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1.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140319, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31740417

RESUMO

The ß-N-acetylhexosaminidase from Penicillium oxalicum (PoHex; EC 3.2.1.52) is a fungal glycosidase with an outstandingly high GalNAcase/GlcNAcase activity ratio. It has a remarkable synthetic capability and can process carbohydrates functionalized at various positions. However, the production in the native fungal host is lengthy, unselective and purification from the fungal medium is complicated and low yielding. We present here a novel production method of this enzyme in the eukaryotic host of Pichia pastoris, followed by elegant one-step purification to homogeneity. The resulting recombinant enzyme has improved biochemical and catalytic properties compared to the fungal wild type. Its good production yield (11 mg/400 mL cultivation medium) greatly expands the scope of synthetic applications. We further demonstrate the synthetic utility and broad acceptor specificity of recombinant PoHex in the glycosylation of a series of challenging acceptors with varying structural architectures, namely secondary and tertiary hydroxyl, aldoxime and a poly-hydroxylated compound.


Assuntos
Pichia/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Penicillium/enzimologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura , beta-N-Acetil-Hexosaminidases/genética
2.
Artigo em Inglês | MEDLINE | ID: mdl-31629812

RESUMO

The present report describes a comprehensive study on comparative biochemical characterization of two lysosomal enzymes, acid phosphatase and ß-hexosaminidase in three different strains of Hydra; Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1 (self-feeder-1). Since morphology and habitat of Hydra effect lysosomal enzymes and their response to environmental pollutants, it would be interesting to identify them in different Hydra strains so as to use them as toxicity testing. Preliminary studies revealed a differential expression of acid phosphatase, ß-hexosaminidase and ß-glucuronidase in three Hydra strains. Expression of all three lysosomal enzymes in H. vulgaris Ind-Pune was low in comparison to H. vulgaris Naukuchiatal and H. magnipapillata sf-1, while their expression is comparable in H. vulgaris Naukuchiatal and H. magnipapillata sf-1. The Michaelis-Menten (KM) values for lysosomal ß-hexosaminidase using 4-nitrophenyl N-acetyl-ß-D-glucosaminide as substrate were found to be 1.3 mM, 1.1 mM and 0.8 mM, respectively for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1. For acid phosphatase using 4-nitrophenyl-phosphate as substrate, the KM values were 0.38 mM, 1.2 mM and 0.52 mM respectively, for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and sf-1 strains. The optimum temperature for ß-hexosaminidase was 60 °C for H. vulgaris Ind-Pune, while 50 °C was observed for H. vulgaris Naukuchiatal and sf-1 strains. The optimum pH for ß-hexosaminidase was found to be 6.0 for H. vulgaris Ind-Pune and H. vulgaris Naukuchiatal, and 5.0 for sf-1. The optimum temperature and pH of acid phosphatase was similar in all three strains, viz., 40 °C and 3.0, respectively. Preliminary localization studies using whole mount in situ hybridization revealed predominant endodermal expression of three enzymes in H. vulgaris Ind-Pune. Our results thus support the conservation of lysosomal hydrolases in Hydra.


Assuntos
Fosfatase Ácida/metabolismo , Hydra/enzimologia , Lisossomos/enzimologia , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Especificidade da Espécie
3.
PLoS One ; 14(12): e0225857, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31790488

