RESUMO
Phospholipases A2 (PLA2) comprise a superfamily of enzymes that specifically catalyze hydrolysis of the ester bond at the sn-2 position of glycerophospholipids, generating lysophospholipids and fatty acids. In Rhodnius prolixus, one of the main vectors of the Chagas's disease etiologic agent Trypanosoma cruzi, it was previously shown that lysophosphatidylcholine, a bioactive lipid, found in the insect's saliva, contributes to the inhibition of platelet aggregation, and increases the production of nitric oxide, an important vasodilator. Due to its role in potentially generating LPC, here we studied the PLA2 present in the salivary glands of R. prolixus. PLA2 activity is approximately 100 times greater in the epithelium than in the contents of salivary glands. Our study reveals the role of the RpPLA2XIIA gene in the insect feeding performance and in the fatty acids composition of phospholipids extracted from the salivary glands. Knockdown of RpPLA2XIIA significantly altered the relative amounts of palmitic, palmitoleic, oleic and linoleic acids. A short-term decrease in the expression of RpPLA2III and RpPLA2XIIA in the salivary glands of R. prolixus was evident on the third day after infection by T. cruzi. Taken together, our results contribute to the understanding of the role of PLA2 in the salivary glands of hematophagous insects and show that the parasite is capable of modulating even tissues that are not colonized by it.
Assuntos
Fosfolipases A2 , Rhodnius , Glândulas Salivares , Trypanosoma cruzi , Animais , Rhodnius/parasitologia , Rhodnius/enzimologia , Rhodnius/genética , Glândulas Salivares/parasitologia , Glândulas Salivares/enzimologia , Glândulas Salivares/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/enzimologia , Fosfolipases A2/metabolismo , Fosfolipases A2/genética , Ácidos Graxos/metabolismo , Doença de Chagas/parasitologia , Insetos Vetores/parasitologia , Insetos Vetores/enzimologiaRESUMO
Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to ß-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon ß-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.
Assuntos
Células Acinares/enzimologia , Antiportadores de Cloreto-Bicarbonato/metabolismo , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Glândulas Salivares/enzimologia , Animais , Células CHO , Antiportadores de Cloreto-Bicarbonato/química , Antiportadores de Cloreto-Bicarbonato/genética , Cricetulus , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Mutação , Fosforilação , Conformação Proteica , Glândulas Salivares/citologia , Relação Estrutura-AtividadeRESUMO
Green plants emit green leaf volatiles (GLVs) as a general damage response. These compounds act as signals for the emitter plant, neighboring plants, and even for insects in the ecosystem. However, when oral secretions from certain caterpillars are applied to wounded leaves, GLV emissions are significantly decreased or modified. We examined four caterpillar species representing two lepidopteran families for their capacity to decrease GLV emissions from Zea mays leaf tissue. We also investigated the source of the GLV modifying components in the alimentary tract of the various caterpillars. In Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), and Manduca sexta (Linnaeus) (Lepidoptera: Sphingidae), we found three distinct mechanisms to modify GLV emission: a heat-stable compound in the gut, a heat-labile enzyme in salivary gland homogenate (previously described in Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae), and an isomerase in the salivary gland homogenate, which catalyzes the conversion of (Z)-3-hexenal to (E)-2-hexenal (previously described in M. sexta). These mechanisms employed by caterpillars to suppress or modify GLV emission suggest a counteraction against the induced indirect volatile defenses of a plant and provides further insights into the ecological functions of GLVs.
Assuntos
Herbivoria , Mariposas/fisiologia , Folhas de Planta/fisiologia , Compostos Orgânicos Voláteis , Aldeídos/metabolismo , Animais , Isomerases/metabolismo , Larva/fisiologia , Glândulas Salivares/enzimologia , Zea maysRESUMO
Octopus bimaculoides is an important commercially fished species in the California Peninsula with aquaculture potential; however, to date limited information is available regarding its digestive physiology. The objective of this study was focused on biochemically characterizing the main enzymes involved in the digestion of O. bimaculoides. Optimum pH, temperature and thermostability were determined for amylases, lipases, trypsin and chymotrypsin; optimum pH and protease inhibitor effect were assessed for acidic and alkaline proteases, and the effect of divalent ions on trypsin and chymotrypsin activity was evaluated in enzymatic extracts from the digestive (DG) and salivary glands (SG) and crop gastric juices (GJ). High amylase activity was detected in GD and GJ whereas this activity is very low in other cephalopods. Salivary glands had the greatest activity in most of the enzyme groups, showing the importance of this organ in digestion. Optimum pH was different depending on the organ and enzyme analyzed. The optimum pH in DG was 3 showing the predominance of acidic proteases in the digestion process. All enzymes were resistant and stable at high temperatures in contrast with other marine species. Trypsin and chymotrypsin activity were highly incremented with the presence of Mg2+, Co2+, Cu2+ and Zn2+ in some tissues. The inhibitor assay showed the importance of serine proteases, metalloproteases and aspartic proteases in the digestive process of this species. This study is the first in assessing the main digestive enzymes of O. bimaculoides and in remarking the importance of other digestive enzyme groups besides proteases in octopuses.
