RESUMO

Mapping B and T cell epitopes constitutes an important action for peptide vaccine design. PLD and CP40 virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a causal agent of Caseous Lymphadenitis, have been evaluated in a murine model as good candidates for vaccine development. Therefore, the goal of this work was to in silico analyze B and T cell epitopes of the PLD and CP40 proteins of a Mexican isolate of Corynebacterium pseudotuberculosis ovis. The Immune Epitope Data Base and Resource website was employed to predict the linear and conformational B-cell, T CD4+, and T CD8+ epitopes of PLD and CP40 proteins of Corynebacterium pseudotuberculosis ovis Mexican strain 2J-L. Fifty B cell epitopes for PLD 2J-L and forty-seven for CP40 2J-L were estimated. In addition, T CD4+ and CD8+ cell epitopes were predicted for PLD 2J-L (MHC I:16 epitopes, MHC II:10 epitopes) and CP40 2J-L (MHC I: 15 epitopes, MHC II: 13 epitopes). This study provides epitopes, paying particular attention to sequences selected by different predictor programs and overlap sequences as B and T cell epitopes. PLD 2J-L and CP40 2J-L protein epitopes may aid in the design of a promising peptide-based vaccine against Caseous Lymphadenitis in Mexico.

Assuntos

Infecções por Corynebacterium , Corynebacterium pseudotuberculosis , Linfadenite , Animais , Camundongos , Ovinos , Epitopos de Linfócito T , México , Biologia Computacional , Infecções por Corynebacterium/prevenção & controle , Vacinas de Subunidades Proteicas

RESUMO

We previously described the participation of canonical phospholipase D isoforms (PLD1 and PLD2) in the inflammatory response of retinal pigment epithelium (RPE) cells exposed to high glucose concentrations (HG). Here, we studied the role of the PLD pathway in RPE phagocytic function. For this purpose, ARPE-19 cells were exposed to HG (33 mM) or to normal glucose concentration (NG, 5.5 mM) and phagocytosis was measured using pHrodo™ green bioparticles® or photoreceptor outer segments (POS). HG exposure for 48 and 72 h reduced phagocytic function of ARPE-19 cells, and this loss of function was prevented when cells were treated with 5 µM of PLD1 (VU0359595 or PLD1i) or PLD2 (VU0285655-1 or PLD2i) selective inhibitors. Furthermore, PLD1i and PLD2i did not affect RPE phagocytosis under physiological conditions and prevented oxidative stress induced by HG. In addition, we demonstrated PLD1 and PLD2 expression in ABC cells, a novel human RPE cell line. Under physiological conditions, PLD1i and PLD2i did not affect ABC cell viability, and partial silencing of both PLDs did not affect ABC cell POS phagocytosis. In conclusion, PLD1i and PLD2i prevent the loss of phagocytic function of RPE cells exposed to HG without affecting RPE function or viability under non-inflammatory conditions.

Assuntos

Fosfolipase D , Linhagem Celular , Células Cultivadas , Glucose/metabolismo , Humanos , Fagocitose/fisiologia , Fosfolipase D/genética , Fosfolipase D/metabolismo , Epitélio Pigmentado da Retina/metabolismo

RESUMO

Classical phospholipase D (PLD) isoforms, PLD1 and PLD2, catalyze the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) which can be further dephosphorylated to diacylglycerol (DAG). Through the generation of these lipid messengers, the PLD pathway can modulate several cellular events, such as proliferation, membrane trafficking, autophagy and the inflammatory response, among many others. This review summarizes the participation of canonical PLD isoforms in physiological and pathological responses in the eye. Although the role of the PLD pathway in ocular and retinal response to stress has not been fully elucidated, pharmacological inhibition of these signaling enzymes seems to be a promising therapeutic tool to avoid inflammatory processes in the retina, abnormal cellular proliferation on the ocular surface and pathological neovascularization. On the contrary, the modulation of classical PLDs may potentiate corneal healing. In summary, the knowledge of the role of PLD1 and PLD2 in the molecular basis of ocular inflammatory and degenerative diseases opens new avenues for potential therapeutic exploration.