RESUMO

Although the high-fat-diet-induced metabolic syndrome (MetS) is a precursor of human cardiac pathology, the myocardial metabolic state in MetS is far from clear. The discrepancies in metabolite handling between human and small animal models and the difficulties inherent in obtaining human tissue complicate the identification of the myocardium-specific metabolic response in patients. Here we use the large animal model of swine that develops the hallmark criteria of human MetS. Our comparative metabolomics together with transcriptomics and computational nonnegative matrix factorization (NMF) interpretation of the data exposes significant decline in metabolites related to the fatty acid oxidation, glycolysis, and pentose phosphate pathway. Behind the reversal lies decreased expression of enzymes that operate in the pathways. We showed that diminished glycogen deposition is a metabolic signature of MetS in the pig myocardium. The depletion of glycogen arises from disbalance in expression of genes that break down and synthesize glycogen. We show robust acetoacetate accumulation and activated expression of key enzymes in ketone body formation, catabolism and transporters, suggesting a shift in fuel utilization in MetS. A contrasting enrichment in O-GlcNAcylated proteins uncovers hexosamine pathway and O-GlcNAcase (OGA) expression involvement in the myocardial response to MetS. Although the hexosamine biosynthetic pathway (HBP) activity and the availability of the UDP-GlcNAc substrate in the MetS myocardium is low, the level of O-GlcNacylated proteins is high as the O-GlcNacase is significantly diminished. Our data support the perception of transcriptionally driven myocardial alterations in expression of standard fatty acids, glucose metabolism, glycogen, and ketone body related enzymes and subsequent paucity of their metabolite products in MetS. This aberrant energy metabolism in the MetS myocardium provide insight into the pathogenesis of CVD in MetS.


Assuntos
Redes e Vias Metabólicas , Síndrome Metabólica/metabolismo , Miocárdio/metabolismo , Animais , Colesterol na Dieta/efeitos adversos , Dieta , Glicosilação , Masculino , Metaboloma , Metabolômica , N-Acetilglucosaminiltransferases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Risco , Suínos , Aprendizado de Máquina não Supervisionado , beta-N-Acetil-Hexosaminidases/metabolismo
4.
Int J Mol Sci ; 20(24)2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31817903

RESUMO

Fungal ß-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The ß-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model ß-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure-activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-ß-d-glucosaminide or 4-nitrophenyl N-acetyl-ß-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the ß(1-4) position, the galacto-acceptor afforded a ß(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on ß-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.


Assuntos
Glicosídeos/metabolismo , Oligossacarídeos/metabolismo , Talaromyces/enzimologia , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/metabolismo , Glicosilação , Cinética , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
5.
Int J Mol Sci ; 20(22)2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31698677

RESUMO

BACKGROUND: Laboratory of allergic diseases 2 (LAD2) human mast cells were developed over 15 years ago and have been distributed worldwide for studying mast cell proliferation, receptor expression, mediator release/inhibition, and signaling. LAD2 cells were derived from CD34+ cells following marrow aspiration of a patient with aggressive mastocytosis with no identified mutations in KIT. Another aspiration gave rise to a second cell line which has recently been re-established (LADR). We queried whether LADR had unique properties for the preclinical study of human mast cell biology. METHODS: LADR and LAD2 cells were cultured under identical conditions. Experiments examined proliferation, beta-hexosaminidase (ß-hex) release, surface receptor and granular protease expression, infectivity with HIV, and gene expression. RESULTS: LADR cells were larger and more granulated as seen with Wright-Giemsa staining and flow cytometry, with cell numbers doubling in 4 weeks, in contrast to LAD2 cells, which doubled every 2 weeks. Both LADR and LAD2 cells released granular contents following aggregation of FcεRI. LADR cells showed log-fold increases in FcεRI/CD117 and expressed CD13, CD33, CD34, CD63, CD117, CD123, CD133, CD184, CD193, and CD195, while LAD2 cells expressed CD33, CD34, CD63, CD117, CD133, CD193 but not CD13, CD123, CD184, or CD195. LADR tryptase expression was one-log-fold increased. LADR cell and LAD2 cell chymase expression were similar. Both cell lines could be infected with T-tropic, M-tropic, and dual tropic HIV. Following monomeric human IgE stimulation, LADR cells showed greater surface receptor and mRNA expression for CD184 and CD195. Expression arrays revealed differences in gene upregulation, especially for the suppressor of cytokine signaling (SOCS) family of genes with their role in JAK2/STAT3 signaling and cellular myelocytomatosis oncogene (c-MYC) in cell growth and regulation. CONCLUSIONS: LADR cells are thus unique in that they exhibit a slower proliferation rate, are more advanced in development, have increased FcεRI/CD117 and tryptase expression, have a different profile of gene expression, and show earlier infectivity with HIV-BAL, LAV, and TYBE when compared to LAD2 cells. This new cell line is thus a valuable addition to the few FcεRI+ human mast cell lines previously described and available for scientific inquiry.


