RESUMO
This study tested the hypothesis that heparanase (HPSE) is related to tumor metastasis and curcumin (CCM) inhibits tumor metastasis by down-regulating HPSE expression. MTT, Transwell assays, and RT-PCR were used to study the effects of CCM on the migration and invasion of CT26 cells and the expression of HPSE. CT26 cells were transfected with lentivirus to establish HPSE-overexpressing cells (OE) and corresponding negative control cells (NC). Signal pathways involved in down-regulating the expression of HPSE and inhibiting the migration and invasion of CT26 cells by CCM were screened by the liquid crystal chip. HPSE promoted CT26 cells migration and invasion, and CCM inhibited the proliferation and metastasis of CT26 cells. The results of RT-PCR indicated that CCM down-regulated HPSE expression. Liquid phase microarray showed that CCM inhibited the phosphorylation of P38 and STAT5 in CT26 cells and NC cells. In contrast, the inhibitory function of CCM was markedly enhanced when HPSE was overexpressed (P < 0.05). In short, HPSE is closely related to metastasis of colon cancer cells. CCM inhibits colon cancer cell migration and invasion by inhibiting HPSE expression, which may be related to P38 MAPK and JAK/STAT5 signal pathways.
Assuntos
Neoplasias do Colo/imunologia , Curcumina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucuronidase/imunologia , Proteínas de Neoplasias/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Metástase NeoplásicaRESUMO
Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid (ARA) to prostaglandins, and COX-mediated metabolites play important roles in the regulation of lipid metabolism and immunity in mammals. However, such roles of COX in fish remain largely unknown. In this study, we designed three semi-purified diets, namely ARA-free (control), ARA, and ARA + acetylsalicylic acid (ASA; a COX inhibitor), and used them to feed grass carp (27.65 ± 3.05 g) for 8 weeks. The results showed that dietary ARA significantly increased the amount of ARA in the hepatopancreas, muscle, and kidney (P < 0.05), whereas this increase was reduced by dietary ASA. The hepatopancreatic prostaglandin E2 content increased in the ARA group, and this increase was inhibited by ASA (P < 0.05). ARA decreased the lipid content in the hepatopancreas, whereas ASA recovered lipid content to a significant level (P < 0.05). ARA significantly decreased the messenger RNA (mRNA) expression levels of fatty acid synthase and stearoyl-CoA desaturase in the hepatopancreas (P < 0.05). However, ASA did not rescue the mRNA expression of these genes (P > 0.05). Interestingly, ARA significantly enhanced the level of peroxisome proliferator-activated receptor α gene expression, and this increase was attenuated by ASA (P < 0.05). Finally, ARA significantly enhanced the mRNA expression of myeloid differentiation factor 88 (MyD88) in the kidney, and ASA attenuated the expression of toll-like receptor 22 and MyD88 (P < 0.05). In conclusion, our findings suggest that COX metabolites play important roles in the inhibition of lipid accumulation in the hepatopancreas of grass carp fed with ARA and that regulation of gene expression promotes lipid catabolism rather than lipogenic activities. Additionally, these eicosanoids might participate in the upregulation of immunity-related genes in the kidney.
Assuntos
Ácido Araquidônico/farmacologia , Carpas/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/fisiologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Ração Animal/análise , Animais , Ácido Araquidônico/administração & dosagem , Carpas/genética , Dieta/veterinária , Suplementos Nutricionais , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Regulação Enzimológica da Expressão Gênica/imunologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Prostaglandina-Endoperóxido Sintases/genéticaRESUMO
Tuberculosis causes severe immunosuppression thereby ensuring the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates TLR-2 receptor down-stream signaling, indicating the possible involvement of TLR-2 in the regulation of the host immune response. Moreover, different PKC isoforms are also involved in the course of infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immuno-modulatory properties which induce the pro-inflammatory responses via induction of TLR-2-mediated signaling. Here, we found that pretreatment of M. tuberculosis-infected macrophages with Ara-LAM caused a significant increase in the conventional PKC expression along with their active association with TLR-2. This association activated the TLR-2 -mediated downstream signaling, facilitating the activation of MAP kinase P38. All these events culminated in the up-regulation of proinflammatory response, which was abrogated by treatment with PKC-α and P38 inhibitors. Moreover, pretreatment of macrophages with Ara-LAM abrogated the IL-10 production while restored MHC-II expression in the infected macrophages. This study demonstrates that Ara-LAM confers protection against tuberculosis via TLR-2/PKC signaling crosstalk which is responsible for the induction of host protective immune response against tuberculosis.
