RESUMO
Native Americans domesticated maize (Zea mays ssp. mays) from lowland teosinte parviglumis (Zea mays ssp. parviglumis) in the warm Mexican southwest and brought it to the highlands of Mexico and South America where it was exposed to lower temperatures that imposed strong selection on flowering time. Phospholipids are important metabolites in plant responses to low-temperature and phosphorus availability and have been suggested to influence flowering time. Here, we combined linkage mapping with genome scans to identify High PhosphatidylCholine 1 (HPC1), a gene that encodes a phospholipase A1 enzyme, as a major driver of phospholipid variation in highland maize. Common garden experiments demonstrated strong genotype-by-environment interactions associated with variation at HPC1, with the highland HPC1 allele leading to higher fitness in highlands, possibly by hastening flowering. The highland maize HPC1 variant resulted in impaired function of the encoded protein due to a polymorphism in a highly conserved sequence. A meta-analysis across HPC1 orthologs indicated a strong association between the identity of the amino acid at this position and optimal growth in prokaryotes. Mutagenesis of HPC1 via genome editing validated its role in regulating phospholipid metabolism. Finally, we showed that the highland HPC1 allele entered cultivated maize by introgression from the wild highland teosinte Zea mays ssp. mexicana and has been maintained in maize breeding lines from the Northern United States, Canada, and Europe. Thus, HPC1 introgressed from teosinte mexicana underlies a large metabolic QTL that modulates phosphatidylcholine levels and has an adaptive effect at least in part via induction of early flowering time.
Assuntos
Adaptação Fisiológica , Flores , Interação Gene-Ambiente , Fosfatidilcolinas , Fosfolipases A1 , Proteínas de Plantas , Zea mays , Alelos , Mapeamento Cromossômico , Flores/genética , Flores/metabolismo , Genes de Plantas , Ligação Genética , Fosfatidilcolinas/metabolismo , Fosfolipases A1/classificação , Fosfolipases A1/genética , Fosfolipases A1/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
INTRODUCTION AND OBJECTIVE: Non-alcoholic fatty liver disease remains as one of the main liver disorders worldwide. It is widely accepted that is the kind of lipid, rather than the amount deposited in the cells that determines cell damage. Cholesterol and saturated free fatty acids are deleterious lipids when accumulated but, in contrast, there are some valuable lipids that could counteract those with harmful properties. Much of this knowledge arises from studies using a single fatty acid, but the effects of a combination of fatty acids, as obtained by diet has been poorly addressed. In the present work, we were focused to figure out the cellular effect of two different mixes of fatty acids, one with high proportion of saturated fatty acids, and another one with high proportion of unsaturated fatty acids (Mediterranean-like) in a cellular model of steatosis. MATERIAL AND METHODS: Primary mouse hepatocytes from animals fed with a western diet (high fat and carbohydrates diet), were treated with both mixes of fatty acids for 24â¯h. RESULTS: Our data clearly show that only the high unsaturated fatty acid mix induced a decrease in triglycerides (47.5%) and cholesterol (59%) content in steatotic hepatocytes mediating cellular protection associated to the decrement of ROS and oxidative damage. The mixture of high saturated fatty acids exhibited no effects, preserving high levels of cholesterol and triglycerides and oxidative damage. In conclusion, our results show that Mediterranean-like mix of fatty acids exerts cellular protection in steatosis by decreasing triglycerides, cholesterol, ROS content and oxidative damage.
Assuntos
Dieta Mediterrânea , Dieta Ocidental , Ácidos Graxos Insaturados/farmacologia , Hepatócitos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Animais , Antígenos de Neoplasias/efeitos dos fármacos , Células Cultivadas , Colesterol/metabolismo , Modelos Animais de Doenças , Hepatócitos/metabolismo , Hepatócitos/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Endogâmicos BALB C , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse Oxidativo/efeitos dos fármacos , Fosfolipases A1/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Triglicerídeos/metabolismoRESUMO
Insect venom can cause systemic allergic reactions, including anaphylaxis. Improvements in diagnosis and venom immunotherapy (VIT) are based on a better understanding of an immunological response triggered by venom allergens. Previously, we demonstrated that the recombinant phospholipase A1 (rPoly p 1) from Polybia paulista wasp venom induces specific IgE and IgG antibodies in sensitized mice, which recognized the native allergen. Here, we addressed the T cell immune response of rPoly p 1-sensitized BALB/c mice. Cultures of splenocytes were stimulated with Polybia paulista venom extract and the proliferation of CD8+ and CD4+ T cells and the frequency of T regulatory cells (Tregs) populations were assessed by flow cytometry. Cytokines were quantified in cell culture supernatants in ELISA assays. The in vitro stimulation of T cells from sensitized mice induces a significant proliferation of CD4+ T cells, but not of CD8+ T cells. The cytokine pattern showed a high concentration of IFN-γ and IL-6, and no significant differences to IL-4, IL-1ß and TGF-ß1 production. In addition, the rPoly p 1 group showed a pronounced expansion of CD4+CD25+FoxP3+ and CD4+CD25-FoxP3+ Tregs. rPoly p 1 sensitization induces a Th1/Treg profile in CD4+ T cell subset, suggesting its potential use in wasp venom immunotherapy.
