RESUMO
Iron accumulation and oxidative stress are hallmarks of retinas from patients with age-related macular degeneration (AMD). We have previously demonstrated that iron-overloaded retinas are a good in vitro model for the study of retinal degeneration during iron-induced oxidative stress. In this model we have previously characterized the role of cytosolic phospholipase A2 (cPLA2) and calcium-independent isoform (iPLA2). The aim of the present study was to analyze the implications of Group V secretory PLA2 (sPLA2), another member of PLA2 family, in cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB) regulation. We found that sPLA2 is localized in cytosolic fraction in an iron concentration-dependent manner. By immunoprecipitation (IP) assays we also demonstrated an increased association between Group V sPLA2 and COX-2 in retinas exposed to iron overload. However, COX-2 activity in IP assays was observed to decrease in spite of the increased protein levels observed. p65 (RelA) NF-κB levels were increased in nuclear fractions from retinas exposed to iron. In the presence of ATK (cPLA2 inhibitor) and YM 26734 (sPLA2 inhibitor), the nuclear localization of both p65 and p50 NF-κB subunits was restored to control levels in retinas exposed to iron-induced oxidative stress. Membrane repair mechanisms were also analyzed by studying the participation of acyltransferases in phospholipid remodeling during retinal oxidation stress. Acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylserine (PS), were observed to show an inhibited acylation profile in retinas exposed to iron while phosphatidylethanolamine (PE) showed the opposite. The use of PLA2 inhibitors demonstrated that PS is actively deacylated during iron-induced oxidative stress. Results from the present study suggest that Group V sPLA2 has multiple intracellular targets during iron-induced retinal degeneration and that the specific role of sPLA2 could be related to inflammatory responses by its participation in NF-κB and COX-2 regulation.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Fosfolipases A2 do Grupo V/fisiologia , Degeneração Macular/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Retina/efeitos dos fármacos , Acetilação , Acetiltransferases/metabolismo , Animais , Western Blotting , Bovinos , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Compostos Ferrosos/toxicidade , Fosfolipases A2 do Grupo V/antagonistas & inibidores , Sobrecarga de Ferro/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A/fisiologia , Retina/metabolismoRESUMO
Introdução: A obesidade se caracteriza como um processo oxidativo e inflamatório, que predispõe adolescentes, de modo precoce, a eventos até recentemente pouco frequentes nessa faixa etária. Assim, a ação da enzima Fosfolipase A associada às lipoproteínas (Lp-PLA ), que reduz fosfolipídios oxidados e gera lisofosfolipídios, bem como a disponibilidade de antioxidantes plasmáticos, representam um importante tema de pesquisa no contexto cardiovascular. Objetivo: Verificar se a atividade da LP-PLA 2 2 e a concentração de antioxidantes lipossolúveis se associam com os principais marcadores de risco cardiovascular em adolescentes. Métodos: Duzentos e quarenta e dois adolescentes (10 a 19 anos), de ambos os sexos foram distribuídos, segundo o índice de massa corporal (IMC), em três grupos: Eutróficos (n=77), Sobrepeso (n=82) e Obesos (n=83). A amostra foi caracterizada através de parâmetros sócio-econômicos, estado de saúde, uso de medicamentos, antedecentes familiares de doenças crônicas e prática de atividade física. Foram avaliados ainda os dados antropométricos (peso, altura e composição corporal - bioimpedância), e o consumo alimentar por meio de três recordatórios 24 h. A partir de uma amostra de sangue coletada após jejum (12h), realizaram-se as análises da atividade da Lp-PLA , LDL(-) e seus auto-anticorpos, perfil lipídico (colesterol total, LDL-C, HDL-C e triglicerídeos), tamanho da HDL, proteína transportadora de éster de colesterol (CETP), ácidos graxos não esterificados (NEFAs), adipocitocinas, assim como antioxidantes (retinol, licopeno, -tocoferol e -caroteno) no plasma. Resultados: Artigo 1: Lp-PLA maybe an important cardiovascular biomarker in obese adolescents. Verificou-se que o perfil lipídico, insulina, HOMA-IR (resistência à insulina) e LDL(-) evidenciaram um maior risco cardiovascular nos adolescentes obesos. A atividade da enzima Lp-PLA 2 mostrou uma variação proporcional ao IMC, circunferência da cintura e porcentagem de gordura.
Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Adulto , Fosfolipases A/fisiologia , Fosfolipases A/sangue , Lipoproteínas/fisiologia , Biomarcadores/sangue , Obesidade , Saúde do Adolescente , Antropometria , Índice de Massa Corporal , Proteção da Criança , Fatores de RiscoRESUMO
A new PLA2 Bj-V from Bothrops jararacussu (14039.49 Da determined by MALDI-TOF mass spectrometry) was isolated in only one chromatographic step by HPLC ion-exchange and its purity was confirmed by reverse phase. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHD...) showed a high degree of homology with basic D49 PLA2 myotoxins from other Bothrops venoms. Bj V showed discrete sigmoidal enzymatic behavior, with maximal activity at pH 8.4 and 35-40 degrees C. Full PLA2 activity required Ca2+ (10 mM) and there was little catalytic activity in the presence of 1 mM Ca2+. The addition of Mn2+ or Mg2+ (10 mM) in the presence of low (1 mM) Ca2+ slightly increased the enzyme activity, whereas Zn2+ and Cu2+ (10 mM) diminished the activity. The substitution of Ca2+ for Mg2+ or Cu2+ also reduced the enzymatic activity. Bj V had PLA2 activity and produced cytotoxicity in murine C2C12 skeletal muscle myoblasts and myotubes. The isolation of these isoforms Bj-IV [1] and Bj-V (described herein) found in a fraction previously described as homogeneous shows us the importance of optimization in purification techniques in order to better understand their biological behavior.
Assuntos
Bothrops/fisiologia , Venenos de Crotalídeos/enzimologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Fosfolipases A/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Isoenzimas/genética , Camundongos , Dados de Sequência Molecular , Fosfolipases A/genética , Fosfolipases A2RESUMO
Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) kill target cells by the granule-exocytosis pathway and by the engagement of molecules belonging to the tumor necrosis factor family. The involvement of secretory phospholipase A(2) (sPLA(2)) in the cytotoxic process has been proposed in NK cells. However, its molecular identity and intracellular localization remain unknown, and its mechanism of action is poorly understood. Here, we have readdressed this issue by studying the cytotoxic activity of whole cell extracts of a CTL line. We observed that inactivation of the perforin-granzyme pathway at 37 degrees C in the presence of 1 mM Ca(2+) enhanced the ability of CTL extracts to induce apoptosis. This potentiation of cell death was Ca(2+)-dependent, thermo-resistant, and inhibited by 4-bromophenacyl bromide and scalaradial (two inhibitors of sPLA(2)). The involvement of an sPLA(2) was confirmed by blocking the pro-apoptotic activity of the Ca(2+)-treated cell extract with an anti-sPLA(2) polyclonal antibody. By cell fractionation assays, we showed that the pro-apoptotic sPLA(2) was localized in the cytoplasmic fraction but not in perforin-rich granules or plasma membrane fractions. Western blotting analysis revealed the presence of four distinct bands of 56, 29.5, 21, and 15 kDa. The highest molecular weight band was consistent with the expression of a group III sPLA2. Taken together, these data indicate that an apoptosis-inducing sPLA(2) is expressed in the cytosol of a CTL cell line and suggest that it plays an effector role in CTL-mediated cytotoxicity.
