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1.
Toxins (Basel) ; 13(8)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437385

RESUMO

Snakebite envenomation is a serious neglected tropical disease, and its management is often complicated by the diversity of snake venoms. In Asia, pit vipers of the Ovophis species complex are medically important venomous snakes whose venom properties have not been investigated in depth. This study characterized the venom proteomes of Ovophis convictus (West Malaysia), Ovophis tonkinensis (northern Vietnam, southern China), and Ovophis okinavensis (Okinawa, Japan) by applying liquid chromatography-tandem mass spectrometry, which detected a high abundance of snake venom serine proteases (SVSP, constituting 40-60% of total venom proteins), followed by phospholipases A2, snake venom metalloproteinases of mainly P-III class, L-amino acid oxidases, and toxins from other protein families which were less abundant. The venoms exhibited different procoagulant activities in human plasma, with potency decreasing from O. tonkinensis > O. okinavensis > O. convictus. The procoagulant nature of venom confirms that consumptive coagulopathy underlies the pathophysiology of Ovophis pit viper envenomation. The hetero-specific antivenoms Gloydius brevicaudus monovalent antivenom (GbMAV) and Trimeresurus albolabris monovalent antivenom (TaMAV) were immunoreactive toward the venoms, and cross-neutralized their procoagulant activities, albeit at variably limited efficacy. In the absence of species-specific antivenom, these hetero-specific antivenoms may be useful in treating coagulotoxic envenomation caused by the different snakes in their respective regions.


Assuntos
Crotalinae , Proteoma , Proteínas de Répteis , Venenos de Víboras , Animais , Antivenenos/imunologia , Coagulantes/análise , Coagulantes/imunologia , Coagulantes/toxicidade , Humanos , L-Aminoácido Oxidase/análise , L-Aminoácido Oxidase/imunologia , L-Aminoácido Oxidase/toxicidade , Metaloproteases/análise , Metaloproteases/imunologia , Metaloproteases/toxicidade , Fosfolipases A2/análise , Fosfolipases A2/imunologia , Fosfolipases A2/toxicidade , Plasma/efeitos dos fármacos , Proteoma/análise , Proteoma/imunologia , Proteoma/toxicidade , Proteômica , Proteínas de Répteis/análise , Proteínas de Répteis/imunologia , Proteínas de Répteis/toxicidade , Serina Proteases/análise , Serina Proteases/imunologia , Serina Proteases/toxicidade , Venenos de Víboras/química , Venenos de Víboras/imunologia , Venenos de Víboras/toxicidade
2.
Dev Comp Immunol ; 119: 104040, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33561521

RESUMO

l-amino acid oxidase (LAAO) is a recently discovered novel fish immune enzyme. To explore the role of LAAO in the immune system of bony fishes, we cloned the full-length coding sequence (CDS) of LAAO of the zebrafish Danio rerio (ZF-LAAO), conducted bioinformatics analysis of ZF-LAAO, and analyzed its expression profile in zebrafish infected with the pathogen Streptococcus agalactiae. The CDS of ZF-LAAO was 1,515 base pairs long, and the encoded protein of ZF-LAAO contained an 18 amino acid signal peptide. ZF-LAAO contained the conserved domains of the LAAO family (dinucleotide binding motif and GG-motif), 2 N-glycosylation sites, and 2 O-glycosylation sites, and it was a stable hydrophilic exocrine protein. Similarity of the amino acid sequence of ZF-LAAO with LAAOs of 14 other bony fish species was >50% in all cases. The greatest similarity (79.45%) was with the LAAO of Anabarilius grahami, and these two LAAOs were grouped together in the phylogenetic tree. In wild-type zebrafish infected with S. agalactiae, changes in ZF-LAAO gene (zflaao) expression occurred mainly in the early stage of infection, and the changes in zflaao expression were more pronounced than those of the immune enzyme lysozyme (LYZ). The expression levels of both LYZ gene of zebrafish (zflyz) and zflaao were significantly elevated at 6 h after infection (p < 0.001), but zflyz expression in the spleen decreased at 12 h whereas zflaao expression in the liver and spleen peaked at 12 h. These results provided a reference for functional studies of the novel immune enzyme LAAO in bony fish.