Assuntos
Alérgenos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Dessensibilização Imunológica , Proteínas de Insetos/farmacologia , Fosfolipases A1/farmacologia , Venenos de Vespas/farmacologia , Alérgenos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Feminino , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Hipersensibilidade/terapia , Mordeduras e Picadas de Insetos/imunologia , Mordeduras e Picadas de Insetos/metabolismo , Mordeduras e Picadas de Insetos/terapia , Proteínas de Insetos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Fosfolipases A1/imunologia , Venenos de Vespas/imunologiaRESUMO
Leishmaniasis is caused by several species of protozoan parasites of the genus Leishmania and represents an important global health problem. Leishmania braziliensis in particular is responsible of cutaneous and mucocutaneous forms of this parasitosis, with prevalence in Latin America. In the present work, we describe in L. braziliensis promastigotes and amastigotes the presence of a Phospholipase A1 (PLA1) activity, an enzyme that catalyses extensive deacylation of phospholipids like phosphatidylcholine. In order to deepen the knowledge about L. braziliensis PLA1, the cloning and expression of the gene that codifies for this enzyme was carried out in a baculovirus expression system with the obtaintion of a purified recombinant protein that displayed PLA1 activity. Given that this is the first molecular and functional protein characterization of a PLA1 in the Leishmania genus, we also performed a phylogenetic analysis of this gene throughout 12 species whose genome sequences were available. The results presented here will contribute to increase the knowledge about trypanosome phospholipases, which could be novel and valuable as potential targets to fight neglected diseases like Leishmaniasis.
Assuntos
Leishmania braziliensis , Fosfolipases A1 , Animais , Baculoviridae/genética , Clonagem Molecular/métodos , Expressão Gênica , Genes de Protozoários , América Latina , Leishmania braziliensis/genética , Leishmania braziliensis/metabolismo , Leishmaniose Cutânea/parasitologia , Fosfolipases A1/genética , Fosfolipases A1/isolamento & purificação , Fosfolipases A1/metabolismo , Filogenia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Sf9RESUMO
Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.
Assuntos
Venenos de Artrópodes/enzimologia , Himenópteros/enzimologia , Mordeduras e Picadas de Insetos/imunologia , Fosfolipases A1/imunologia , Fosfolipases A2/imunologia , Sequência de Aminoácidos , Animais , Venenos de Artrópodes/imunologia , Humanos , Mordeduras e Picadas de Insetos/enzimologia , Fosfolipases A1/química , Fosfolipases A1/metabolismo , Fosfolipases A2/química , Fosfolipases A2/metabolismoRESUMO
Lipids from microorganisms are ligands of Toll like receptors (TLRs) and modulate the innate immune response. Herein, we analyze in vitro the effect of total lipid extracts from Trypanosoma cruzi amastigotes of RA and K98 strains (with polar biological behavior) on the induction of the inflammatory response and the involvement of TLRs in this process. We demonstrated that total lipid extracts from both strains induced lipid body formation, cyclooxygenase-2 expression and TNF-α and nitric oxide release in macrophages, as well as NF-κB activation and IL-8 release in HEK cells specifically through a TLR2/6 dependent pathway. We also evaluated the inflammatory response induced by total lipid extracts obtained from lysed parasites that were overnight incubated to allow the action of parasite hydrolytic enzymes, such as Phospholipase A1, over endogenous phospholipids. After incubation, these total lipid extracts showed a significantly reduced pro-inflammatory response, which could be attributed to the changes in the content of known bioactive lipid molecules like lysophospholipids and fatty acids, here reported. Moreover, analyses of total fatty acids in each lipid extract were performed by gas chromatography-mass spectrometry. Our results indicate a relevant role of T. cruzi lipids in the induction of a pro-inflammatory response through the TLR2/6 pathway that could contribute to the modulation of the immune response and host survival.