Assuntos
Antituberculosos/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/microbiologia , Proteína Quinase C/fisiologia , Tuberculose/imunologia , Animais , Arabinose , Células Cultivadas , Citocinas/biossíntese , Avaliação Pré-Clínica de Medicamentos/métodos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Mediadores da Inflamação/metabolismo , Isoenzimas/biossíntese , Isoenzimas/genética , Macrófagos Peritoneais/enzimologia , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas Quinases Ativadas por Mitógeno/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/biossíntese , Nitritos/metabolismo , Proteína Quinase C/biossíntese , Proteína Quinase C/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/metabolismo , Tuberculose/enzimologia , Tuberculose/patologia , Regulação para Cima/efeitos dos fármacosRESUMO
The objective of this study was to evaluate the effect of dietary supplementation of a Rubus coreanus ethanolic extract on immunostimulatory response in white leg shrimp Penaeus vannamei. Shrimps with an average initial weight of 0.5 ± 0.04 g were collected and acclimatized for 10 days. Four experimental diets including a control diet, a probiotic diet and 0.25 and 0.5% of R. coreanus ethanolic extract (RcEE) diets were used to feed the shrimps. After 8 weeks of culture, shrimp fed with probiotic and 0.25% RcEE diet had showed significant enhancement in the growth while shrimp fed with 0.5% RcEE diet showed significantly increased expression of immune genes and antioxidant enzymes activities. One week of challenge experiments for all the four diets fed shrimps showed decreased cumulative mortality in the 0.5% RcEE diets fed shrimps, when compared with the probiotic and 0.25% RcEE diet fed shrimp groups. The results indicates that R. coreanus ethanolic extract could be used as a herbal immunostimulant for shrimps to increase its immunity and disease resistance against the bacterial pathogen, Vibrio alginolyticus.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Penaeidae/imunologia , Penaeidae/metabolismo , Extratos Vegetais/farmacologia , Piretrinas/química , Rosaceae/química , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Ração Animal/análise , Animais , Antocianinas/química , Antocianinas/farmacologia , Catalase/genética , Catalase/metabolismo , Dieta , Regulação Enzimológica da Expressão Gênica/imunologia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Penaeidae/enzimologia , Extratos Vegetais/química , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Matrix metalloproteinase-9 (MMP-9) belongs to a family of zinc-dependent endopeptidases and is associated with vital inflammatory processes. Here, we isolated and characterized MMP-9 cDNA from grass carp (Ctenopharyngodon idella) (designated as CiMMP-9). The cDNA was 2880 bp long and encoded a putative protein of 675 amino acids, with a predicted molecular mass of 75.816 kDa and an isoelectric point (pI) of 5.25. CiMMP-9 contained all three classical MMP-9 family signatures. The mRNA of CiMMP-9 was constitutively expressed in all tested tissues of untreated grass carp, with the highest expression levels in the blood, trunk kidney, head kidney and spleen. CiMMP9 transcript was present in unfertilized eggs, which suggests that CiMMP9 transcription is maternally inherited. Fluorescent real-time quantitative RT-PCR was used to examine the expression of the CiMMP-9 gene in C. idella after being challenged with Aeromonas hydrophila. A clear time-dependent expression pattern of CiMMP-9 was found after the bacterial challenge, and mRNA expression reached a maximum level at 7 days post challenge. This indicates that MMP-9 is inducible and is involved in immune responses, thus suggesting that CiMMP-9 plays an important role in A. hydrophila-related diseases and in early embryonic development stages in C. idella.