Assuntos
Linhagem Celular/citologia , Mastócitos/citologia , Antígenos CD/metabolismo , Degranulação Celular , Proliferação de Células , Quimases/metabolismo , Regulação da Expressão Gênica , Infecções por HIV/patologia , Humanos , Mastócitos/fisiologia , Transdução de Sinais , Triptases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
6.
Nat Struct Mol Biol ; 26(11): 1071-1077, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31695185

RESUMO

Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GlcNAc, catalyzed by O-GlcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GlcNAcase (OGA), precise functional dissection of site-specific O-GlcNAc modification in vivo is currently not possible without affecting the entire O-GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GlcNAc is a hydrolytically stable and accurate structural mimic of O-GlcNAc that can be encoded in mammalian systems with CRISPR-Cas9 in an otherwise unperturbed O-GlcNAcome. Using this approach, we target an elusive Ser 405 O-GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.


Assuntos
Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/genética , Animais , Sistemas CRISPR-Cas , Glicosilação , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , N-Acetilglucosaminiltransferases/química , Processamento de Proteína Pós-Traducional , Especificidade por Substrato , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/genética
7.
J Immunol ; 203(10): 2724-2734, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31586037

RESUMO

Alternatively activated macrophages are essential effector cells during type 2 immunity and tissue repair following helminth infections. We previously showed that Ym1, an alternative activation marker, can drive innate IL-1R-dependent neutrophil recruitment during infection with the lung-migrating nematode, Nippostrongylus brasiliensis, suggesting a potential role for the inflammasome in the IL-1-mediated innate response to infection. Although inflammasome proteins such as NLRP3 have important proinflammatory functions in macrophages, their role during type 2 responses and repair are less defined. We therefore infected Nlrp3 -/- mice with N. brasiliensis Unexpectedly, compared with wild-type (WT) mice, infected Nlrp3 -/- mice had increased neutrophilia and eosinophilia, correlating with enhanced worm killing but at the expense of increased tissue damage and delayed lung repair. Transcriptional profiling showed that infected Nlrp3 -/- mice exhibited elevated type 2 gene expression compared with WT mice. Notably, inflammasome activation was not evident early postinfection with N. brasiliensis, and in contrast to Nlrp3 -/- mice, antihelminth responses were unaffected in caspase-1/11-deficient or WT mice treated with the NLRP3-specific inhibitor MCC950. Together these data suggest that NLRP3 has a role in constraining lung neutrophilia, helminth killing, and type 2 immune responses in an inflammasome-independent manner.


Assuntos
Inflamassomos/fisiologia , Pneumopatias Parasitárias/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Nippostrongylus/imunologia , Infecções por Strongylida/imunologia , Animais , Caspase 1/fisiologia , Quimiotaxia de Leucócito , Eosinofilia/etiologia , Eosinofilia/imunologia , Furanos/farmacologia , Imunidade Inata , Interleucina-4/farmacologia , Lectinas/biossíntese , Lectinas/genética , Pulmão/patologia , Pulmão/fisiologia , Pneumopatias Parasitárias/complicações , Pneumopatias Parasitárias/patologia , Pneumopatias Parasitárias/fisiopatologia , Macrófagos Alveolares/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Neutrófilos/imunologia , Regeneração , Infecções por Strongylida/complicações , Infecções por Strongylida/patologia , Infecções por Strongylida/fisiopatologia , Sulfonamidas/farmacologia , Transcrição Genética , beta-N-Acetil-Hexosaminidases/biossíntese , beta-N-Acetil-Hexosaminidases/genética
8.
Appl Microbiol Biotechnol ; 103(19): 7869-7881, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401752