Assuntos
Lipídeos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 6 Toll-Like/imunologia , Receptores Toll-Like/imunologia , Trypanosoma cruzi/metabolismo , Animais , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Ácidos Graxos/imunologia , Células HEK293 , Humanos , Imunidade Inata , Interleucina-8/metabolismo , Gotículas Lipídicas , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Fosfolipases A1/genética , Fosfolipases A1/metabolismo , Proteínas Recombinantes , Trypanosoma cruzi/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.
Assuntos
Venenos de Formiga/imunologia , Venenos de Abelha/imunologia , Hipersensibilidade/imunologia , Proteínas de Insetos/imunologia , Fosfolipases A1/imunologia , Venenos de Vespas/imunologia , Alérgenos/imunologia , Animais , Formigas/enzimologia , Formigas/imunologia , Abelhas/enzimologia , Abelhas/imunologia , Brasil , Reações Cruzadas , Europa (Continente) , Feminino , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/etiologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Testes Intradérmicos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/imunologia , Vespas/enzimologia , Vespas/imunologiaRESUMO
Two commercially available and widely used enzymes, the parent Thermomyces lanuginosus lipase (TLL) and the shuffled phospholipase A1 Lecitase (Lecitase Ultra), were encapsulated in AOT/isooctane reverse micelles and evaluated regarding their structure and activity. Preparations were also tested as effective biocatalysts. Small-angle X-ray scattering (SAXS), electronic paramagnetic resonance (EPR), and fluorescence spectroscopy were the techniques applied to assess the effects of enzyme incorporation to a reverse micellar nanostructure. SAXS analysis showed that the radius of gyration (Rg) changed from 16 to 38 Å, as the water content (w0) increased. Elongated shapes were more commonly observed than spherical shapes after enzyme encapsulation. EPR studies indicated that enzymes do not participate in the interface, being located in the aqueous center. Fluorescence energy transfer showed that TLL is located in the water core, whereas Lecitase Ultra is closer to the interface. Enzymatic activity toward a standard esterification reaction endured after the enzyme was incorporated into the micelles. The activity of TLL for systems with w0 15 showed the highest conversion yield, 38% in 2 h, while the system with w0 10 showed the highest initial velocity, 0.43 µM/min. This last system had a Rg of 19.3 Å, similar to that of the TLL monomer. Lecitase Ultra showed the highest conversion yields in systems with w0 10, 55% in 2 h. However, the initial rate was much lower than that of TLL, suggesting less affinity for the substrates, which is expected since Lecitase Ultra is a phospholipase. In summary, we here used several spectroscopic and scattering techniques to reveal the shape and stability of TTL and Lecitase Ultra encapsulated systems, which allowed the selection of w0 values to provide optimized enzymatic activity.
Assuntos
Ascomicetos/enzimologia , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Micelas , Fosfolipases A1/química , Espectroscopia de Ressonância de Spin Eletrônica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Difração de Raios XRESUMO
Este estudo objetivou compreender as práticas de cuidado dos profissionais de saúde que assistem os idosos Kaingang. Estudo qualitativo, apoiado na etnografia, realizado com dez profissionais à que atuam na atenção primária saúde da Terra Indígena Faxinal, Paraná, Brasil. Os dados foram coletados no período de novembro de 2010 a fevereiro de 2012 por meio da observação participante e entrevistas, e, analisados à luz da Teoria Transcultural do Cuidado. Identificaram-se como práticas de cuidado a medicação e imunização, bem como, cuidados da medicina tradicional. Para realização destes cuidados, os profissionais dispunham de estratégias que proporcionavam manutenção dos idosos na assistência. Conclui-se que valores culturais e científicos necessitam integrar a assistência para melhoria da saúde dos idosos indígenas.
This research aims to understand the care practices of health professionals who assist the elderly Kaingang. It is a qualitative study, supported in ethnography, conducted by ten professionals working in primary health care in the indigenous land of Faxinal, Paraná, Brazil. The data was collected from November 2010 to February 2012 by participant observation and interviews, and analyzed based on the Transcultural Care Theory. Was identified the preoccupation of the carers practices with the medication and immunization, as well as traditional medical care. To achieve these, care professionals had strategies that implemented maintenance of older people in care. We conclude that cultural values and integrate scientific need assistance to improve the health of elderly indigenous.