Assuntos
Aeromonas hydrophila/fisiologia , Carpas/metabolismo , Doenças dos Peixes/metabolismo , Regulação Enzimológica da Expressão Gênica/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Metaloproteinase 9 da Matriz/metabolismo , Sequência de Aminoácidos , Animais , Carpas/embriologia , Carpas/genética , Carpas/imunologia , Clonagem Molecular , DNA Complementar/genética , Embrião não Mamífero/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/metabolismo , Infecções por Bactérias Gram-Negativas/microbiologia , Larva/metabolismo , Metaloproteinase 9 da Matriz/genética , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de SequênciaRESUMO
Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cytMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of the above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus. A phylogenetic analysis of the amino acid sequences showed that C. quadricarinatus proPO and cytMnSOD were more closely related to the proPO and cytMnSOD of other crayfish than to those of penaeids, crabs, lobsters, or freshwater prawns. A tissue distribution analysis revealed that proPO was primarily expressed in hemocytes, gills, and the heart, while cytMnSOD was detected in all tissues examined. All of the crayfish artificially infected with white spot syndrome virus (WSSV) died within 4 days. According to a non-lethal dose, there was no mortality in crayfish when infected deliberately with Aeromonas hydrophila. Total hemocyte counts (THCs) had significantly decreased in crayfish at 48 and 72 h after infection with WSSV compared to the control group. In contrast, THCs of crayfish after A. hydrophila challenge had recovered by 48 and 72 h from a lower level at 24 h. There were similar responses in enzyme activities toward WSSV and A. hydrophila infection. Phenoloxidase (PO) and superoxide dismutase (SOD) activities per hemocyte significantly increased from 48 to 72 h compared to the control group. After WSSV challenge, expressions of proPO and cytMnSOD transcripts in hemocytes significantly decreased at 12h, then had respectively recovered and increased at 24 h. At 48-72 h, transcript levels were finally downregulated. No significant differences in the expression profiles of proPO and cytMnSOD were observed between the A. hydrophila-infected and control groups, besides the significant upregulation at 24h post-infection. These results implicate proPO and cytMnSOD in the immune response, and they presented similar expression patterns, although different defense mechanisms may exist for crayfish induced by WSSV and A. hydrophila.
Assuntos
Aeromonas hydrophila/imunologia , Astacoidea/imunologia , Catecol Oxidase/imunologia , Precursores Enzimáticos/imunologia , Superóxido Dismutase/imunologia , Vírus da Síndrome da Mancha Branca 1/imunologia , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Astacoidea/microbiologia , Astacoidea/virologia , Sequência de Bases , Catecol Oxidase/genética , Catecol Oxidase/metabolismo , Clonagem Molecular , Citosol/enzimologia , Citosol/imunologia , DNA Complementar/química , DNA Complementar/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Água Doce , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/imunologia , Hemócitos/imunologia , Hemócitos/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Análise de Sobrevida , Fatores de Tempo , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
Cinnamomum cambodianum has been used as a traditional medicine in Cambodia. Its effect on the bone marrow-derived mast cells (BMMCs) mediated allergic response remains unknown. In this study, a chloroform-soluble extract of C. cambodianum was evaluated for its effect on allergic mediators, including prostaglandin D2 (PGD2), leukotriene C4 (LTC4), ß-hexosaminidase and cyclooxygenase-2 (COX-2) protein, in phorbol 12-myristate 13-acetate (PMA) plus calcimycin-stimulated BMMCs. The results revealed that the chloroform-soluble extract inhibited the production of interleukin-6, PGD2 and LTC4, and the expression of COX-2 in PMA plus calcimycin-stimulated BMMCs, implying a potential benefit of C. cambodianum in the treatment of allergy.
Assuntos
Células da Medula Óssea/imunologia , Clorofórmio/química , Cinnamomum/química , Misturas Complexas , Hipersensibilidade/tratamento farmacológico , Mastócitos/imunologia , Animais , Células da Medula Óssea/metabolismo , Calcimicina/farmacologia , Ionóforos de Cálcio/farmacologia , Carcinógenos/farmacologia , Misturas Complexas/química , Misturas Complexas/farmacologia , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/imunologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Leucotrieno C4/imunologia , Leucotrieno C4/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Prostaglandina D2/imunologia , Prostaglandina D2/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Glutathione peroxidases (GPx) are key enzymes in the antioxidant systems of living organisms by catalyzing the reduction of peroxides to non-reactive products. In the present study, the full-length cDNA encoding a selenium-dependent GPx was identified from Venerupis philippinarum (designated as VpSe-GPx), and the spatial and temporal expression patterns post-Vibrio anguillarum, heavy metals and benzo[a]pyrene (B[a]P) challenge were also investigated. VpSe-GPx possessed all the conserved features critical for the fundamental structure and function of glutathione peroxidase. The VpSe-GPx mRNA was found to be most abundantly expressed in hepatopancreas. Vibrio challenge could significantly up-regulate the mRNA expression of VpSe-GPx, and the highest expression level was detected at 24 h post-infection with 6.5-fold increase compared with that in the control group. For heavy metals exposure, the expression of VpSe-GPx was significantly induced by 20, 40 µg L(-1) Cd and 10, 20 µg L(-1) Cu but depressed by 10 µg L(-1) Cd and 40 µg L(-1) Cu. With regards to B[a]P exposure, the expression of VpSe-GPx mRNA was significantly induced at 48 and 96 h post challenge. All these results suggested that VpSe-GPx was potentially involved in mediating the immune response and antioxidant defense in V. Philippinarum.