RESUMO

ß-N-Acetylhexosaminidases (EC 3.2.1.52) are a unique family of glycoside hydrolases with dual substrate specificity and a particular reaction mechanism. Though hydrolytic enzymes per se, their good stability, easy recombinant production, absolute stereoselectivity, and a broad substrate specificity predestine these enzymes for challenging applications in carbohydrate synthesis. This mini-review aims to demonstrate the catalytic potential of ß-N-acetylhexosaminidases in a range of unusual reactions, processing of unnatural substrates, formation of unexpected products, and demanding reaction designs. The use of unconventional media can considerably alter the progress of transglycosylation reactions. By means of site-directed mutagenesis, novel catalytic machineries can be constructed. Glycosylation of difficult substrates such as sugar nucleotides was accomplished, and the range of afforded glycosidic bonds comprises unique non-reducing sugars. Specific functional groups may be tolerated in the substrate molecule, which makes ß-N-acetylhexosaminidases invaluable allies in difficult synthetic problems.


Assuntos
Biocatálise , Proteínas Mutantes/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Glicosilação , Proteínas Mutantes/genética , beta-N-Acetil-Hexosaminidases/genética
9.
Biosci Biotechnol Biochem ; 83(12): 2280-2287, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31412751

RESUMO

The increasing number of patients suffering from allergic diseases is a global health problem. Grifola frondosa is an edible mushroom consumed as a health food in Asia, and has recently been reported to have anti-allergic effects. We previously reported that G. frondosa extract (GFE) and its active components, ergosterol and its derivatives, inhibited the antigen-induced activation of RBL-2H3 cells. Here, we demonstrated that GFE and ergosterol also had an inhibitory effect on the degranulation of bone marrow-derived mast cells (BMMCs) and alleviated anaphylactic cutaneous responses in mice. Using an air pouch-type allergic inflammation mouse model, we confirmed that oral administration of GFE and ergosterol suppressed the degranulation of mast cells in vivo. Our findings suggest that G. frondosa, including ergosterol as its active component, reduces type I allergic reactions by suppressing mast cell degranulation in mice, and might be a novel functional food that prevents allergic diseases.


Assuntos
Degranulação Celular/efeitos dos fármacos , Ergosterol/farmacologia , Grifola/química , Hipersensibilidade/prevenção & controle , Mastócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Alimento Funcional , Liberação de Histamina/efeitos dos fármacos , Hipersensibilidade/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores
10.
J Ethnopharmacol ; 244: 112136, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31377261

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia lactiflora Pall. (peony) is a medicinal plant used in the Xiaoqinglong decoction, a commonly prescribed traditional Chinese medicine for asthma. The main active ingredients of peony roots-described as the total glucosides of peony (TGP)-have anti-inflammatory, immunomodulatory, and protective effects on endothelial cells, and they are known to improve rheumatoid arthritis. This study explored the underlying mechanism of TGP activity in the treatment of allergic asthma. MATERIALS AND METHODS: Allergic asthma was induced in BALB/c mice by administering injections of ovalbumin (OVA) mixed with aluminum hydroxide gel and inhaling nebulized OVA. The OVA-sensitized mice were treated with TGP by oral gavage, and the potentially anti-asthmatic treatment effect was studied by testing airway hyperresponsiveness, classifying and counting of leukocytes, performing cytokine assays, and analyzing the lung histopathology. The ß-hexosaminidase activity was assayed as a biomarker to evaluate the effect of TGP on mast cell degranulation. The mechanism of TGP was explored by monitoring the Ca2+ influx level in mast cells (RBL-2H3) using a Ca2+ fluorescent probe technique. RESULTS: In mice with OVA-induced allergic asthma, TGP reduced airway hyperresponsiveness and improved lung tissue pathology, which included a decrease in inflammatory cell infiltration and collagen deposition. TGP also significantly lowered BALF leukocyte, eosinophil, and neutrophil counts, along with chemokines and cytokines, such as eotaxin, TNF-α, IL-4, and MIP-1α, in serum and lungs of OVA-challenged mice. These effects were further confirmed with the decrease of ß-hexosaminidase release and the inhibition of Ca2+ influx in mast cell degranulation. CONCLUSIONS: Our findings suggest that TGP improved OVA-induced allergic asthma in mice mainly by suppressing Ca2+ influx-dependent mast cell degranulation.


Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Glucosídeos/uso terapêutico , Mastócitos/efeitos dos fármacos , Paeonia , Animais , Antiasmáticos/farmacologia , Asma/induzido quimicamente , Asma/imunologia , Asma/fisiopatologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Cálcio/metabolismo , Degranulação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/sangue , Citocinas/imunologia , Glucosídeos/farmacologia , Contagem de Leucócitos , Masculino , Mastócitos/fisiologia , Camundongos Endogâmicos BALB C , Ovalbumina , Ratos , beta-N-Acetil-Hexosaminidases/metabolismo
11.
Mediators Inflamm ; 2019: 9086758, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31360120

RESUMO

Macrophages contribute to a continuous increase in blood pressure and kidney damage in hypertension, but their polarization status and the underlying mechanisms have not been clarified. This study revealed an important role for M2 macrophages and the YM1/Chi3l3 protein in hypertensive nephropathy in a mouse model of hypertension. Bone marrow cells were isolated from the femurs and tibia of male FVB/N (control) and transgenic hypertensive animals that overexpressed the rat form of angiotensinogen (TGM(rAOGEN)123, TGM123-FVB/N). The cells were treated with murine M-CSF and subsequently with LPS+IFN-γ to promote their polarization into M1 macrophages and IL-4+IL-13 to trigger the M2 phenotype. We examined the kidneys of TGM123-FVB/N animals to assess macrophage polarization and end-organ damage. mRNA expression was evaluated using real-time PCR, and protein levels were assessed through ELISA, CBA, Western blot, and immunofluorescence. Histology confirmed high levels of renal collagen. Cells stimulated with LPS+IFN-γ in vitro showed no significant difference in the expression of CD86, an M1 marker, compared to cells from the controls or the hypertensive mice. When stimulated with IL-4+IL-13, however, macrophages of the hypertensive group showed a significant increase in CD206 expression, an M2 marker. The M2/M1 ratio reached 288%. Our results indicate that when stimulated in vitro, macrophages from hypertensive mice are predisposed toward polarization to an M2 phenotype. These data support results from the kidneys where we found an increased infiltration of macrophages predominantly polarized to M2 associated with high levels of YM1/Chi3l3 (91,89%), suggesting that YM1/Chi3l3 may be a biomarker of hypertensive nephropathy.


Assuntos
Hipertensão/metabolismo , Nefropatias/metabolismo , Lectinas/metabolismo , Macrófagos/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Biomarcadores/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Rim/metabolismo , Nefropatias/genética , Lectinas/genética , Ativação de Macrófagos/fisiologia , Masculino , Camundongos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , beta-N-Acetil-Hexosaminidases/genética
12.
Food Chem ; 300: 125209, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31344629

RESUMO

Turbot can induce allergy in susceptible individuals due to the presence of parvalbumin (PV), a major fish allergen. This study aimed at evaluating the digestibility and the ability of PV to elicit the release of cellular degranulation, following treatment with tyrosinase (PV-Tyr), caffeic acid (PV-CA) and in combination (PV-Tyr/CA), using in vitro digestion and RBL-2H3 (passive rat basophil leukemia) cell line. The digestion assay products revealed that the stability of PV in simulated gastric fluid (SGF) was stronger, while in simulated intestinal fluid (SIF) was rather weak. Western blot analysis revealed that the IgG-binding abilities of the cross-linked PV were markedly reduced. Moreover, crosslinking hampered the release of cellular degranulation process in RBL-2H3 cell lines. PV-Tyr/CA showed highly significant reduction in the release rate of ß-hexosaminidase (66.02%), histamine (35.01%), tryptase (29.25%), cysteinyl leukotrienes (29.72%), prostaglandin D2 (34.96%), IL-4 (43.99%) and IL-13 (38.93%) and shown potential in developing hypoallergenic fish products.