Assuntos
Doenças dos Peixes/imunologia , L-Aminoácido Oxidase/imunologia , Streptococcus agalactiae/imunologia , Transcriptoma/imunologia , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Interações Hospedeiro-Patógeno/imunologia , L-Aminoácido Oxidase/classificação , L-Aminoácido Oxidase/genética , Modelos Moleculares , Filogenia , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Streptococcus agalactiae/fisiologia , Fatores de Tempo , Peixe-Zebra/genética , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
3.
Front Immunol ; 11: 600012, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33343572

RESUMO

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro, thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Memória Imunológica , L-Aminoácido Oxidase/imunologia , Ativação Linfocitária , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Doença Aguda , Animais , Linfócitos T CD8-Positivos/patologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Sinapses Imunológicas/genética , Sinapses Imunológicas/imunologia , Sinapses Imunológicas/patologia , L-Aminoácido Oxidase/genética , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica/genética , Camundongos , Camundongos Knockout
4.
Fish Shellfish Immunol ; 106: 685-690, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32822860

RESUMO

An l-amino acid oxidase (LAO) is an amino acid metabolism enzyme that also performs a variety of biological activities. Recently, LAOs have been discovered to be deeply involved in innate immunity in fish because of their antibacterial and antiparasitic activity. The determinant of potent antibacterial/antiparasitic activity is the H2O2 byproduct of LAO enzymatic activity that utilizes the l-amino acid as a substrate. In addition, fish LAOs are upregulated by pathogenic bacteria or parasite infection. Furthermore, some fish LAOs show that the target specificity depends on the virulence of the bacteria. All results reflect that LAOs are new innate immune molecules. This review also describes the potential of the immunomodulatory functions of fish LAOs, not only the innate immune function by a direct oxidation attack of H2O2.


Assuntos
Peixes/imunologia , L-Aminoácido Oxidase/imunologia , Animais , Peixes/genética , Brânquias/imunologia , Imunomodulação , Intestinos/imunologia , L-Aminoácido Oxidase/sangue , L-Aminoácido Oxidase/genética , Pele/imunologia
5.
Fish Shellfish Immunol ; 69: 85-89, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28818617

RESUMO

Cryptocaryon irritans is an important protozoan parasite which infects almost all kinds of marine teleosts, causing heavy economic losses. In our previous studies, we found that rabbitfish (Siganus oramin) displayed high resistance to C. irritans infection, and a novel protein, l-amino acid oxidase (LAAO), was identified from the serum that was lethal to C. irritans. In this study, the rabbitfish were firstly infected with a high dose of C. irritans, then the LAAO mRNA expression pattern and the activity of three enzymes [superoxide dismutase (SOD), Na+/K+-ATPase and Ca2+/Mg2+-ATPase] were measured in various tissues. The results indicated that, after infection, the feeding and swimming of rabbitfish was normal, and the infection intensity in the host was low. Tissue distribution analysis showed that LAAO mRNA was most pronounced in the head kidney and gill, with lower expression observed in the muscle. After infection with C. irritans, the LAAO mRNA was up-regulated early post infection (from 6 to 24 h) in both gill and spleen, but then returned to normal levels, implying that LAAO may play an important role in the host's early immune response. The SOD activity in the liver was significantly higher in the infection group than in the control group by 48 h post infection, while Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities in the gill were decreased by 12 and 24 h after infection; no significant difference was detected at the other time points throughout the experiment. Together, these results suggest that biochemical responses of rabbitfish are relatively mild after infection with a high dose of parasite, and the LAAO may play an important role in the host's defense against C. irritans.


Assuntos
Infecções por Cilióforos/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Animais , Cilióforos/fisiologia , Brânquias/metabolismo , Rim Cefálico/metabolismo , L-Aminoácido Oxidase/genética , L-Aminoácido Oxidase/imunologia , Perciformes , Distribuição Aleatória
6.
Sci Rep ; 7: 42673, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28205610

RESUMO

L-amino acid oxidases from snake venoms have been described to possess various biological functions. In this study, we investigated the inflammatory responses induced in vivo and in vitro by CR-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodostoma venom, and its antitumor potential. CR-LAAO induced acute inflammatory responses in vivo, with recruitment of neutrophils and release of IL-6, IL-1ß, LTB4 and PGE2. In vitro, IL-6 and IL-1ß production by peritoneal macrophages stimulated with CR-LAAO was dependent of the activation of the Toll-like receptors TLR2 and TLR4. In addition, CR-LAAO promoted apoptosis of HL-60 and HepG2 tumor cells mediated by the release of hydrogen peroxide and activation of immune cells, resulting in oxidative stress and production of IL-6 and IL-1ß that triggered a series of events, such as activation of caspase 8, 9 and 3, and the expression of the pro-apoptotic gene BAX. We also observed that CR-LAAO modulated the cell cycle of these tumor cells, promoting delay in the G0/G1 and S phases. Taken together, our results suggest that CR-LAAO could serve as a potential tool for the development of novel immunotherapeutic strategies against cancer, since this toxin promoted apoptosis of tumor cells and also activated immune cells against them.