Assuntos
Bivalves/enzimologia , Bivalves/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Vibrio/imunologia , Animais , Sequência de Bases , Benzo(a)pireno/toxicidade , Bivalves/microbiologia , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hepatopâncreas/metabolismo , Metais Pesados/toxicidade , Dados de Sequência Molecular , Análise de Sequência de DNA , Fatores de TempoRESUMO
We investigated the effects of Pycnogenol supplementation on the arachidonic acid pathway in human polymorphonuclear leukocytes (PMNL) in response to an inflammatory stimulus. Pycnogenol is a standardised extract of French maritime pine bark consisting of procyanidins and polyphenolic monomers. Healthy volunteers aged 35 to 50 years were supplemented with 150 mg Pycnogenol a day for five days. Before and after the final day of supplementation, blood was drawn and PMNL were isolated. PMNL were primed with lipopolysaccharide (LPS) and stimulated with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP) to activate the arachidonic acid pathway and the biosynthesis of leukotrienes, thromboxane and prostaglandins. Pycnogenol supplementation inhibited 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) gene expression and phospholipase A2 (PLA2) activity. This effect was associated with a compensatory up-regulation of COX-1 gene expression. Interestingly, Pycnogenol suspended the interdependency between 5-LOX and 5-lipoxygenase activating protein (FLAP) expression. Pycnogenol supplementation reduced leukotriene production but did not leave prostaglandins unaltered, which we attribute to a decline of COX-2 activity in favour of COX-1. Here we show for the first time that Pycnogenol supplementation simultaneously inhibits COX-2 and 5-LOX gene expression and reduces leukotriene biosynthesis in human PMNL upon pro-inflammatory stimulation ex vivo.
Assuntos
Anti-Inflamatórios/administração & dosagem , Araquidonato 5-Lipoxigenase/metabolismo , Ciclo-Oxigenase 2/metabolismo , Flavonoides/administração & dosagem , Neutrófilos/efeitos dos fármacos , Adulto , Araquidonato 5-Lipoxigenase/genética , Araquidonato 5-Lipoxigenase/imunologia , Ácidos Araquidônicos/metabolismo , Células Cultivadas , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/imunologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Lipopolissacarídeos/imunologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/patologia , Fosfolipases A2/genética , Fosfolipases A2/imunologia , Fosfolipases A2/metabolismo , Extratos Vegetais , RNA Mensageiro/análiseRESUMO
Synthesis of nitric oxide (NO) is one of the important effector functions of innate immune cells. Although several reports have indicated mistletoe lectins induce immune cells to produce cytokines, studies regarding the activities of the lectins in the production of NO have been very limited. Here, we report on the induction of NO synthesis in a murine macrophage cell line, RAW264.7, by Korean mistletoe lectin (KML-IIU). When the macrophage cells were treated with KML-IIU in the presence of a suboptimal concentration of IFN-gamma, NO production was induced in a concentration-dependent manner. Significantly higher levels of NO were induced by subchains of the KML-IIU (A and B), which have lower toxicities, as compared to the hololectin. Furthermore, expression of the inducible nitric oxide synthase (iNOS) gene was elevated in accordance with the level of NO production. When the synthase was inhibited by iNOS inhibitors (L-NIL and L-NAME), NO production was specifically reduced in a concentration-dependent manner. Our studies demonstrate that the KML-IIU and its subchains induce NO production in murine macrophage cells via activation of the iNOS gene expression, suggesting that the KML-IIU subchains may be used as an immunomodulator to enhance the effector functions of innate immune cells.