kDa em larvas de segundo estádio e 63 a 21 kDa em larvas de terceiro estádio (27) , resultados que se assemelham aos valores de pesos moleculares encontrados para os produtos E/S de larvas de C. hominivorax. Estudos com produtos E/S de outros dípteros, como S. magellanica, revelaram o perfil de proteínas produzidas por larvas de terceiro estádio caracterizado por bandas de 63 kDa a 23 kDa (28) .…”

Section: Discussionunclassified

“…Estudos realizados por Cuervo et al (22) identificaram também que as principais proteases expressas por O. thornax pertencem à classe de serina proteases. Pires et al (27) detectaram atividades de cisteína e serina proteinases em larvas de segundo e terceiro estádios de D. hominis e mostraram uma maior atividade de serina proteinase quando comparada à atividade de cisteína proteinase para esta espécie.…”

Section: Discussionunclassified

CARACTERIZAÇÃO BIOQUÍMICA DO PRODUTO DE EXCREÇÃO/SECREÇÃO DE LARVAS DE Cochliomyia hominivorax (DIPTERA: CALLIPHORIDAE)

et al. 2016

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ResumoA espécie Cochliomyia hominivorax, conhecida popularmente como mosca da bicheira, é um parasita obrigatório de animais de sangue quente e sua distribuição geográfica estende-se por toda a América do Sul, excetuando-se o Chile. O parasitismo por esta mosca provoca perdas econômicas significativas e tem grande importância no Brasil. São poucos os estudos com foco nos produtos de excreção e secreção desta espécie e este trabalho teve como objetivo estudar as enzimas presentes no produto de secreção e excreção (E/S) dos três estádios larvais de C. hominivorax. O perfil de proteínas foi obtido por eletroforese em gel de poliacrilamida e a atividade proteolítica foi analisada utilizando-se gelatina, azocaseína e Na-benzoil-arginina-nitroanilida (BAPNA) como substrato. Nos produtos de E/S dos três estádios, as proteínas foram detectadas com um peso molecular aparente que variou entre 116 e 20 kDa. No ensaio de azocaseína, em diferentes faixas de pH, a maior atividade proteolítica ocorreu em pH 7,5 para todos os estádios larvais. Os ensaios foram realizados usandose estes mesmos substratos e as amostras foram tratadas com os inibidores Benzamidina, Pepstatin A, 4-(2-aminoetil)benzenosulfonil fluoreto hidrocloreto (AEBSF), N-α-tosil-L-lisina clorometil cetona (TLCK), N-α-tosil-L-fenilalanina clorometil cetona (TPCK), Ácido etilenodiamino tetra acético (EDTA), Leupeptina e Trans-epoxysuccinyl L-leucylamido-4-guanidino butano (E-64). As proteinases presentes nos produtos E/S de L1 são em sua maioria serina proteases do tipo tripsina e quimotripsina, enquanto que para os produtos E/S de L2 e L3 foi evidenciada a presença de serina proteases e aspartil proteases. Palavras-chave: enzima; miíase; mosca; parasitas. AbstractThe species Cochliomyia hominivorax, also known as screwworm fly, is an obligate parasite of warmblooded animals and its geographic range extends thoughout South America, except Chile. This fly causes significant economic losses and has great importance in Brazil. Few studies have focused on the excretion and secretion products of this species, and this research aimed to study the enzymes present in the secretion and excretion (E/S) products of the three larval instars of C. hominivorax. The E/S profile of proteins was obtained by polyacrylamide gel electrophoresis and proteolytic activity was analyzed using gelatin, azocasein and Na-benzoyl-arginine-nitroanilide as substrates. In E/S products of the three instars, proteins were detected with an apparent molecular weight

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“…Although molecules from D. hominis LSP have not been identified, our results confirm that cellular suppression occurs in mice with human bot fly myiasis. Based on the results of studies using crude extracts of D. hominis L 2 and L 3 , it has been suggested that serine- proteases occur in such larvae 23 . Our data reveal a reduction in spleen cell numbers during D. hominis myiasis.…”

Section: Discussionmentioning

confidence: 99%

Spleen cell proliferation during and after skin myiasis by human bot fly Dermatobia hominis

Gonçalves¹,

Nascimento²,

Breyner³

et al. 2009

Rev. Inst. Med. trop. S. Paulo

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SUMMARYSpleen cells from mice were examined at 5, 10, 15, 20 and 25 days post-infection (dpi) with Dermatobia hominis larva and at 5, 10, 15, 30 and 60 days post-larval emergence (dple). Cell proliferation in vitro assays were carried out with RPMI-1640 medium and larval secretory product (LSP) of D. hominis at 5, 10, 15, 20 and 25 days. When each group of mice was tested against each medium, significance was only seen for 25 dpi, with increasing order: LSP-10 d, -25 d, -5 d, -20 d, -15 d and RPMI. Significant results were also observed when each medium was tested against mice at each dpi or dple. Each dple group vs. each medium produced significant results only for 10 dple, with increasing order: LSP-5 d, -20 d, -25 d, -10 d, -15 d and RPMI. Comparative tests were also carried out between groups to refine certain observations. The LSPs were also analyzed using SDS-PAGE. The results prove that myiasis caused depletion of spleen cells, particularly under the effect of the LSP-10 and -15, but the cells tended to increase up to 60 dple. This in vitro assay may represent the real systemic immune response in the relationship LSP-D. hominis-host.

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The main proteinases in Dermatobia hominis second and third instars larvae are serine-proteinases

Cited by 10 publications

References 22 publications

Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

Serine protease activities in Oxysarcodexia thornax (Walker) (Diptera: Sarcophagidae) first instar larva

CARACTERIZAÇÃO BIOQUÍMICA DO PRODUTO DE EXCREÇÃO/SECREÇÃO DE LARVAS DE Cochliomyia hominivorax (DIPTERA: CALLIPHORIDAE)

Spleen cell proliferation during and after skin myiasis by human bot fly Dermatobia hominis

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