Assuntos
Fator de Indução de Apoptose/fisiologia , Apoptose/fisiologia , Fosfolipases A/fisiologia , Linfócitos T Citotóxicos/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , Sistema Livre de Células , Citosol/fisiologia , Citotoxicidade Imunológica , Proteína Ligante Fas/fisiologia , Fosfolipases A2 do Grupo II , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Proteínas Citotóxicas Formadoras de Poros/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Linfócitos T Citotóxicos/enzimologia , Temperatura , Receptores Chamariz do Fator de Necrose Tumoral/deficiência , Receptores Chamariz do Fator de Necrose Tumoral/genéticaRESUMO
This study was designed to elucidate the signalling pathways by which secretory phospholipases A2 (sPLA2s) induce in vitro neutrophil migration. The cell migration assays were performed with Naja mocambique venom PLA2 (sPLA2 with high catalytic activity), bothropstoxin-I (sPLA2 devoid of catalytic activity) and platelet-activating factor (PAF), using a 48-well microchemotaxis chamber. Both the non-selective protein kinase inhibitor staurosporine (30-300 nM) and the selective protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpyperazine (H7; 50-200 microM) as well as the Gi inactivator pertussis toxin (30-300 nM) caused a concentration-dependent inhibition of the neutrophil migration induced by either N. mocambique venom PLA2 (100 microg/ml) or bothropstoxin-I (100 microg/ml). Pertussis toxin nearly abolished PAF-induced migration, while staurosporine and H7 partly (but significantly) inhibited the chemotactic responses to PAF. The dual inhibitor of cytosolic PLA2 and Ca2+ -independent PLA2 (iPLA2), arachidonil-trifluoromethyl-ketone (ATK; 0.2-20 microM), or the specific iPLA2 inhibitor bromoenol lactone (1-30 microM) caused a concentration-dependent inhibition of the migration induced by either sPLA2s. At the maximal concentration used for each compound, the migration was almost suppressed. In contrast, both of these compounds caused only slight inhibitions of PAF-induced migration. No rise in intracellular Ca2+ was observed in neutrophil-stimulated sPLA2, as determined in cells preloaded with fura 2-AM. In the experimental condition used, pertussis toxin, staurosporine, H7, ATK or bromoenol lactone did not induce cytotoxic effects, according to MTT assay. Our results suggest that activation of an endogenous PLA2 through activation of GTP-binding protein and PKC is the main mechanism by which exogenous sPLA2s cause neutrophil migration.
Assuntos
Movimento Celular/fisiologia , Neutrófilos/citologia , Fosfolipases A/fisiologia , Transdução de Sinais/fisiologia , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citosol/metabolismo , Venenos Elapídicos/enzimologia , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Líquido Intracelular/metabolismo , Naftalenos/farmacologia , Neutrófilos/efeitos dos fármacos , Toxina Pertussis/farmacologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Fator de Ativação de Plaquetas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Pironas/farmacologiaRESUMO
Considerable progress has been made in characterizing the individual participant enzymes and their relative contributions in the generation of eicosanoids, lipid mediators derived from arachidonic acid, such as prostaglandins and leukotrienes. However, the role of individual phospholipase (PL) A(2) enzymes in providing arachidonic acid to the downstream enzymes for eicosanoid generation in biologic processes has not been fully elucidated. In this review, we will provide an overview of the classification of the families of PLA(2) enzymes, their putative mechanisms of action, and their role(s) in eicosanoid generation and inflammation.
Assuntos
Fosfolipases A/fisiologia , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Ácidos Araquidônicos/biossíntese , Ácidos Araquidônicos/metabolismo , Membrana Celular/enzimologia , Inflamação/enzimologia , Inflamação/prevenção & controle , Camundongos , Camundongos Knockout , Neoplasias/enzimologia , Neoplasias/prevenção & controle , Fosfolipases A/classificação , Fosfolipases A/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismoRESUMO
In Brazil, the genus Crotalus is responsible for approximately 1500 cases of snakebite annually. The most common complication in the lethal cases is acute renal failure, although the mechanisms of the damaging effects are not totally understood. In this work, we have examined the renal effects caused by a supernatant of macrophages stimulated by Crotalus durissus cascavella venom as well the potential role of phospholipase A2 and cyclo-oxygenase. Rat peritoneal macrophages were collected and placed in a RPMI medium and stimulated by crude Crotalus durissus cascavella venom (1, 3 or 10 microg/ml) for 1 hr. They were then washed and kept in a culture for 2 hr. The supernatant (1 ml) was tested in an isolated perfused rat kidney. The first 30 min. of each experiment were used as an internal control, and the supernatant was added to the system after this period. All experiments lasted 120 min. A study of toxic effect on perfusion pressure, glomerular filtration rate, urinary flow percent of sodium tubular transport and percent of proximal tubular sodium transport was made. The lowest concentration of venom (1 microg/ml) was not statistically different from the control values. The most intense effects were seen at 10 microg/ml for all renal parameters. The infusion of the supernatant of macrophages stimulated with crude venom (3 or 10 microg/ml) increased the perfusion pressure, glomerular filtration rate and urinary flow, decreased the percent of sodium tubular transport and percent of proximal tubular sodium transport. Dexamethasone (10 microM) and quinacrine (10 microM) provided protection against the effect of the venom on glomerular filtration rate, urinary flow, percent of sodium tubular transport, percent of proximal tubular sodium transport and perfusion pressure. Indomethacin (10 microM) and nordiidroguaretic acid (1 microM) reversed almost all functional changes, except those of the perfusion pressure. These results suggest that macrophages stimulated with Crotalus durissus cascavella venom release mediators capable of promoting nephrotoxicity in vitro. Moreover, phospholipase A2 and cyclooxygenase products are involved in these biologic effects.