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Macrófagos/enzimologia , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico/biossíntese , Preparações de Plantas/farmacologia , Proteínas de Plantas/farmacologia , Proteínas Inativadoras de Ribossomos/farmacologia , Toxinas Biológicas/farmacologia , Animais , Antivirais/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/imunologia , Imunidade Inata/efeitos dos fármacos , Interferon gama/farmacologia , Lisina/análogos & derivados , Lisina/farmacologia , Macrófagos/imunologia , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/imunologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/imunologia , Proteínas Inativadoras de Ribossomos Tipo 2RESUMO
OBJECTIVE: Our laboratory has previously created 2 strains of rabbits with genetically determined high-atherosclerotic response (HAR) and low-atherosclerotic response (LAR). The aim of the present study was to identify new genes of atherosclerosis susceptibility in macrophages from the 2 strains. METHODS AND RESULTS: Suppression subtractive hybridization was used to screen for genes with higher expression in macrophages from LAR rabbits. We identified a cDNA fragment with high homology to human arginase I (AI; 91%) and subsequently cloned the full-length cDNA of the rabbit homologue. Quantitative RT-PCR revealed a significantly higher macrophage AI mRNA expression in LAR rabbits than in HAR rabbits (77428+/-10941 versus 34344+/-4538; P=0.002; copies/10(6) copies beta-actin), which also correlated with a significantly higher arginase enzyme activity. Northern blot analysis led to the identification of a size polymorphism of AI mRNA. This was because of a 413 bp C-repeat insertion in the 3' untranslated region. The shorter transcript variant was predominantly expressed in LAR rabbits and associated with significantly higher AI mRNA expression levels. Transfection experiments indicated decreased mRNA stability of the long AI variant. CONCLUSIONS: High expression of arginase I in macrophages may contribute to atherosclerosis resistance of LAR rabbits, possibly by conferring antiinflammatory effects in the vessel wall.
Assuntos
Arginase/genética , Aterosclerose/genética , Aterosclerose/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Macrófagos Peritoneais/enzimologia , Animais , Arginase/metabolismo , DNA Complementar/genética , Suscetibilidade a Doenças , Macrófagos Peritoneais/imunologia , Regiões Promotoras Genéticas/genética , Estabilidade de RNA , RNA Mensageiro/genética , CoelhosRESUMO
A copper/zinc superoxide dismutase (Cu,Zn-SOD) cDNA was cloned from the hepatopancreas of giant freshwater prawn Macrobrachium rosenbergii using reverse transcription-polymerase chain reaction (RT-PCR) by degenerate primers. Both 3'- and 5'-regions were isolated by the rapid amplification of cDNA ends method. Analysis of nucleotide sequence revealed that the Cu,Zn-SOD cDNA clone consists of 845 bp with an open reading frame of 603 bp encoding a protein of 201 amino acids with a 22 amino acid signal peptide. The calculated molecular mass of the mature proteins (179 amino acids) is 21 kDa with an estimated pI of 4.75. Two putative N-glycosylation sites, NXT and NXS, were observed in the Cu,Zn-SOD. Four conserved amino acids responsible for binding copper (H86, H89, H106 and H163) and four conserved amino acids responsible for binding zinc (H106, H114, H123 and D126) were observed. Sequence comparison showed that the Cu,Zn-SOD deduced amino acid sequence of M. rosenbergii has similarity of 60% and 64% to that of freshwater crayfish Pacifastacus leniusculus ecCu,Zn-SOD and blue crab Callinectes sapidus ecCu,Zn-SOD, respectively. Quantitative real-time RT-PCR analysis showed that Cu,Zn-SOD transcripts in haemocytes of M. rosenbergii increased 3h and 6h after injection of Lactococcus garvieae, whereas Cu,Zn-SOD transcripts decreased in the hepatopancreas 3h after L. garvieae injection.