Assuntos
Ácidos Cafeicos/química , Citocinas/metabolismo , Hipersensibilidade Alimentar/imunologia , Monofenol Mono-Oxigenase/química , Parvalbuminas/química , Alérgenos/química , Alérgenos/farmacocinética , Animais , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Digestão , Proteínas de Peixes da Dieta/química , Linguados , Suco Gástrico , Histamina/metabolismo , Humanos , Parvalbuminas/imunologia , Parvalbuminas/farmacologia , Ratos , beta-N-Acetil-Hexosaminidases/metabolismo
13.
J Food Biochem ; 43(1): e12674, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-31353487

RESUMO

Mung bean seed is a well-known plant protein consumed in Asian countries but the protein is usually retrieved as a waste product during starch production. This study investigated the anti-allergic property of mung bean protein hydrolysates (MBPH) produced by enzymatic hydrolysis using non-gastrointestinal (non-GI), GI and a combination of non-GI+GI enzymes. The hydrolysates were investigated for any anti-allergic property by detecting the amount of ß-hexosaminidase released in RBL-2H3 cells, and complemented with the MTT assay to show cell viability. It was found that MBPH hydrolyzed by a combination of flavourzyme (non-GI enzyme) and pancreatin (GI enzyme) exhibited the highest anti-allergic activity (135.61%), followed by those produced with alcalase, a non-GI enzyme (121.74%) and 80.32% for pancreatin (GI enzyme). Minimal toxicity (<30%) of all hydrolysates on RBL-2H3 cells line was observed. The results suggest that MBPH can potentially serve as a hypoallergenic food ingredient or supplement. PRACTICAL APPLICATIONS: Mung bean (Vigna radiata L. (Wilczek)) is also known as "green gram" and it is an excellent source of protein. The major mung bean storage proteins are the globulin, albumin and legumin, which are also referred to as legume allergens. Our study showed that mung bean peptides obtained after enzymatic hydrolysis influenced ß-hexosaminidase inhibition without any toxic effect on RBL-2H3 cells. This indicates that mung bean allergenicity can be reduced after enzymatic hydrolysis and the protein hydrolysates could be as a hypoallergic food, ingredient, supplement and/or protein substitute in the formulation of food products.


Assuntos
Antialérgicos/farmacologia , Endopeptidases/metabolismo , Trato Gastrointestinal/enzimologia , Pancreatina/metabolismo , Subtilisinas/metabolismo , Vigna/química , Sequência de Aminoácidos , Animais , Antialérgicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeos/química , Peptídeos/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Proteólise , Ratos , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo
14.
Basic Res Cardiol ; 114(4): 28, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31152247