Assuntos
Venenos de Crotalídeos/farmacologia , Rim/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Fosfolipases A/fisiologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Frações Subcelulares/fisiologia , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Feminino , Técnicas In Vitro , Indicadores e Reagentes , Macrófagos/química , Masculino , Fosfolipases A2 , Ratos , Ratos Wistar , Frações Subcelulares/química , Urodinâmica/efeitos dos fármacosRESUMO
The purpose of this study was to examine the ability of type I- (porcine pancreas and Naja mocambique mocambique venom), type II- (bothropstoxin-I, bothropstoxin-II, and piratoxin-I), and type III- (Apis mellifera venom) secretory phospholipases A2 (sPLA2s) to induce human neutrophil chemotaxis, and the role of the cell surface proteoglycans, leukotriene B4 (LTB4), and platelet-activating factor (PAF), in mediating this migration. The neutrophil chemotaxis assays were performed by using a 48-well microchemotaxis chamber. Piratoxin-I, bothropstoxin-I, N. m. mocambique venom PLA2 (10-1000 microg/mL each), bothropstoxin-II (30-1000 microg/mL), porcine pancreas PLA2 (0.3-30 microg/mL), and A. mellifera venom PLA2 (30-300 microg/mL) caused concentration-dependent neutrophil chemotaxis. Heparin (10-300 U/mL) concentration-dependently inhibited the neutrophil migration induced by piratoxin-I, bothropstoxin-II, and N. m. mocambique and A. mellifera venom PLA2s (100 microg/mL each), but failed to affect the migration induced by porcine pancreas PLA2. Heparan sulfate (300 and 1000 microg/mL) inhibited neutrophil migration induced by piratoxin-I, whereas dermatan sulfate and chondroitin sulfate (30-1000 microg/mL each) had no effect. Heparitinase I and heparinase (300 mU/mL each) inhibited by 41.5 and 47%, respectively, piratoxin-I-induced chemotaxis, whereas heparitinase II and chondroitinase AC failed to affect the chemotaxis. The PAF receptor antagonist WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thienol-[3,2-f] -triazolo-[4,3-a] -diazepine-2-yl]-1-(4-morpholynil)-1-propionate) (0.1-10 microM) and the LTB4 synthesis inhibitor AA-861 [2-(12-hydroxydodeca-5,10-diynyl)-3,5,6-trimethyl-1,4-benzoquinone] (0.1-10 microM) significantly inhibited the piratoxin-I-induced chemotaxis. Piratoxin-I (30-300 microg/mL) caused a concentration-dependent release of LTB4. Our results suggest that neutrophil migration in response to sPLA2s is independent of PLA activity, and involves an interaction of sPLA2s with cell surface heparin/heparan binding sites triggering the release of LTB4 and PAF.