Assuntos
Regulação Enzimológica da Expressão Gênica/imunologia , Palaemonidae/enzimologia , Palaemonidae/genética , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Estruturas Animais/imunologia , Animais , Sequência de Bases/genética , Clonagem Molecular/métodos , Primers do DNA/química , DNA Complementar/química , DNA Complementar/genética , Água Doce , Hemócitos/imunologia , Hemócitos/fisiologia , Hepatopâncreas/enzimologia , Hepatopâncreas/imunologia , Lactococcus/imunologia , Dados de Sequência Molecular , Palaemonidae/imunologia , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência/veterinária , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/química , Superóxido Dismutase/imunologia , Fatores de TempoRESUMO
Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.
Assuntos
Antineoplásicos/farmacologia , Ceramidas/metabolismo , Interleucina-2/farmacologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Regulação Enzimológica da Expressão Gênica/imunologia , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Humanos , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , RNA Mensageiro/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Transcrição Gênica/imunologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Tirfostinas/farmacologiaRESUMO
OBJECTIVE: Antibodies directed to citrulline-containing proteins are highly specific for rheumatoid arthritis (RA) and can be detected in up to 80% of patients with RA. Citrulline is a nonstandard amino acid that can be incorporated into proteins only by posttranslational modification of arginine by peptidylarginine deiminase (PAD) enzymes. The objective of this study was to investigate the presence of anticitrulline antibodies, PAD enzymes, and citrullinated antigens in mouse models of both acute and chronic destructive arthritis: streptococcal cell wall (SCW)-induced arthritis and collagen-induced arthritis (CIA), respectively. METHODS: Synovial tissue biopsy specimens were obtained from naive mice, mice with CIA, and mice with SCW-induced arthritis. The expression of messenger RNA (mRNA) for PAD enzymes was analyzed by reverse transcriptase-polymerase chain reaction; the presence of PAD proteins and their products (citrullinated proteins) was analyzed by Western blotting and by immunolocalization. The presence of anticitrullinated protein antibodies was investigated by an anti-cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) and an ELISA using in vitro citrullinated fibrinogen. RESULTS: In both mouse models, PAD type 2 (PAD2) mRNA was present in the synovium but was not translated into PAD2 protein. In contrast, PAD4 mRNA, although absent from healthy synovium, was readily transcribed and translated by polymorphonuclear neutrophils infiltrating the synovial tissue during inflammation. As a consequence, several synovial proteins were subjected to citrullination. One of these proteins was identified as fibrin, which has been reported to be citrullinated also in synovium of patients with RA. Although generation of citrullinated antigens during synovial inflammation in the mice was eminent, no anti-CCP antibodies could be detected. CONCLUSION: Citrullination of synovial antigens is an active process during joint inflammation in both mice and humans, but the induction of autoantibodies directed to these proteins is a more specific phenomenon, detectable only in human RA patients.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Citrulina/metabolismo , Membrana Sinovial/imunologia , Animais , Especificidade de Anticorpos , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Autoanticorpos/sangue , Biópsia , Citrulina/imunologia , Modelos Animais de Doenças , Epitopos , Regulação Enzimológica da Expressão Gênica/imunologia , Hidrolases/genética , Hidrolases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteína-Arginina Desiminase do Tipo 2 , Proteína-Arginina Desiminase do Tipo 3 , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Proteínas/imunologia , Proteínas/metabolismo , RNA Mensageiro/análise , Líquido Sinovial/imunologia , Membrana Sinovial/enzimologia , Membrana Sinovial/patologiaAssuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Adjuvantes Imunológicos/metabolismo , Amilose/biossíntese , Solanum tuberosum/metabolismo , Amido/biossíntese , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/imunologia , Adjuvantes Imunológicos/genética , Amilose/análise , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Camelídeos Americanos/imunologia , Camelídeos Americanos/metabolismo , Inibidores Enzimáticos/imunologia , Inibidores Enzimáticos/metabolismo , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação da Expressão Gênica de Plantas/imunologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Solanum tuberosum/imunologia , Amido/química , Amido/imunologiaRESUMO