Assuntos

Fosfolipase D , Olho/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/metabolismo , Isoformas de Proteínas/metabolismo , Transdução de Sinais

RESUMO

BACKGROUND: Phospholipase D (PLD) is used as the biocatalyst for phosphatidylserine (PS) production. In general, PLD was expressed in insoluble form in Escherichia coli. High-level soluble expression of PLD with high activity in E. coli is very important for industrial production of PLD. RESULTS: Streptomyces chromofuscus PLD coding gene was codon-optimized, cloned without signal peptide, and expressed in E. coli. The optimal recombinant E. coli pET-28a+PLD/BL21(DE3) was constructed with pET-28a without His-tag. The highest PLD activity reached 104.28 ± 2.67 U/mL in a 250-mL shake flask after systematical optimization. The highest PLD activity elevated to 122.94 ± 1.49 U/mL by feeding lactose and inducing at 20 C after scaling up to a 5.0-L fermenter. Substituting the mixed carbon source with 1.0 % (w/v) of cheap dextrin and adding a feeding medium could still attain a PLD activity of 105. 81 ± 2.72 U/mL in a 5.0-L fermenter. Fish peptone from the waste of fish processing and dextrin from the starch are both very cheap, which were found to benefit the soluble PLD expression. CONCLUSIONS: After combinatorial optimization, the high-level soluble expression of PLD was fulfilled in E. coli. The high PLD activity along with cheap medium obtained at the fermenter level can completely meet the requirements of industrial production of PLD.

Assuntos

Fosfolipases/metabolismo , Streptomyces/enzimologia , Solubilidade , Streptomyces/genética , Temperatura , Códon , Técnicas de Química Combinatória , Escherichia coli

RESUMO

Chronic hyperglycemia, oxidative stress and inflammation are key players in the pathogenesis of diabetic retinopathy (DR). In this work we study the role of phospholipase D (PLD) pathway in an in vitro model of high glucose (HG)-induced damage. To this end, we exposed human retinal pigment epithelium (RPE) cell lines (ARPE-19 and D407) to HG concentrations (16.5 or 33 mM) or to normal glucose concentration (NG, 5.5 mM) for 4, 24 or 72 h. Exposure to HG increased reactive oxygen species levels and caspase-3 cleavage and reduced cell viability after 72 h of incubation. In addition, short term HG exposure (4 h) induced the activation of early events, that involve PLD and ERK1/2 signaling, nuclear factor kappa B (NFκB) nuclear translocation and IκB phosphorylation. The increment in pro-inflammatory interleukins (IL-6 and IL-8) and cyclooxygenase-2 (COX-2) mRNA levels was observed after 24 h of HG exposure. The effect of selective pharmacological PLD1 (VU0359595) and PLD2 (VU0285655-1) inhibitors demonstrated that ERK1/2 and NFκB activation were downstream events of both PLD isoforms. The increment in IL-6 and COX-2 mRNA levels induced by HG was reduced to control levels in cells pre-incubated with both PLD inhibitors. Furthermore, the inhibition of PLD1, PLD2 and MEK/ERK pathway prevented the loss of cell viability and the activation of caspase-3 induced by HG. In conclusion, our findings demonstrate that PLD1 and PLD2 mediate the inflammatory response triggered by HG in RPE cells, pointing to their potential use as a therapeutic target for DR treatment.