Assuntos
Movimento Celular/fisiologia , Glicosaminoglicanos/fisiologia , Neutrófilos/enzimologia , Fosfolipases A/fisiologia , Azepinas/farmacologia , Benzoquinonas/farmacologia , Movimento Celular/efeitos dos fármacos , Quimiotaxia/fisiologia , Condroitina Liases/farmacologia , Flavobacterium/enzimologia , Heparina/farmacologia , Heparina Liase/farmacologia , Humanos , Técnicas In Vitro , Leucotrieno B4/metabolismo , Inibidores de Lipoxigenase/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/fisiologia , Fosfolipases A2 , Inibidores da Agregação Plaquetária/farmacologia , Polissacarídeo-Liases/farmacologia , Triazóis/farmacologiaRESUMO
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
We have shown previously that exposure to microcystin-LR (MCLR) causes renal toxic effects in isolated perfused rat kidney. That study was extended further to approach the perspective of pharmacological blockade of renal toxic effects by MCLR through the use of experimental therapeutic agents. An isolated kidney perfusion system was utilized and samples of urine and perfusate were collected at 10min intervals to determine the levels of inulin, sodium, potassium and osmolality. Dexamethasone (20microg ml(-1)) and indomethacin (10microg ml(-1)) were administered in the beginning of the perfusion and MCLR was employed in a dose of 1microg ml(-1) after an internal control of 30min to evaluate the perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR) and urinary flow (UF). Dexamethasone and indomethacin antagonized the toxic effects of MCLR on PP, RVR, GFR and UF. Histologic analysis of dexamethasone and indomethacin treated groups did not show any vascular or interstitial alterations. MCLR potentially impairs the renal function, probably causing vascular and glomerular lesions and, promoting renal alterations through direct or indirect actions. These data seem to indicate that the renal alterations promoted by MCLR involves also phospholipase A(2) and arachidonic acid-derived mediators.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Dexametasona/uso terapêutico , Indometacina/uso terapêutico , Rim/efeitos dos fármacos , Peptídeos Cíclicos/antagonistas & inibidores , Fosfolipases A/fisiologia , Prostaglandina-Endoperóxido Sintases/fisiologia , Análise de Variância , Animais , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Inulina/metabolismo , Rim/enzimologia , Rim/metabolismo , Masculino , Toxinas Marinhas , Microcistinas , Peptídeos Cíclicos/toxicidade , Potássio/metabolismo , Ratos , Ratos Wistar , Sódio/metabolismoRESUMO
The S100 proteins are a family of calcium-binding proteins found in the central and peripheral nervous systems of vertebrates. S100beta, the most abundant member of this family in the CNS, mediates calcium signal transduction, and shows neurotrophic, gliotrophic and mitogenic actions that influence the development and maintenance of the nervous system. Another member of the S100 family (S100A10) was found to modulate phospholipid turnover by inhibiting the activity of enzyme phospholipase A2 (PLA2). We determined the concentration of S100beta protein in the plasma of 23 medicated schizophrenic patients and 23 healthy controls. S100beta protein accounts for 96% of the total S100 in the brain. Schizophrenic patients showed reduced S100beta concentrations (p=0.003), and this finding was not related to clinical variables or to intake of antipsychotic medication. Decreased S100beta could be related to the findings of increased PLA2 activity and to brain maldevelopment in schizophrenia. These results are discussed further with respect to the role of adenosine in S100beta release.
Assuntos
Proteínas S100/sangue , Esquizofrenia/fisiopatologia , Adulto , Encéfalo/fisiopatologia , Citosol/enzimologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Crescimento Neural , Fosfolipases A/fisiologia , Fosfolipases A2 , Escalas de Graduação Psiquiátrica , Valores de Referência , Subunidade beta da Proteína Ligante de Cálcio S100 , Esquizofrenia/diagnósticoRESUMO
Phospholipase A2 (PLA2), its localization on human sperm and its involvement in sperm-egg interaction, was investigated. Sperm-egg interaction was examined using an in vitro assay of the interaction between human sperm and zona-free or zona-intact hamster egg. PLA2- specific antibodies and/or lysophosphatidylcholine (LPC) were added to the coincubation medium. PLA2 was localized on the anterior tip of the human sperm head by an immunogold silver staining method in light microscopy (IGSS) and TEM. PLA2-specific antibodies inhibited human sperm-zona-free oocyte fusion significantly. LPC treatment allows interspecies fertilization of zona-intact hamster oocytes. PLA2 plays an important role in membrane-fusion events. This statement is supported by the fact that PLA2 is localized in the region where an exocytotic event, such as the acrosome reaction, occurs in the spermatozoon. PLA2-specific antibodies inhibited sperm-egg fusion, but not sperm-oolemma adhesion. LPC may stimulate the fertilizing ability of spermatozoa and induce changes on the zona pellucida and on the oolemma promoting in sperm-egg fusion. Based on these findings, it is suggested that sperm PLA2 and one of its modulators, the LPC, may contribute to membrane-fusion events in mammalian fertilization.