Immunomodulation involves the use of antibodies to alter the function of molecules and is an emerging tool for manipulating both plant and animal systems. To realize the full potential of this technology, two major obstacles must be overcome. First, most antibodies do not function well intracellularly because critical disulfide bonds cannot form in the reducing environment of the cytoplasm or because of difficulties in targeting to subcellular organelles. Second, few antibodies bind to the active sites of enzymes and thus they generally do not neutralize enzyme function. Here we show that the unique properties of single-domain antibodies from camelids (camels and llamas) can circumvent both these obstacles. We demonstrate that these antibodies can be correctly targeted to subcellular organelles and inhibit enzyme function in plants more efficiently than antisense approaches. The use of these single-domain antibody fragments may greatly facilitate the successful immunomodulation of metabolic pathways in many organisms.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Adjuvantes Imunológicos/metabolismo , Cadeias Pesadas de Imunoglobulinas/imunologia , Solanum tuberosum/metabolismo , Amido/biossíntese , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/imunologia , Adjuvantes Imunológicos/genética , Amilose/análise , Amilose/biossíntese , Animais , Camelídeos Americanos/imunologia , Camelídeos Americanos/metabolismo , Inibidores Enzimáticos/imunologia , Inibidores Enzimáticos/metabolismo , Regulação Enzimológica da Expressão Gênica/imunologia , Regulação da Expressão Gênica de Plantas/imunologia , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Tubérculos/enzimologia , Tubérculos/genética , Tubérculos/imunologia , Tubérculos/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Solanum tuberosum/imunologia , Amido/química , Amido/imunologiaRESUMO
Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high levels of PG production during inflammation and immune responses. Previous studies with pharmacological inhibitors suggested a role for protein kinase C (PKC) in PG production possibly by regulating COX-2 expression. In this study, we addressed the role of PKC-alpha in the modulation of COX-2 expression and PGE2 synthesis by the overexpressing of a dominant-negative (DN) mutant of this isoenzyme in the mouse macrophage cell line RAW 264.7. We investigated the effect of various stimuli on COX-2 expression, namely, LPS, IFN-gamma, and the intracellular parasite Leishmania donovani. Whereas LPS-induced COX-2 mRNA and protein expression were down-regulated in DN PKC-alpha-overexpressing clones, IFN-gamma-induced COX-2 expression was up-regulated in DN PKC-alpha-overexpressing clones with respect to normal RAW 264.7 cells. Measurements of PGE2 levels revealed a strong correlation between PGE2 secretion and IFN-gamma-induced COX-2 mRNA and protein levels in DN PKC-alpha-overexpressing clones. Taken together, these results suggest a role for PKC-alpha in the modulation of LPS- and IFN-gamma-induced COX-2 expression, as well as in IFN-gamma-induced PGE2 secretion.
Assuntos
Adjuvantes Imunológicos/fisiologia , Isoenzimas/biossíntese , Isoenzimas/fisiologia , Macrófagos/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Proteína Quinase C/fisiologia , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Animais , Linhagem Celular , Células Clonais , Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Genes Dominantes , Interferon gama/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Fagocitose/imunologia , Regiões Promotoras Genéticas/imunologia , Prostaglandina-Endoperóxido Sintases/genética , Prostaglandina-Endoperóxido Sintases/metabolismo , Proteína Quinase C/biossíntese , Proteína Quinase C/genética , Proteína Quinase C-alfaRESUMO
In response to endotoxemia induced by administration of lipopolysaccharide, a complex series of reactions occurs in mammalian tissues. During this inflammation response, cells produce different mediators, such as reactive oxygen species, a number of arachidonic acid metabolites, and cytokines. The reactive oxygen species thus generated have been suggested to produce tissue injury as a result of macromolecular damage or by interfering with regulatory processes. They may also act as important signaling molecules to induce redox-sensitive genes. We report here that transgenic mice overexpressing 2 major forms of human glutathione peroxidases (GPs), intra- and extracellular GP, are able to modulate host response during endotoxemic conditions. We show that these animals have a decreased hypotension and increased survival rate after administration of a high dosage of lipopolysaccharide. Overexpression of GPs alters vascular permeability and production of cytokines (interleukin-1 beta and tumor necrosis factor-alpha) and NO, affects arachidonic acid metabolism, and inhibits leukocyte migration. These results suggest an important role for peroxides in pathogenesis during endotoxemia, and GPs, by regulating their level, may prove to be good candidates for antioxidant therapy to protect against such injury.