Assuntos
L-Aminoácido Oxidase/metabolismo , Venenos de Serpentes/enzimologia , Viperidae/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Citocinas/metabolismo , Humanos , Imunoterapia , Mediadores da Inflamação/metabolismo , L-Aminoácido Oxidase/imunologia , L-Aminoácido Oxidase/farmacologia , L-Aminoácido Oxidase/uso terapêutico , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Venenos de Serpentes/imunologia , Venenos de Serpentes/farmacologia , Venenos de Serpentes/uso terapêutico , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
7.
Genes Immun ; 17(2): 148-52, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26673964

RESUMO

The immunosuppressive phenylalanine oxidase interleukin 4-induced gene 1 (IL4I1), primarily produced by antigen-presenting cells, inhibits T-cell proliferation and promotes the generation of Foxp3(+) regulatory T cells in vitro. Highly expressed by tumour-associated macrophages from human cancers, IL4I1 has a potential role in immune evasion from the anti-tumour immune response. We have reviewed single-nucleotide polymorphisms (SNPs) and mutations described for the exon 4 of the IL4I1 isoform 1, which is expressed in lymphoid tissue. Two of them were expressed in an exogenous system to analyse their effect on the enzymatic activity. The N92D SNP leads to a hyperactive enzyme, while the R102G mutation is hypomorphic. Moreover, we show that IL4I1 activity is not only directed against phenylalanine, as initially described, but also at a lower level against arginine. These data pave the way to more extensive analyses of the mutational state of IL4I1 in pathological conditions such as cancer, where its participation in immune system dysfunctions may have therapeutic implications.


Assuntos
L-Aminoácido Oxidase/química , Mutação , Neoplasias Ovarianas/genética , Polimorfismo de Nucleotídeo Único , Evasão Tumoral/genética , Animais , Arginina/química , Arginina/metabolismo , Éxons , Feminino , Expressão Gênica , Células HEK293 , Humanos , Íntrons , L-Aminoácido Oxidase/genética , L-Aminoácido Oxidase/imunologia , Monoaminoxidase/química , Monoaminoxidase/genética , Monoaminoxidase/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Fenilalanina/química , Fenilalanina/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Homologia Estrutural de Proteína , Viperidae/metabolismo
8.
Eur J Immunol ; 45(2): 474-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25446972

RESUMO

IL4I1 encodes an L-phenylalanine oxidase that inhibits T-cell proliferation. It has been recently reported that IL4I1 is expressed in TH17 cells as part of a mechanism that limits their pathogenicity. We have previously identified a population of human FOXP3(+) Treg cells that secrete IL-17 ex vivo; here, we addressed the expression of IL4I1 in that Treg-cell population. We found that in ex vivo isolated circulating Treg cells, IL4I1 expression is induced by activation. Moreover, IL4I1 expression is restricted to cells that do not express Helios, a transcription factor that characterizes natural Treg cells, but that express Aiolos, which is involved in the differentiation of TH17 and induced Treg cells. We also showed that conversion of Treg cells under inflammatory conditions increases IL4I1 expression, likely as part of a regulatory loop that attempts to limit the pathogenicity resulting from their conversion into TH17. The specific expression of IL4I1 in TH17 and iTreg cells may provide insights into approaches that aim at modulating these populations in different pathological conditions involving inflammation-mediated immunosuppression.


Assuntos
Fatores de Transcrição Forkhead/genética , Fator de Transcrição Ikaros/genética , L-Aminoácido Oxidase/genética , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Comunicação Celular , Diferenciação Celular , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Fator de Transcrição Ikaros/imunologia , Tolerância Imunológica , Interleucina-17/genética , Interleucina-17/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Ionomicina/farmacologia , L-Aminoácido Oxidase/imunologia , Ativação Linfocitária , Transdução de Sinais , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Células Th17/citologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/imunologia
9.
J Interferon Cytokine Res ; 35(1): 42-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25007029