Assuntos
Lisofosfatidilcolinas/farmacologia , Fusão de Membrana/fisiologia , Oócitos/fisiologia , Fosfolipases A/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/enzimologia , Animais , Cricetinae , Feminino , Fertilização in vitro/efeitos dos fármacos , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Fusão de Membrana/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Fosfolipases A/imunologia , Fosfolipases A2 , Espermatozoides/imunologiaRESUMO
We examined the role of IFN gamma in protection against Toxoplasma gondii in the monocytoid cell line THP1. The addition of IFN gamma to cultured infected THP1 cells reduced the number of parasitized cells without altering intracellular multiplication during the first 24 hr. This reduction was potentiated by bacterial lipopolysaccharide (LPS). We also examined the role of an enzyme important for T. gondii cellular invasion, secretory phospholipase-A2 (sPLA2) and its relation with IFN gamma-induced protection. Treatment of cells or parasites with a specific inhibitor of sPLA2 significantly reduced the number of infected cells at 6 hr. The addition of exogenous sPLA2 from Naja naja venom did not interfere with the protective effect of IFN gamma and conferred protection when used alone. PLA2 activity was measured in supernatants of parasites maintained in the presence of IFN gamma, and the results suggested that IFN gamma opposes cell invasion by T. gondii by suppressing parasite production of PLA2.
Assuntos
Interferon gama/uso terapêutico , Monócitos/parasitologia , Fosfolipases A/fisiologia , Toxoplasma/enzimologia , Toxoplasma/patogenicidade , Toxoplasmose/prevenção & controle , Animais , Compostos de Boro/farmacologia , Linhagem Celular , Humanos , Líquido Intracelular/parasitologia , Monócitos/imunologia , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Fosfolipases A2 , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/enzimologia , Toxoplasmose/imunologiaRESUMO
Antecedentes: La actividad de fosfolipasa A2 (FLA2) ha sido relacionada con el incremento en los niveles de prostaglandina E2 (PGE2) en el líquido amniótico (LA). Altas concentraciones de este compuesto se encuentran en el LA en las siguientes condiciones: trabajo de parto, infección y ruptura prematura de membrana (RPM). Sin embargo la asociación entre este incremento y la FLA2 nunca ha sido demostrada. Objetivo: Estudiar la asociación entre la actividad FLA2 y la patogénesis de la RPM. Material y métodos: Se analizó la actividad de esta enzima en 3 grupos de LA: 1-Seguno trimestre 16-22 semanas de gestación (SG), 2- término 36-40 SG y 3- Ruptura Prematura de Membranas 28-36 SG, con y sin presencia de infección. Resultados: La actividad de FLA2 en los LA de segundo trimestre fue de 1.782 ñ 1.31 nanomolos de Fosfatidil-Colina degradada/mg proteína, mientras de RPM no infectadas tuvieron valores de 12.357 ñ 5.96 nm/mg y las infectadas de 29.077 ñ 17.91 nm/mg. Se estableció diferencias significativas entre las muestras de segundo trimestre y todos los demás grupos (p< 0.0001), entre RPM infectada y todos los grupos (p< 0.0001); y no se encontró diferencia entre los LA de término y de RPM no infectados. Discusión: Sugerimos la participación de la Fosfolipasa A2 en el mecanismo fisiológico de trabajo de parto y en el mecanismo patogénico de la RPM
Assuntos
Humanos , Feminino , Gravidez , Ruptura Prematura de Membranas Fetais/fisiopatologia , Líquido Amniótico/enzimologia , Fosfolipases A/análise , Fosfolipases A/fisiologiaRESUMO
In the present paper the properties of a phospholipase A2 (PLA2) activity associated with rod outer segments (ROS) have been studied. Under adequate experimental conditions, ROS PLA2 activity presented a maximum at pH 9.0. When using 1-palmitoyl-2[1- 14C]arachidonoyl-sn-glycero-3-phosphocholine as substrate, the apparent Km value was 36.5 microM and the Vmax value was 0.612 nanomol per hour per mg protein. The enzyme was fully activated at free calcium concentrations in the range 100- 300 microM. Concentrations of CaCl2 above 1 milliM inhibited its activity as a function of the ion concentration. The presence of EGTA or EDTA caused a 73% inhibition of PLA2 activity in ROS relative to the activity observed when no calcium was added. Treatment of the membranes with different kinds of detergents (Triton X-100, sodium deoxycholate and CHAPS) at concentrations below and above