Assuntos
Amilases/metabolismo , Quimotripsina/metabolismo , Lipase/metabolismo , Octopodiformes/metabolismo , Tripsina/metabolismo , Animais , Suco Gástrico/enzimologia , Glândulas Salivares/enzimologiaRESUMO
Background: The salivary glands of Lucilia sericata are the first organs to express specific endopeptidase enzymes. These enzymes play a central role in wound healing, and they have potential to be used therapeutically. Methods: Rapid amplification of cDNA ends and rapid amplification of genomic ends were used to identify the coding sequence of MMP-1 from L. sericata. Different segments of MMP1 gene, namely the middle part, 3' end, and 5' end, were cloned, sequenced, and analyzed using bioinformatics tools to determine the distinct features of MMP-1 protein. Results: Assembling the different segments revealed that the complete mRNA sequence of MMP-1 is 1932 bp long. CDS is 1212 bp long and is responsible for the production of MMP-1 of 404 amino acid residues with a predicted molecular weight of 45.1 kDa. The middle part, 3' end, and 5' end sequences were 933, 503, and 496 bp. In addition, it was revealed that the MMP-1 genomic sequence includes three exons and two introns. Furthermore, the three-dimensional structure of L. sericata MMP-1 protein was evaluated, and its alignment defined that it has high similarity to chain A of human MMP-2 with 100% confidence, 72% coverage, and 38% identity according to the SWISS-MODEL modeling analysis. Conclusions: MMP-1 of L. sericata has a close relationship with its homologs in invertebrates and other insects. The present study significantly contributes to understanding the function, classification, and evolution of the characterized MMP-1 from L. sericata and provides basic required information for the development of an effective medical bioproduct.
Assuntos
Glândulas Salivares/enzimologia , Metaloproteinase 1 da Matriz/genética , Dípteros/enzimologia , Dípteros/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase , Análise de Sequência de RNA , DNA Complementar/genética , Biologia Computacional , LarvaRESUMO
Mg2+-ATPase activity was detected in the three salivary glands of adult triatomines, males and females, of Triatoma infestans (Klug, 1834) and Panstrongylus megistus (Burmeister, 1835) (Heteroptera, Triatominae). A predominance of binucleated cells in D1 and D2 and mononucleated in D3 was observed, with bulky and polyploidy nuclei. ATPase activity was detected in the nuclei, possibly in euchromatin and nucleolus, where this enzyme probably acts in the transcription process. ATPase reaction was also evidenced in the nuclear membrane, which is probably associated with nuclear-cytoplasmatic transport. These characteristics indicate a high metabolism and protein synthesis, which must be essential to saliva production as well as in maintaining the hematophagy of triatomines.
A atividade da enzima ATPase dependente de Mg2+ foi detectada nas três glândulas salivares de triatomíneos adultos, machos e fêmeas, de Triatoma infestans (Klug, 1834) e Panstrongylus megistus (Burmeister, 1835) (Heteroptera, Triatominae). Observou-se um predomínio de células binucleadas em D1 e D2, e mononucleadas em D3, com núcleos volumosos e poliplóides. Atividade da ATPase foi detectada no núcleo, provavelmente na eucromatina e no nucléolo, onde esta enzima atuaria no processo de transcrição. A atividade também foi evidenciada na membrana nuclear e, possivelmente, associada ao transporte núcleo-citoplasmático. Essas características indicam alto metabolismo e elevada síntese proteica, essenciais para a produção de saliva e manutenção da hematofagia de triatomíneos.