Este estudio tuvo como objetivo entender las prácticas de cuidado de los profesionales de la salud que asisten a los ancianos Kaingang. Estudio cualitativo, apoyado en la etnografía, llevado a cabo con diez profesionales que trabajan en la atención primaria de la salud de la tierra indígena de Faxinal, Paraná, Brasil. Los datos fueron recogidos a partir de noviembre 2010 a febrero 2012 a través de la observación participante y las entrevistas, y analizado con base en la Teoría del Cuidado Transcultural. Se identificaron las prácticas de atención médica y imunizacion,el cuidado de la medicina, así tradicional. Para lograrlo, los profesionales tenían estrategias que proporcionaban el mantenimiento de las personas mayores en su atención. Se concluye que los valores culturales y científicos necesitan ayuda para mejorar la salud de los ancianos indígenas.
Assuntos
Animais , Ratos , Fígado/enzimologia , Lisossomos/enzimologia , Fosfolipases A/metabolismo , Fosfolipases/metabolismo , Inibidores de Proteases/farmacologia , Células Cultivadas , Quimotripsina/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Leucina/análogos & derivados , Leucina/farmacologia , Leupeptinas/farmacologia , Oligopeptídeos/farmacologia , Pepstatinas/farmacologia , Fosfolipases A1 , Fatores de TempoRESUMO
Lecitase Ultra has been immobilized on cyanogen bromide agarose (via covalent attachment) and on octyl agarose (via physical adsorption on the hydrophobic support by interfacial activation). Both immobilized preparations have been incubated in dextran sulfate (DS) or polyethylenimine (PEI) solutions to coat the enzyme surface. Then, the activity versus different substrates and under different experimental conditions was evaluated. The PEI coating generally produced a significant increase in enzyme activity, in some cases even by more than a 30-fold factor (using the octyl-Lecitase at pH 5 in the hydrolysis of methyl phenyl acetate). In opposition, the DS coating usually produced some negative effects on the enzyme activity. The rate of irreversible inhibition of the covalent preparation using diethyl p-nitrophenylphosphate did not increase after PEI coating suggesting that the increase in Lecitase activity is not a consequence of the stabilization of the open form of Lecitase. Moreover, the coating greatly increased the stability of the immobilized Lecitase, for example using DS and the covalent preparation, the half-life was increased by a 30-fold factor in 30% acetonitrile. The stabilizing effect was not found in all cases, in certain cases even a certain destabilization is found (e.g., octyl-Lecitase-DS at pH 7). Thus, the effects of the ionic polymer coating strongly depend on the substrate, experimental conditions and immobilization technique employed.
Assuntos
Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Fosfolipases A1/química , Fosfolipases A1/metabolismo , Biotecnologia , Catálise , Materiais Revestidos Biocompatíveis/química , Brometo de Cianogênio , Sulfato de Dextrana , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Polietilenoimina , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , SefaroseRESUMO
Chihuahua cheese or Mennonite cheese is one of the most popular and consumed cheeses in Mexico and by the Hispanic community in the United States. According to local producers the yield of Chihuahua cheese ranges from 9 to 9.5 kg of cheese from 100 kg of milk. Cheese yield is a crucial determinant of profitability in cheese-manufacturing plants; therefore, different methods have been developed to increase it. In this work, a miniature Chihuahua-type cheese model was used to assess the effect of a phospholipase A1 (PL-A1) and exopolysaccharide (EPS)-producing bacteria (separately and in combination) on the yield, microstructure, and texture of cheese. Four different cheeses were manufactured: cheese made with PL-A1, cheese made with EPS-producing bacteria, cheese with both PL-A1 and EPS-producing bacteria, and a cheese control without PL-A1 or EPS-producing bacteria. The compositional analysis of cheese was carried out using methods of AOAC International (Washington, DC). The actual yield and moisture-adjusted yield were calculated for all cheese treatments. Texture profile analyses of cheeses were performed using a texture analyzer. Micrographs were obtained by electron scanning microscopy. Fifty panelists carried out sensorial analysis using ranking tests. Incorporation of EPS-producing bacteria in the manufacture of cheese increased the moisture content and water activity. In contrast, the addition of PL-A1 did not increase fat retention or cheese yield. The use of EPS alone improved the cheese yield by increasing water and fat retention, but also caused a negative effect on the texture and flavor of Chihuahua cheese. The use of EPS-producing bacteria in combination with PL-A1 improved the cheese yield and increased the moisture and fat content. The cheeses with the best flavor and texture were those manufactured with PL-A1 and the cheeses manufactured with the combination of PL-A1 and EPS-producing culture.