Assuntos
Endotoxemia/imunologia , Endotoxemia/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biossíntese , Animais , Pressão Sanguínea , Permeabilidade Capilar/fisiologia , Movimento Celular/imunologia , Eicosanoides/sangue , Endotoxemia/induzido quimicamente , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Interleucina-1/genética , Leucócitos/citologia , Leucócitos/imunologia , Leucócitos/metabolismo , Leucotrieno B4/biossíntese , Peroxidação de Lipídeos/imunologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Óxido Nítrico/biossíntese , Estresse Oxidativo/imunologia , Ativação Plaquetária/fisiologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Delta opioid receptors (DOR) are G-protein coupled 7-transmembrane receptors (GPCR), expressed by thymic and splenic T cells, that modulate interleukin (IL)-2 production and proliferation in response to concanavalin A or crosslinking the TCR. Mitogen-activated protein kinases (MAPKs) are involved in mediating intracellular responses to TCR crosslinking. In addition, MAPKs can be activated by signaling cascades that are initiated by the release of G-proteins from GPCRs. To determine whether DORs expressed by T cells signal through the MAPKs, extracellular-regulated kinases (ERKs) 1 and 2, two delta opioid peptides, deltorphin and [D-Ala2,D-Leu5]-enkephalin (DADLE), were studied in Jurkat cells that had been stably transfected with DOR (DOR-Ju.1). These peptides rapidly and dose-dependently induced ERK phosphorylation; pretreatment with naltrindole (NTI), a selective DOR antagonist, abolished this. Pertussis toxin (PTX) also inhibited phosphorylation, indicating the involvement of the Gi/o proteins. Herbimycin A, a protein tyrosine kinase (PTK) inhibitor, reduced the DADLE-induced ERK phosphorylation by 68%. ERK phosphorylation was inhibited by Bisindolylmaleimide 1 (GF109203X), an inhibitor of PKC, and by pretreatment with PMA prior to DADLE. A GTP/GDP exchange assay was used to assess the potential role of Ras in the pathway leading to ERK phosphorylation; DADLE failed to stimulate GTP/GDP exchange in comparison to PMA. Additional studies showed that DADLE stimulated an increase in cfos mRNA; this was reduced by the inhibitor of MAPK/ERK kinase (MEK), PD98059. Therefore, in DOR-Ju.1 cells, DOR agonists stimulate ERK phosphorylation in a Ras independent and PKC-dependent manner; PTKs appear to be involved. MAPKs mediate the increase in cfos mRNA induced by DOR agonists.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Jurkat/química , Proteínas Quinases Ativadas por Mitógeno , Proteínas/metabolismo , Receptores Opioides delta/genética , Proteínas ras/metabolismo , Adjuvantes Imunológicos/metabolismo , Animais , Benzoquinonas , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Carcinógenos/farmacologia , Leucina Encefalina-2-Alanina/farmacologia , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Proteínas Ativadoras de GTPase , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Células Jurkat/enzimologia , Células Jurkat/imunologia , Lactamas Macrocíclicas , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno , Proteína Quinase 3 Ativada por Mitógeno , Oligopeptídeos/farmacologia , Toxina Pertussis , Fosforilação , Proteínas Proto-Oncogênicas c-fos/genética , Quinonas/farmacologia , RNA Mensageiro/análise , Receptores Opioides delta/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rifabutina/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Virulência de Bordetella/farmacologia , Proteínas Ativadoras de ras GTPaseRESUMO
OBJECTIVE: To investigate the role of nitric oxide (NO) production and NO synthase (NOS) induction during adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) in Dark Agouti rats. METHODS: Urinary nitrate excretion and immune NOS (INOS) messenger RNA (mRNA) expression were measured in the joint, lymph node, spleen, and liver tissues following the induction of either AIA or CIA. RESULTS: Urinary nitrate excretion and iNOS mRNA expression increased substantially during joint inflammation in both models of arthritis. However, the increases in urinary nitrate excretion and iNOS mRNA expression observed in the joint, liver, and spleen tissues during AIA were greater than those observed during CIA, although iNOS induction in the lymph nodes was similar for both models. A prior injection with Mycobacterium bovis heat-shock protein resulted in suppression of arthritis and NO production in AIA, but not in CIA. CONCLUSION: Differences in NO production during AIA versus CIA are a reflection of the fundamental pathophysiologic differences between these 2 models of arthritis. Thus, NO production in these 2 models could not be merely a nonspecific reaction to the adjuvant injection, nor simply a byproduct of local inflammation in the joint.