Assuntos

Retinopatia Diabética/metabolismo , Glucose/farmacologia , Fosfolipase D/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Interleucina-6/genética , Interleucina-8/genética , Microscopia Confocal , Microscopia de Fluorescência , Estresse Oxidativo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/metabolismo , Quinase Induzida por NF-kappaB

RESUMO

Linoleic acid (LA) is an essential and omega-6 polyunsaturated fatty acid that mediates a variety of biological processes, including migration and invasion in breast cancer cells. Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. Increases of expression and activity of PLD are reported in several human cancers, including gastric, colorectal, renal, stomach, lung and breast. In this article, we demonstrate that LA induces an increase of PLD activity in MDA-MB-231 breast cancer cells. Particularly, PLD1 and/or PLD2 mediate migration and invasion induced by LA. Moreover, LA induces increases in number and size of spheroids via PLD activity. FFAR1 also mediates migration and invasion, whereas PLD activation induced by LA requires the activities of FFAR1, FFAR4 and EGFR in MDA-MB-231 cells. In summary, PLD plays a pivotal role in migration and invasion induced by LA in MDA-MB-231 breast cancer cells.

Assuntos

Neoplasias da Mama/enzimologia , Movimento Celular/efeitos dos fármacos , Ácido Linoleico/farmacologia , Proteínas de Neoplasias/metabolismo , Fosfolipase D/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7 , Invasividade Neoplásica

RESUMO

BACKGROUND: Human Papillomavirus (HPV) infection is the main risk factor for the development and progression of cervical cancer. HPV-16 E6 and E7 expression is essential for induction and maintenance of the transformed phenotype. These oncoproteins interfere with the function of several intracellular proteins, including those controlling the PI3K/AKT/mTOR pathway in which Phospolipase D (PLD) and Phosphatidic acid (PA) play a critical role. METHODS: PLD activity was measured in primary human keratinocytes transduced with retroviruses expressing HPV-16 E6, E7 or E7 mutants. The cytostatic effect of rapamycin, a well-known mTOR inhibitor with potential clinical applications, was evaluated in monolayer and organotypic cultures. RESULTS: HPV-16 E7 expression in primary human keratinocytes leads to an increase in PLD expression and activity. Moreover, this activation is dependent on the ability of HPV-16 E7 to induce retinoblastoma protein (pRb) degradation. We also show that cells expressing HPV-16 E7 or silenced for pRb acquire resistance to the antiproliferative effect of rapamycin. CONCLUSION: This is the first indication that HPV oncoproteins can affect PLD activity. Since PA can interfere with the ability of rapamycin to bind mTOR, the use of combined strategies to target mTOR and PLD activity might be considered to treat HPV-related malignancies.

Assuntos

Papillomavirus Humano 16/genética , Proteínas E7 de Papillomavirus/genética , Fosfolipase D/metabolismo , Proteína do Retinoblastoma/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Expressão Gênica , Humanos , Queratinócitos/metabolismo , Queratinócitos/virologia , Modelos Biológicos , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Fosfolipase D/genética , Ligação Proteica , Sirolimo/farmacologia

RESUMO

Endocannabinoids (eCBs) and acylethanolamides (AEs) have lately received more attention due to their neuroprotective functions in neurological disorders. Here we analyze the alterations induced by perinatal asphyxia (PA) in the main metabolic enzymes and receptors of the eCBs/AEs in the dorsal striatum of rats. To induce PA, we used a model developed by Bjelke et al. (1991). Immunohistochemical techniques were carried out to determine the expression of neuronal and glial markers (NeuN and GFAP), eCBs/AEs synthesis and degradation enzymes (DAGLα, NAPE-PLD and FAAH) and their receptors (CB1 and PPARα). We found a decrease in NAPE-PLD and PPARα expression. Since NAPE-PLD and PPARα take part in the production and reception of biochemical actions of AEs, such as oleoylethanolamide, these results may suggest that PA plays a key role in the regulation of this system. These data agree with previous results obtained in the hippocampus and encourage us to develop further studies using AEs as potential neuroprotective compounds.

Assuntos

Asfixia Neonatal/metabolismo , Corpo Estriado/metabolismo , Endocanabinoides/metabolismo , Lipase Lipoproteica/metabolismo , PPAR alfa/metabolismo , Fosfolipase D/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Ratos , Ratos Sprague-Dawley

RESUMO

Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/ßII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and ß protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, ß and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage.