Assuntos
Queijo/análise , Microbiologia de Alimentos , Fosfolipases A1/análise , Polissacarídeos Bacterianos/metabolismo , Animais , Bactérias/metabolismo , Queijo/microbiologia , México , Leite/química , PaladarRESUMO
Phospholipase A1 (PLA1) has been described in the infective stages of Trypanosoma cruzi as a membrane-bound/secreted enzyme that significantly modified host cell lipid profile with generation of second lipid messengers and concomitant activation of protein kinase C. In the present work we determined higher levels of PLA1 expression in the infective amastigotes and trypomastigotes than in the non-infective epimastigotes of lethal RA strain. In addition, we found similar expression patterns but distinct PLA1 activity levels in bloodstream trypomastigotes from Cvd and RA (lethal) and K98 (non-lethal) T. cruzi strains, obtained at their corresponding parasitemia peaks. This fact was likely due to the presence of different levels of anti-T. cruzi PLA1 antibodies in sera of infected mice, that modulated the enzyme activity. Moreover, these antibodies significantly reduced in vitro parasite invasion indicating the participation of T. cruzi PLA1 in the early events of parasite-host cell interaction. We also demonstrated the presence of lysophospholipase activity in live infective stages that could account for self-protection against the toxic lysophospholipids generated by T. cruzi PLA1 action. At the genome level, we identified at least eight putative genes that codify for T. cruzi PLA1 with high amino acid sequence variability in their amino and carboxy-terminal regions; a putative PLA1 selected gene was cloned and expressed as a recombinant protein that possessed PLA1 activity. Collectively, the results presented here point out at T. cruzi PLA1 as a novel virulence factor implicated in parasite invasion.
Assuntos
Fosfolipases A1/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/patogenicidade , Fatores de Virulência/metabolismo , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Doença de Chagas/imunologia , Doença de Chagas/parasitologia , Chlorocebus aethiops , Clonagem Molecular , DNA de Protozoário/química , DNA de Protozoário/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Interações Hospedeiro-Parasita , Camundongos , Dados de Sequência Molecular , Parasitemia/imunologia , Parasitemia/parasitologia , Fosfolipases A1/genética , Análise de Sequência de DNA , Trypanosoma cruzi/genética , Células Vero , Fatores de Virulência/genéticaRESUMO
The phospholipases A(1) (PLA(1) s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2-D, MALDI-TOF-TOF MS and classical protocols of protein chemistry, which included N- and C-terminal sequencing. The existence of an intact form of PLA(1) and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific-IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA(1) , combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA(2) s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.
Assuntos
Fosfolipases A1/análise , Venenos de Vespas/análise , Vespas/química , Animais , Glicosilação , Imunoglobulina E/imunologia , Isoenzimas/análise , Isoenzimas/química , Isoenzimas/imunologia , Espectrometria de Massas , Fosfolipases A1/química , Fosfolipases A1/imunologia , Proteômica , Análise de Sequência de Proteína , Venenos de Vespas/imunologiaRESUMO
The aim of the present research was to analyse the pathways for phosphatidic acid metabolism in purified nuclei from cerebellar cells. Lipid phosphate phosphatase and diacylglyceride lipase activities were detected in nuclei from cerebellar cells. It was observed that DAGL activity makes up 50% of LPP activity and that PtdOH can also be metabolised to lysophosphatidic acid. With a nuclear protein content of approximately 40 µg, the production of diacylglycerol and monoacylglycerol was linear for 30 min and 5 min, respectively, whereas it increased with PtdOH concentrations of up to 250 µM. LysoPtdOH, sphingosine 1-phosphate and ceramide 1-phosphate, which are alternative substrates for LPP, significantly reduced DAG production from PA. DAG and MAG production increased in the presence of Triton X-100 (1 mM) whereas no modifications were observed in the presence of ionic detergent sodium deoxycholate. Ca²+ and Mg²+ stimulated MAG production without affecting DAG formation whereas fluoride and vanadate inhibited the generation of both products. Specific PtdOH-phospholipase A1 and PtdOH-phospholipase A2 were also detected in nuclei. Our findings constitute the first reported evidence of active PtdOH metabolism involving LPP, DAGL and PtdOH-selective PLA activities in purified nuclei prepared from cerebellar cells.