Assuntos
Animais , Adenosina Trifosfatases/análise , Glândulas Salivares/enzimologia , Magnésio , Triatominae/fisiologia , Eucromatina , Membrana Nuclear , Monoéster Fosfórico HidrolasesRESUMO
Mg2+-ATPase activity was detected in the three salivary glands of adult triatomines, males and females, of Triatoma infestans (Klug, 1834) and Panstrongylus megistus (Burmeister, 1835) (Heteroptera, Triatominae). A predominance of binucleated cells in D1 and D2 and mononucleated in D3 was observed, with bulky and polyploidy nuclei. ATPase activity was detected in the nuclei, possibly in euchromatin and nucleolus, where this enzyme probably acts in the transcription process. ATPase reaction was also evidenced in the nuclear membrane, which is probably associated with nuclear-cytoplasmatic transport. These characteristics indicate a high metabolism and protein synthesis, which must be essential to saliva production as well as in maintaining the hematophagy of triatomines.(AU)
A atividade da enzima ATPase dependente de Mg2+ foi detectada nas três glândulas salivares de triatomíneos adultos, machos e fêmeas, de Triatoma infestans (Klug, 1834) e Panstrongylus megistus (Burmeister, 1835) (Heteroptera, Triatominae). Observou-se um predomínio de células binucleadas em D1 e D2, e mononucleadas em D3, com núcleos volumosos e poliplóides. Atividade da ATPase foi detectada no núcleo, provavelmente na eucromatina e no nucléolo, onde esta enzima atuaria no processo de transcrição. A atividade também foi evidenciada na membrana nuclear e, possivelmente, associada ao transporte núcleo-citoplasmático. Essas características indicam alto metabolismo e elevada síntese proteica, essenciais para a produção de saliva e manutenção da hematofagia de triatomíneos.(AU)
Assuntos
Animais , Triatominae/fisiologia , Glândulas Salivares/enzimologia , Adenosina Trifosfatases/análise , Magnésio , Monoéster Fosfórico Hidrolases , Eucromatina , Membrana NuclearRESUMO
Alkaline phosphatase activity was detected in salivary gland cells of the Rhodnius neglectus Lent, 1954, and R. prolixus Stal, 1859, vectors of Trypanosoma cruzi Chagas, 1909 (etiological agent of Chagas disease) and T. rangeli Tejera, 1920 (pathogenic to insect). The Gomori technique was used to demonstrate alkaline phosphatase activity. Alkaline phosphatase activity was observed throughout the entire gland, with an increased activity in the posterior region of the principal gland. In particular, phosphatase activity was found in the nucleolar corpuscles, suggesting a relationship with the rRNA transcription and ribosomal biogenesis. Alkaline phosphatase was also detected in the nuclear membrane and nuclear matrix, suggesting an association with the nucleo-cytoplasmic transport of ribonucleoproteins and the mechanisms of cell cycle and DNA replication, respectively. This study highlights the importance of alkaline phosphatase in the salivary gland of R. prolixus and R. neglectus and emphasizes its importance in secretory activity. Secretory activity is directly involved in hematophagy and, consequently, in development during metamorphosis. The observed presence of alkaline phosphatase suggests its involvement in the production of saliva allowing feeding of these insects that are important vectors of Chagas disease.
Assuntos
Fosfatase Alcalina/metabolismo , Proteínas de Insetos/metabolismo , Insetos Vetores/enzimologia , Rhodnius/enzimologia , Glândulas Salivares/enzimologia , Animais , Doença de Chagas/parasitologia , Doença de Chagas/transmissão , Feminino , Insetos Vetores/parasitologia , Masculino , Rhodnius/parasitologia , Glândulas Salivares/parasitologia , Trypanosoma cruzi/fisiologiaRESUMO
Metalloproteases (MPs) have been considered essential for blood feeding and other physiological functions in several hematophagous animals, including ticks. We report the characterization of MP sequences of three important ticks from Asia, Africa and America: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) and R. microplus (BrRm-MPs). Amino acid sequence identity between R. microplus and R. sanguineus MPs ranged from 76 to 100 %, and identities among I. persulcatus, I. ricinus and I. scapularis MP sequences ranged from 88 to 97 %. This high sequence identity and typical functional motifs show that all sequences are MPs. The presence of a zinc binding site, a Met-turn and cysteine rich domain at the C-terminal region indicates that these proteins belong to the reproplysin family of MPs. Differences in amino acid sequences of BrRm-MP1, BrRm-MP2, BrRm-MP4 and BrRm-MP5 (from Porto Alegre strain ticks) were 6, 2, 7 and 5 %, respectively, when compared with sequences deposited in GenBank for the same genes from other R. microplus isolates. Analyses of MPs predicted that they have various highly antigenic regions. Semi-quantitative RT-PCR analysis revealed the presence of transcripts in salivary glands of partially and fully fed female ticks. None of these transcripts were observed in males (except BrRm-MP4) and eggs. These enzymes may be functional components required during tick feeding to manipulate host defenses and support tick hematophagy.
Assuntos
Ixodidae/enzimologia , Metaloproteases/genética , Filogenia , Glândulas Salivares/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Ixodidae/genética , Masculino , Dados de Sequência Molecular , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The chemotherapeutic agent 5-Fluorouracil (5-FU) can induce salivary gland hypofunction (SGH); however, previous studies did not reach final conclusions on the influence of this drug on glandular tissue. Thus, the aim of this study was to investigate the effect of 5-FU on submandibular (SMs) and sublingual glands (SLs), as well as, the effect of laser phototherapy (LPT) on SGH induced by 5-FU. Eighty-five hamsters were divided into three groups: control (C), chemotherapy (CT) and laser (L), and the SGH was induced by two injections of 5-FU in groups CT and L. The irradiation was performed using a diode (λ780 nm/20 mW/5 J cm(-2)/0.2 J and 10 s per point/spot size of 0.04 cm(2)) and applied daily. On the euthanasia day, SMs and SLs were removed and biochemical analyses were carried out. The lactate dehydrogenase activity was increased in group CT when compared with group C for SLs and SMs (P < 0.05). In addition, the peroxidase and catalase activities were increased and superoxide dismutase was decreased by 5-FU (P < 0.05). However, LPT appears to be a protective mechanism against oxidative stress, tending to alter the activity of these antioxidant enzymes, suggesting LPT as a promising therapy to modulate the 5-FU harmful effect.