RESUMO

Allergic diseases, such as allergic rhinitis, caused by an immunoglobulin E (IgE)-mediated reaction to specific allergens are a common chronic condition worldwide. Interleukin-21 (IL-21), a type I cytokine that is produced by T cells, exerts regulatory effects on a variety of immune cells. In our previous study, we found that serum levels of IL-21 were significantly decreased in patients with severe atopic dermatitis, suggesting that IL-21 might play a role in allergic reactions. In this study, we investigated the role of IL-21/IL-21 receptor (IL-21R) in patients with allergic rhinitis. Our results demonstrated that there was no difference in IL-21 serum levels between allergic rhinitis patients and controls. However, allergic patients had significantly increased expression of IL-21R on naive and memory B cells. IL-21R was upregulated through stimulation by the combination of CD40 ligand (CD40L) and IL-4. IL-21 alone neither induced nor inhibited IgE secretion from CD40L-stimulated B cells. However, IL-21 inhibited IgE secretion of B cells that were induced by the combination of CD40L and IL-4 in allergic patients. Moreover, a negative correlation between the expression of IL-21R and serum levels of IgE was found in patients with allergy. These results suggest that the role of IL-21 in an ongoing allergic reaction is to downregulate the IgE level by binding to IL-21R on B cells, which increases the expression in allergic patients.


Assuntos
Linfócitos B/imunologia , Imunoglobulina E/biossíntese , Subunidade alfa de Receptor de Interleucina-21/biossíntese , Interleucinas/imunologia , Rinite Alérgica/imunologia , Adulto , Ligante de CD40/imunologia , Regulação para Baixo , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Subunidade alfa de Receptor de Interleucina-21/imunologia , Interleucinas/sangue , Interleucinas/metabolismo , L-Aminoácido Oxidase/imunologia , Masculino , Ligação Proteica , Regulação para Cima , Adulto Jovem
10.
Fish Shellfish Immunol ; 41(2): 356-61, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25238719

RESUMO

Siganus oraminl-amino acid oxidase is a novel natural protein (named SR-LAAO) isolated from serum of the rabbitfish (S. oramin), which showed antibacterial activity against both Gram-positive and Gram-negative bacteria and had a lethal effect on the parasites Cryptocaryon irritans, Trypanosoma brucei brucei and Ichthyophthirius multifiliis. In order to test whether recombinant SR-LAAO (rSR-LAAO) produced by the eukaryotic expression system also has antimicrobial activity, the yeast Pichia pastoris was used as the expression host to obtain rSR-LAAO in vitro. Crude rSR-LAAO produced by P. pastoris integrated with the SR-LAAO gene had antibacterial activity against both Gram-positive and Gram-negative bacteria as shown by inhibition zone assay of the antibacterial spectrum on agar plates. The average diameter of the inhibition zone of crude rSR-LAAO against the Gram-positive bacteria Staphylococcus aureus and Streptococcus agalactiae was 1.040 ± 0.045 cm and 1.209 ± 0.085 cm, respectively. For the Gram-negative bacteria Aeromonas sobria, Escherichia coli, Vibrio alginolyticus, Vibrio cholera and Photobacterium damselae subsp. piscicida, the average diameter of inhibition zone was 1.291 ± 0.089 cm, 0.943 ± 0.061 cm, 0.756 ± 0.057 cm, 0.834 ± 0.023 cm and 1.211 ± 0.026 cm, respectively. These results were obtained at the logarithmic growth phase of S. agalactiae and A. sobria cell suspensions after incubation with 0.5 mg/mL crude rSR-LAAO for 24 h. The final bacterial growth rate was decreased significantly. The relative inhibition rate can reach 50% compared to crude products from P. pastoris integrated with an empty vector at the same concentration of protein. The antimicrobial activity of crude rSR-LAAO was likely associated with H2O2 formation, because its inhibition zones were disturbed significantly by catalase. Scanning electron microscopy results showed crude rSR-LAAO-treated bacterial surfaces became rough and particles were attached, cell walls were retracted and cell membranes were ruptured. Together, the results of this study indicated rSR-LAAO from the P. pastoris expression system is a potential antibiotic for application as a therapeutic agent against bacterial diseases.


Assuntos
Bactérias/imunologia , Cilióforos/imunologia , L-Aminoácido Oxidase/imunologia , Perciformes/imunologia , Proteínas Recombinantes/imunologia , Animais , Western Blotting , Catalase/imunologia , Primers do DNA/genética , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Vetores Genéticos/genética , Peróxido de Hidrogênio/imunologia , L-Aminoácido Oxidase/genética , Microscopia Eletrônica de Varredura , Perciformes/metabolismo , Pichia , Proteínas Recombinantes/genética
11.
Appl Microbiol Biotechnol ; 97(21): 9323-41, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24077723

RESUMO

L-amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the "true" LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with "narrow" or "strict" substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.