Assuntos
Núcleo Celular/metabolismo , Cerebelo/citologia , Redes e Vias Metabólicas , Ácidos Fosfatídicos/metabolismo , Animais , Cálcio/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Ceramidas/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/enzimologia , Cerebelo/metabolismo , Detergentes/farmacologia , Diglicerídeos/biossíntese , Diglicerídeos/metabolismo , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Lisofosfolipídeos/metabolismo , Magnésio/farmacologia , Masculino , Monoglicerídeos/biossíntese , Monoglicerídeos/metabolismo , Fosfolipases A1/metabolismo , Fosfolipases A2/metabolismo , Ratos , Ratos Wistar , Fluoreto de Sódio/farmacologia , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de Tempo , Vanadatos/farmacologiaRESUMO
The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 Da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients.
Assuntos
Fosfolipases A1/química , Venenos de Vespas/enzimologia , Vespas/enzimologia , Sequência de Aminoácidos , Animais , Humanos , Immunoblotting , Imunoglobulina E , Modelos Moleculares , Dados de Sequência Molecular , Fosfolipases A1/metabolismo , Conformação ProteicaRESUMO
Here we have studied phospholipase A1 (Plase A1) from Trypanosoma cruzi infective stages and it's possible role regarding the interaction with mammalian host cells. Plase A1 was mainly detected as a membrane-bound activity in the infective amastigote and trypomastigote stages, being remarkably higher with respect to the non-infective epimastigotes. It is noteworthy that only the infective stages secreted Plase A1. Moreover, along the differentiation process from epimastigotes into metacyclic trypomastigotes, the secreted enzyme activity increased simultaneously with the appearance of metacyclic forms, as expected. Since this enzyme is predominantly membrane-associated and secreted by the infective stages, Vero cell lipid profile modifications were analysed after interaction with either intact infective parasites or purified T. cruzi Plase A1. Significant changes in Vero cell lipid composition were observed, with the appearance of free fatty acids, diacylglycerol and lysophosphatidylcholine. Concomitantly with the generation of second lipid messengers, host cell protein kinase C activation was demonstrated. These results indicate that T. cruzi Plase A1 could play a critical role in the early events of parasite-host cell interaction that precede invasion.
Assuntos
Metabolismo dos Lipídeos , Fosfolipases A/metabolismo , Proteína Quinase C/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/fisiologia , Animais , Chlorocebus aethiops , Ativação Enzimática , Fosfolipases A1 , Células VeroRESUMO
We found that, as in African trypanosomes, endogenous phospholipase A(1) (Plase A(1)) activity can catalyse extensive deacylation of phospholipids upon cell death in all life stages of Trypanosoma cruzi. A major lysosomal Plase A(1) was purified and characterized. The enzyme products can explain the lesions surrounding degenerating T. cruzi cells in host tissues. Thus Plase A(1) emerges as a target to block pathogenesis in trypanosomal infections.
Assuntos
Doença de Chagas/parasitologia , Lisossomos/enzimologia , Fosfolipases A/metabolismo , Fosfolipídeos/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Catálise , Doença de Chagas/metabolismo , Humanos , Fosfolipases A/isolamento & purificação , Fosfolipases A/fisiologia , Fosfolipases A1 , Trypanosoma cruzi/patogenicidadeRESUMO
A rapid and economical method for the purification of phospholipase A1 (PLA1) from the extracellular medium of the ciliate Tetrahymena thermophila is presented. Essentially, the procedure, here designated as purification by selective interaction (PSI), entails the incubation of media containing PLA1 with liposomes made of soy bean phospholipids. The PLA1-lipid complexes are precipitated by the addition of CaCl2 and collected by centrifugation. Elution of the PLA1 is effected by treating the complexes with 40% dimethylformamide, a reversible inhibitor of this enzyme, which is easily removed by dialysis. In combination with DEAE cellulose ion exchange chromatography, PSI yielded homogeneous PLA1 preparations with a 14% recovery and a 416-fold increase in specific activity. This procedure, which can be completed within 1 day, may prove useful for the isolation of phospholipases from other sources. This practical method for the purification of a microbial PLA1 opens the way to large-scale production of these types of enzyme, which are not as yet commercially available.