RESUMO

Several post-translational modifications figure prominently in ventricular remodeling. The beta-O-linkage of N-acetylglucosamine (O-GlcNAc) to proteins has emerged as an important signal in the cardiovascular system. Although there are limited insights about the regulation of the biosynthetic pathway that gives rise to the O-GlcNAc post-translational modification, much remains to be elucidated regarding the enzymes, such as O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which regulate the presence/absence of O-GlcNAcylation. Recently, we showed that the transcription factor, E2F1, could negatively regulate OGT and OGA expression in vitro. The present study sought to determine whether E2f1 deletion would improve post-infarct ventricular function by de-repressing expression of OGT and OGA. Male and female mice were subjected to non-reperfused myocardial infarction (MI) and followed for 1 or 4 week. MI significantly increased E2F1 expression. Deletion of E2f1 alone was not sufficient to alter OGT or OGA expression in a naïve setting. Cardiac dysfunction was significantly attenuated at 1-week post-MI in E2f1-ablated mice. During chronic heart failure, E2f1 deletion also attenuated cardiac dysfunction. Despite the improvement in function, OGT and OGA expression was not normalized and protein O-GlcNAcyltion was not changed at 1-week post-MI. OGA expression was significantly upregulated at 4-week post-MI but overall protein O-GlcNAcylation was not changed. As an alternative explanation, we also performed guided transcriptional profiling of predicted targets of E2F1, which indicated potential differences in cardiac metabolism, angiogenesis, and apoptosis. E2f1 ablation increased heart size and preserved remote zone capillary density at 1-week post-MI. During chronic heart failure, cardiomyocytes in the remote zone of E2f1-deleted hearts were larger than wildtype. These data indicate that, overall, E2f1 exerts a deleterious effect on ventricular remodeling. Thus, E2f1 deletion improves ventricular remodeling with limited impact on enzymes regulating O-GlcNAcylation.


Assuntos
Fator de Transcrição E2F1/deficiência , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Capilares/metabolismo , Capilares/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Fator de Transcrição E2F1/genética , Feminino , Deleção de Genes , Glicosilação , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
15.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(4): 289-295, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-31167686

RESUMO

Objective To investigate the effect of asthmatic mouse spleen-derived CD4+ T cells on the polarization of bone marrow-derived macrophages (BMDMs) in vitro and its mechanism. Methods An animal model of allergic asthma induced by Dermatophagoides farinae allergen was established in mice. After the last challenge lasting 24 hours, the middle lobe of mouse lung was taken and HE staining was used to observe its inflammatory changes. The levels of miR-155-5p in the lung and spleen as well as spleen CD4+ T cells were detected by real-time quantitative PCR (qRT-PCR). The proportions of CD4+IFN-γ+ Th1 cells and CD4+IL-4+ Th2 cells in the spleen of asthmatic mice were detected by flow cytometry. The mRNA expression levels of M2 macrophage marker genes arginase 1 (Arg1), chitinase-like molecule 3 (YM1/Chi3l3) and resistance-like α (Retnlα/FIZZ1) in the lung were examined by qRT-PCR. Spleen-derived CD4+ T cells from the asthmatic mice were co-cultured in vitro with BMDMs for 48 hours, and then the mRNA expression levels of Arg1, YM1, and FIZZ1 in the BMDMs were detected by qRT-PCR. The spleen CD4+ T cells of the asthmatic mice were transfected with miR-155-5p inhibitor or the negative control, and then co-cultured with BMDM for 48 hours. The qRT-PCR was used to further determine the expression levels of Arg1, YM1, FIZZ1 in BMDMs. Results Compared with the control group, the levels of miR-155-5p in the lung, spleen and spleen CD4+ T cells of asthmatic mice increased, and the proportion of Th2 cells in asthmatic mouse spleen also increased. The expression levels of the M2 macrophage marker genes Arg1, YM1 and FIZZ1 were up-regulated in the lung of asthmatic mice compared to the control group. After co-culture of spleen CD4+ T cells from asthmatic mice with BMDMs in vitro, the mRNA expression levels of M2 marker genes Arg1, YM1 and FIZZ1 of BMDMs were up-regulated. While transfected with miR-155-5p-inhibitor, the spleen CD4+ T cells of asthmatic mice did not significantly affect the M2 marker gene expression of the BMDMs. Conclusion The spleen-derived CD4+ T cells of asthmatic mice can promote the polarization of co-cultured macrophages towards M2 phenotype in vitro.