Assuntos
Aminoácidos/metabolismo , Cetoácidos/metabolismo , L-Aminoácido Oxidase/metabolismo , Amônia/metabolismo , Bactérias/enzimologia , Biotecnologia/métodos , Clonagem Molecular , Fungos/enzimologia , Expressão Gênica , Peróxido de Hidrogênio/metabolismo , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/imunologia , Engenharia de Proteínas , Venenos de Serpentes/enzimologia , Especificidade por Substrato
12.
PLoS One ; 8(1): e54589, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23355881

RESUMO

L-amino acid oxidases (LAAO) are flavoproteins that catalyze the oxidative deamination of L-amino acids to a keto-acid along with the production of H2O2 and ammonia. Interleukin 4 induced gene 1 (IL4I1) is a secreted LAAO expressed by macrophages and dendritic cells stimulated by microbial derived products or interferons, which is endowed with immunoregulatory properties. It is the first LAAO described in mammalian innate immune cells. In this work, we show that this enzyme blocks the in vitro and in vivo growth of Gram negative and Gram positive bacteria. This antibiotic effect is primarily mediated by H2O2 production but is amplified by basification of the medium due to the accumulation of ammonia. The depletion of phenylalanine (the primary amino acid catabolized by IL4I1) may also participate in the in vivo inhibition of staphylococci growth. Thus, IL4I1 plays a distinct role compared to other antibacterial enzymes produced by mononuclear phagocytes.


Assuntos
Células Dendríticas/metabolismo , Escherichia coli/crescimento & desenvolvimento , Peróxido de Hidrogênio/metabolismo , L-Aminoácido Oxidase/metabolismo , Macrófagos/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Escherichia coli/imunologia , Humanos , Peróxido de Hidrogênio/imunologia , L-Aminoácido Oxidase/imunologia , Macrófagos/imunologia , Fenilalanina/imunologia , Fenilalanina/metabolismo , Staphylococcus aureus/imunologia
13.
Dev Comp Immunol ; 39(4): 456-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23295245

RESUMO

The rockfish Sebastes schlegeli skin mucus contains a potent antibacterial protein, SSAP (S. schlegeli antibacterial protein), a novel l-amino acid oxidase with strict substrate specificity that acts against water-borne Gram-negative bacteria. We previously demonstrated that SSAP distributes in the skin and gills. Here we investigated the intra-tissue localization of SSAP in the tissues by in situ hybridization. Skin and gill sections were hybridized with digoxigenin-conjugated SSAP-specific RNA probe. SSAP mRNA-positive cells located near the basal membrane of skin epidermis and the gill epithelium. Furthermore, skin section was analyzed by immunohistochemistry and reacted with anti-SSAP antiserum as a primary antibody. The mucus layer and mucous cells in the skin were immunopositive. Skin and gill extracts produced hydrogen peroxide, responsible for antibacterial activity, in the presence of l-lysine. These results suggested that SSAP functions locally as a humoral defense factor in S. schlegeli skin and gills and prevents pathogenic bacterial invasion.


Assuntos
Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Peixes/imunologia , Bactérias Gram-Negativas/imunologia , L-Aminoácido Oxidase/imunologia , L-Aminoácido Oxidase/metabolismo , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Peixes/química , Peixes/metabolismo , L-Aminoácido Oxidase/química
14.
FASEB J ; 23(8): 2514-20, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19276171

RESUMO

The innate immune system plays an important role in protecting organs that are continuous with the outer surface of the body from bacterial infection. The antibacterial factors involved in this system have been sought in exocrine glands, particularly in the mammary glands. Because milk produced in the mammary glands is enriched in various nutrients, supporting the proliferation of bacteria, mammary glands appear to be at the greatest risk of bacterial infection and proliferation. Here, we show that mouse milk contains L-amino acid oxidase (LAO), a lactating mammary gland-specific protein that displays antibacterial activity in vitro through the production of hydrogen peroxide from free amino acids. We produced LAO-disrupted mouse lines to define the physiological properties and importance of the protein in vivo. The LAO-knockout mice were healthy and had normal mammary gland development; however, the antibacterial activity normally observed in milk from wild-type mice was absent from the milk of knockout mice. The content of free amino acids targeted by LAO was very low in wild-type milk, whereas these amino acids were abundant in LAO-knockout milk. Knockout mice exhibited weak resistance to an intramammary bacterial challenge compared to their wild-type counterparts. Further, preadministration of wild-type milk whey reduced the severity of bacterial infection in LAO-knockout mice. These results demonstrate that milk LAO protects the mammary gland against bacterial infection, and this antibacterial effect may be due to the generation of hydrogen peroxide by using free amino acids abundantly present in milk.