Assuntos

Fosfolipase D/metabolismo , Proteína Quinase C-épsilon/metabolismo , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/patologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Diglicerídeos/metabolismo , Humanos , Inflamação/enzimologia , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C-épsilon/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos

RESUMO

BACKGROUND: Corynebacterium xerosis is a commensal organism found in skin and mucous membranes of humans. It is considered an unusual pathogen, and it is rarely found in human and animal clinical samples. Here we describe the isolation of C. xerosis from a 4-months-old Pelifolk lamb located in Tesistán, central western Mexico. This microorganism should be considered for differential diagnosis in cutaneous abscessed lesions in sheep, as it represents a zoonotic risk factor for human infection in sheep farms. CASE PRESENTATION: The animal exhibited a hard-consistency, 5 cm diameter abscess, without drainage, in the neck. The presumptive clinical diagnosis was caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis. Samples were obtained by puncture and cultured in 8 % sheep blood agar under microaerophilic conditions. Colonies were non-haemolytic, brown-yellowish and showed microscopic and biochemical features similar to C. pseudotuberculosis, except for the urea test. A multiplex-PCR for the amplification of partial sequences of the pld, rpoB and intergenic fragment from 16S to 23S genes suggested that isolate could be C. xerosis, which was later confirmed by sequencing analysis of the rpoB gene. CONCLUSIONS: This study shows for the first time isolation and molecular characterization of C. xerosis from a clinical sample of an ovine cutaneous abscess in Mexico. This finding highlights the need for differential diagnosis of this pathogen in ovine skin abscesses, as well as epidemiological and control studies of this pathogen in sheep farms.

Assuntos

Abscesso/diagnóstico , Infecções por Corynebacterium/diagnóstico , Corynebacterium/isolamento & purificação , Genes Bacterianos , Linfadenite/diagnóstico , Doenças dos Ovinos/diagnóstico , Abscesso/microbiologia , Abscesso/patologia , Abscesso/veterinária , Animais , Corynebacterium/classificação , Corynebacterium/genética , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/patologia , Infecções por Corynebacterium/veterinária , Diagnóstico Diferencial , Linfadenite/microbiologia , Linfadenite/patologia , Linfadenite/veterinária , Masculino , México , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNA , Ovinos , Doenças dos Ovinos/microbiologia , Doenças dos Ovinos/patologia , Carneiro Doméstico , Pele/microbiologia , Pele/patologia

RESUMO

BACKGROUND: Although in vivo studies have implicated endocannabinoids in metabolic dysfunction, little is known about direct, chronic activation of the endocannabinoid system (ECS) in human islets. Therefore, this study investigated the effects of prolonged exposure to cannabinoid agonists on human islet gene expression and function. METHODS: Human islets were maintained for 2 and 5 days in the absence or presence of CB1r (ACEA) or CB2r (JWH015) agonists. Gene expression was quantified by RT-PCR, hormone levels by radioimmunoassay and apoptosis by caspase activities. RESULTS: Human islets express an ECS, with mRNAs encoding the biosynthetic and degrading enzymes NAPE-PLD, FAAH and MAGL being considerably more abundant than DAGLα, an enzyme involved in 2-AG synthesis, or CB1 and CB2 receptor mRNAs. Prolonged activation of CB1r and CB2r altered expression of mRNAs encoding ECS components, but did not have major effects on islet hormone secretion. JWH015 enhanced insulin and glucagon content at 2 days, but had no effect after 5 days. Treatment with ACEA or JWH015 for up to 5 days did not have marked effects on islet viability, as assessed by morphology and caspase activities. CONCLUSIONS: Maintenance of human islets for up to 5 days in the presence of CB1 and CB2 receptor agonists causes modifications in ECS element gene expression, but does not have any major impact on islet function or viability. GENERAL SIGNIFICANCE: These data suggest that the metabolic dysfunction associated with over-activation of the ECS in obesity and diabetes in humans is unlikely to be secondary to impaired islet function.