their critical micelle concentration resulted in an inhibition of PLA2 activity. However, when Triton X-100 was present at a concentration of 0.05 milliM, no significant change in enzymatic activity could be observed. Maximum inhibition was observed in the presence of CHAPS 25 milliM (87%). Seventy-five percent of PLA2 activity was recovered in ROS membranes after extraction of soluble and peripheral proteins. When retina phospholipids labelled with [3H]oleic acid and [3H]arachidonic acid were used as substrates, (diradyl)- ethanolamine glycerophospholipids (EtnGpl) were hydrolysed more efficiently than phosphatidylcholine (PtdCho). Moreover, hydrolysis of both phospholipids was stimulated when the substrates presented a higher degree of unsaturation in their fatty acyl components.
Assuntos
Fosfolipases A/química , Fosfolipases A/fisiologia , Segmento Externo da Célula Bastonete/enzimologia , Animais , Bovinos , Cromatografia em Camada Fina , Ativação Enzimática , Estabilidade Enzimática , Ácidos Graxos/análise , Concentração de Íons de Hidrogênio , Lipídeos de Membrana/análise , Fosfolipases A/isolamento & purificação , Fosfolipases A2 , Fosfolipídeos/análise , Retina/enzimologia , Especificidade por SubstratoRESUMO
Recently, we have shown that soluble factors released by human lymphocytes after lectin stimulation could increase the contractile tension of rat atria "in vitro" and that interleukin-2 (IL-2) could be part of this reaction. The effect of IL-2 was potentiated by the Ca2+ ionophore A23187 or free arachidonic acid (AA). In this study we demonstrate that the action of IL-2 can be prevented by pre-incubation of the heart tissue with monoclonal anti-IL-2 receptor (anti-p55), suggesting that binding to the IL-2 receptor is necessary for the induction of the biologic effect. In the presence of A23187 or AA, the effect of the synthetic diacylglyceride oleoyl-acetyl-glycerol (OAG) was similar to that of IL-2. Elimination of phospholipase C activity by pre-incubation of the atria with 2-nitro-carboxyphenyl,N,N'-diphenylcarbamate (NCDC) abrogated the effects of IL-2 in the presence of A23187 or AA, but was ineffective when OAG + A23187 or OAG + AA was used. Inhibition of atrial phospholipase A2 activity with p-bromo-phenacylbromide (BPB) blocked the response of atria to either IL-2 + A23187 or OAG + A23187 but was not effective when AA was used as second signal (IL-2 + AA or OAG + AA). Both the OAG and the IL-2 positive inotropic effects could be prevented by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H7) but were poorly inhibited by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), an inhibitor of the cyclic nucleotide-dependent protein kinases.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Interleucina-2/farmacologia , Contração Miocárdica/efeitos dos fármacos , Fosfolipases A/fisiologia , Proteínas Quinases/fisiologia , Fosfolipases Tipo C/fisiologia , Animais , Ácidos Araquidônicos/metabolismo , Átrios do Coração/efeitos dos fármacos , Lipoxigenase/metabolismo , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Prostaglandina-Endoperóxido Sintases/metabolismo , Inibidores de Proteínas Quinases , Ratos , Receptores de Interleucina-2/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
We briefly reviewed some ionic mechanisms participating in the fulfillment of the acrosome reaction processes, indicating the results obtained by the simultaneous and dynamic quantitation of sodium, potassium, calcium and hydrogen concentrations in the in incubation medium of human spermatozoa induced to achieve the acrosome reaction through the addition of cAMP and follicular fluid. At the same time, the participation of zinc and some enzymatic activities, like phospholipase A2, in the occurrence of this process, is indicated. A study is done about the sperm nuclear decondensation mechanisms, pointing at the importance of various participants in this event, like the disulfide groups reducing agents, some metal ions and the glycosaminoglycans, as well as a proposal of a mechanism which could be physiologically functional for the in vivo occurrence of this phenomenon. Finally we present some results explaining the DNA synthesis activation, which is repressed in the spermatozoa since the spermiogenesis final stages, and is indispensable for the chromosomic duplication required during egg segmentation.