Assuntos
Imunidade Inata , L-Aminoácido Oxidase/imunologia , Glândulas Mamárias Animais/enzimologia , Glândulas Mamárias Animais/imunologia , Animais , Infecções Bacterianas/enzimologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/prevenção & controle , Feminino , L-Aminoácido Oxidase/deficiência , L-Aminoácido Oxidase/genética , Lactação/imunologia , Lactação/metabolismo , Glândulas Mamárias Animais/microbiologia , Mastite/enzimologia , Mastite/imunologia , Mastite/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Leite/química , Leite/enzimologia , Leite/imunologia , Infecções Estafilocócicas/enzimologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia , Staphylococcus aureus/patogenicidade
15.
Toxicon ; 53(3): 330-41, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19101583

RESUMO

Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined.


Assuntos
Bothrops , L-Aminoácido Oxidase/farmacologia , Venenos de Víboras/enzimologia , Venenos de Víboras/toxicidade , Animais , Anticorpos/sangue , Bioensaio , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Cavalos , L-Aminoácido Oxidase/imunologia , L-Aminoácido Oxidase/isolamento & purificação , Dose Letal Mediana , Testes de Neutralização , Staphylococcus aureus/efeitos dos fármacos
16.
J Proteome Res ; 7(6): 2445-57, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18444672

RESUMO

We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.


Assuntos
Antivenenos/análise , Venenos de Crotalídeos/metabolismo , Proteoma/análise , Viperidae/metabolismo , Animais , Antivenenos/imunologia , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/química , Venenos de Crotalídeos/imunologia , Eletroforese em Gel de Poliacrilamida , L-Aminoácido Oxidase/análise , L-Aminoácido Oxidase/imunologia , Lectinas Tipo C/análise , Metaloproteases/análise , Metaloproteases/imunologia , Oligopeptídeos/análise , Fosfolipases A2 Secretórias/análise , Proteoma/imunologia , Serina Endopeptidases/análise , Inibidores de Serina Proteinase/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Fator A de Crescimento do Endotélio Vascular/análise
17.
Blood ; 110(1): 220-7, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17356132

RESUMO

Interleukin-4-induced gene 1 (IL4I1) was first described as a B-cell IL4-inducible gene and is highly expressed in primary mediastinal B-cell lymphomas. We established stable HEK293 clones expressing human and mouse IL4I1 to examine their biochemical properties and function. Both proteins were secreted into the culture medium, and we observed the secretion of endogenous human IL4I1 (hIL4I1) protein in a mediastinal lymphoma B-cell line, MedB-1. We showed that IL4I1 has l-amino acid oxidase activity, optimal at physiological pH and primarily directed toward phenylalanine. Immunohistochemical analysis of secondary lymphoid organs showed staining of germinal center macrophages and inflammatory myeloid cells. In vitro, functional enzyme was highest in mature dendritic cells (DCs), suggesting a role in antigen-presenting cell/T-lymphocyte cross-talk. Indeed, hIL4I1 inhibited the proliferation of CD3-stimulated T lymphocytes with a similar effect on CD4(+) and CD8(+) T cells. In contrast, memory T cells were more strongly affected by hIL4I1 and its catabolite H(2)O(2) than naive T cells. hIL4I1 inhibitory effect was dependent on enzymatic activity and H(2)O(2) production and associated with a transient down-regulation of TCRzeta expression. Altogether these data suggest IL4I1 as a new immunomodulatory enzyme produced by DCs.


Assuntos
Aminoácido Oxirredutases/imunologia , Proliferação de Células , Células Dendríticas/química , L-Aminoácido Oxidase/imunologia , Linfócitos T/citologia , Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Peróxido de Hidrogênio/metabolismo , Imuno-Histoquímica , L-Aminoácido Oxidase/biossíntese , L-Aminoácido Oxidase/metabolismo , Linfoma de Células B/patologia , Macrófagos/química , Camundongos , Células Mieloides/química
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