RESUMO

This article presents additional data regarding the study "The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium" [1]. The new data presented here show that short exposure of RPE cells to lipopolysaccharide (LPS) induces an early and transient activation of the extracellular signal-regulated kinase (ERK1/2). This early ERK1/2 activation is dependent on phosphatidylinositol bisphosphate-phospholipase C (PIP2-PLC). On the contrary, neither the phospholipase D 1 (PLD1) nor the PLD2 inhibition is able to modulate the early ERK1/2 activation induced by LPS in RPE cells.

RESUMO

Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.

Assuntos

Arginina Vasopressina/farmacologia , Glucose/metabolismo , Hiperglicemia/enzimologia , Complexos Multiproteicos/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Cromonas/farmacologia , Glucose/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Morfolinas/farmacologia , Miócitos de Músculo Liso/enzimologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Ratos

RESUMO

Loxosceles venom comprises a mixture of diverse toxins that induces intense local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure. Among several toxins in the venom, phospholipases D (PLDs), also called dermonecrotic toxins, are the most important and best studied, since they account for the main effects observed in loxoscelism. Despite their importance, biological analysis of PLDs is hampered by the minute amounts normally purified from the venom, and therefore many efforts have been made to clone those toxins. However, to date, no PLD from Loxosceles gaucho has been obtained in a heterologous system. Thus, in this work we show the cloning of a PLD from L. gaucho venom gland, named LgRec1, which was successfully expressed in a bacterial system. LgRec1 evoked local reaction (edema, erythema, ecchymosis, and paleness), dermonecrosis and hemolysis. It was also able to hydrolyze sphingomyelin and promote platelet aggregation. ELISA and Western blot analysis showed that LgRec1 was recognized by an anti-L. gaucho venom serum, a commercial arachnidic antivenom as well as a monoclonal antibody raised against the dermonecrotic fraction of L. gaucho venom. In addition, LgRec1 demonstrated to be highly immunogenic and antibodies raised against this recombinant toxin inhibited local reaction (~65%) and dermonecrosis (~100%) elicited by L. gaucho whole venom. Since PLDs are considered the major components accounting for the local and systemic envenomation effects caused by spiders from genus Loxosceles, the information provided here may help to understand the mechanisms behind clinical symptomatology.

Assuntos

Fosfolipase D/genética , Venenos de Aranha/genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Sequência de Bases , Clonagem Molecular , Reações Cruzadas , Expressão Gênica , Hemólise/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Fosfolipase D/imunologia , Fosfolipase D/metabolismo , Fosfolipase D/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Coelhos , Alinhamento de Sequência , Esfingomielina Fosfodiesterase/metabolismo , Relação Estrutura-Atividade

RESUMO

This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT(®). A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.

Assuntos

Proteínas de Bactérias/análise , Corynebacterium pseudotuberculosis/química , Corynebacterium pseudotuberculosis/classificação , Proteoma , Animais , Proteínas de Bactérias/química , Infecções por Corynebacterium/microbiologia , Infecções por Corynebacterium/veterinária , Eletroforese em Gel Bidimensional/métodos , Linfadenite/microbiologia , Linfadenite/veterinária , Proteômica/métodos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos

RESUMO

BACKGROUND: Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation. METHODS: J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases. RESULTS: ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP. CONCLUSIONS: Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin. GENERAL SIGNIFICANCE: ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.

Assuntos

Quinase 2 de Adesão Focal/metabolismo , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Estresse Oxidativo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores Purinérgicos P2X7/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , Humanos , Transporte de Íons , Macrófagos/enzimologia , Camundongos

RESUMO

Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).

Assuntos

Animais , Bovinos , Coelhos , Infecções por Corynebacterium , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/isolamento & purificação , Fosfolipase D/genética , Fosfolipase D/isolamento & purificação , Regulação da Expressão Gênica , Técnicas In Vitro , Linfadenite , RNA , Búfalos , Eletroforese , Ativação Enzimática , Métodos , Coelhos , Ovinos

RESUMO

Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).

RESUMO

Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).