Assuntos
Fertilização/fisiologia , Mamíferos/fisiologia , Acrossomo/fisiologia , Animais , Cátions/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Replicação do DNA , Feminino , Heparina/farmacologia , Humanos , Masculino , Fosfolipases A/fisiologia , Fosfolipases A2RESUMO
Because previous investigations have suggested that platelet activating factor and tumor necrosis factor-alpha (TNF-alpha) are important mediators of experimental necrotizing enterocolitis in the rat, we measured platelet activating factor, acetylhydrolase (the platelet activating factor breakdown enzyme), and TNF-alpha in the plasma of 12 human neonates with necrotizing enterocolitis and eight age-matched control subjects with similar gestational ages, postnatal ages, and weights. Almost all patients with necrotizing enterocolitis had elevated plasma platelet activating factor values (18.1 +/- 3.6 ng/ml vs. 3.1 +/- 0.9 ng/ml in control subjects, p less than 0.01). Plasma acetylhydrolase activity was lower in patients than in control subjects (10.6 +/- 0.7 nmol/ml/min vs 23.0 +/- 1.4 nmol/ml/min, p less than 0.01). Plasma TNF-alpha concentration was significantly elevated in patients with necrotizing enterocolitis (136 +/- 75 U/ml vs 1.5 +/- 0.8 U/ml, p less than 0.05), although the individual variation was high. There was no correlation between individual TNF-alpha and platelet activating factor levels. We conclude that platelet activating factor and TNF-alpha are elevated in patients with necrotizing enterocolitis and that suppressed platelet activating factor degradation contributes to the increased platelet activating factor levels; platelet activating factor and TNF-alpha may contribute to the pathophysiology of necrotizing enterocolitis.
Assuntos
Enterocolite Pseudomembranosa/fisiopatologia , Fator de Ativação de Plaquetas/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , 1-Alquil-2-acetilglicerofosfocolina Esterase , Enterocolite Pseudomembranosa/sangue , Seguimentos , Idade Gestacional , Humanos , Recém-Nascido , Fosfolipases A/sangue , Fosfolipases A/fisiologia , Fator de Ativação de Plaquetas/análise , Contagem de Plaquetas , Fator de Necrose Tumoral alfa/análiseAssuntos
Bungarotoxinas/toxicidade , Crotoxina/toxicidade , Junção Neuromuscular/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Acetilcolina/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Dose Letal Mediana , Camundongos , Fosfolipases A/fisiologia , Potássio/metabolismoRESUMO
Scanning electron microscopy was used to study the surface morphology of neural retina cells from fetal rats. Dissociated cells were plated on polyornithine and examined after 1.5-3.0 hr in vitro. A quantitative analysis of the proportion of cells with and without processes was made and the former were classified according to the length and number of their processes. Treatment with p-bromophenacyl bromide (BPB), a selective inhibitor of phospholipase A2 (PLA2), induced significant changes on the early surface activity of retinal cells. An inhibitory effect on cell process formation was observed in monolayers grown for 2 hr in the presence of BPB: process formation was also inhibited when high concentrations (10(-6) M or more) were applied as a 30 min pulse, whereas a similar pulse of a lower concentration (10(-7) M) stimulated the appearance of cells with short processes. These observations suggest that PLA2 or some other BPB-reactive substance is involved in the extension of neural cell processes.