Assuntos
Asma/imunologia , Polaridade Celular , Macrófagos/citologia , Baço/citologia , Células Th1/citologia , Células Th2/citologia , Animais , Arginase/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lectinas/metabolismo , Camundongos , MicroRNAs/metabolismo , Baço/imunologia , beta-N-Acetil-Hexosaminidases/metabolismo
16.
Food Chem ; 297: 124972, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253320

RESUMO

The aim of the present study was to evaluate Paralichthys olivaceus parvalbumin (PV) following treatment by laccase (LAC) in the presence of propyl gallate (PG) on the structure and potential allergenicity. The structure of LAC + PG treated PV was analyzed through SDS-PAGE, CD, fluorescence, and allergenicity was analyzed by immunological and cell model. Our results showed that LAC + PG treatment can induce structural changes through PV cross-linking. Western blotting and indirect ELISA analysis revealed the decrease in IgG binding capacity of PV, corresponding with the structural changes. The results of in vitro digestion illustrate that LAC + PG treated PV showed more resistance to gastrointestinal digestion compared to untreated PV. The release rate of ß-hexosaminidase and histamine decreased by 35.6% and 66.9%, respectively, with LAC + PG treatment by RBL-2H3 cell assay. Considering the wide utilization of LAC in food industry, our treatment reveals its potential for creation of hypoallergenic fish products under mild reaction conditions.


Assuntos
Alérgenos/imunologia , Proteínas de Peixes/imunologia , Linguados/imunologia , Lacase/metabolismo , Parvalbuminas/imunologia , Galato de Propila/química , Animais , Catálise , Reagentes para Ligações Cruzadas/química , Digestão , Ensaio de Imunoadsorção Enzimática , Proteínas de Peixes/química , Indústria Alimentícia , Histamina/metabolismo , Parvalbuminas/química , beta-N-Acetil-Hexosaminidases/metabolismo
17.
Appl Biochem Biotechnol ; 189(4): 1262-1273, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31240546

RESUMO

As a type of ß-N-acetyl-D-hexosaminidase enzyme purified from the Ostriniafurnacalis (Asian corn borer), OfHex1 has been previously reported to participate in chitin degradation, indicating that it may be an ideal target for designing new environmentally friendly pesticides. Besides, a natural product, TMG-chitotriomycin, has been found to be an effective inhibitor of OfHex1, and some studies have shown that the interactions between TMG unit and residues in - 1 subsite of OfHex1 are very conservative and important, inspiring us to design new inhibitors of ß-N-acetyl-D-hexosaminidase with a new strategy. In the present study, the virtual screening of TMG unit as the core fragment was conducted using the combined computational methods, such as docking, molecular dynamics, pharmacophore model, and pesticide-likeness rule. Nine compounds with the binding free energy lower than TMG-ß-(GlcNAc)2 were obtained. According to the decomposition energy and the interactions analysis, compounds 2, 3, 6 and 8, forming the hydrogen bonds with Val327 and Trp490, were considered as the possible lead structures for the further study. Our findings indicated that fragment-based lead discovery strategy might provide valuable insights into designing novel potential OfHex1 inhibitors.


Assuntos
Inibidores Enzimáticos/química , Proteínas de Insetos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mariposas/enzimologia , beta-N-Acetil-Hexosaminidases , Animais , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Praguicidas/química , Álcoois Açúcares/química , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/isolamento & purificação
18.
Int J Mol Med ; 44(2): 363-374, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31198979

RESUMO

Chronic, low­grade inflammation associated with obesity and diabetes result from the infiltration of adipose and vascular tissue by immune cells and contributes to cardiovascular complications. Despite an incomplete understanding of the mechanistic underpinnings of immune cell differentiation and inflammation, O­GlcNAcylation, the addition of O­linked N­acetylglucosamine (O­GlcNAc) to cytoplasmic, nuclear and mitochondrial proteins by the two cycling enzymes, the O­linked N­acetylglucosamine transferase (OGT) and the O­GlcNAcase (OGA), may contribute to fine­tune immunity and inflammation in both physiological and pathological conditions. Early studies have indicated that O­GlcNAcylation of proteins play a pro­inflammatory role in diabetes and insulin resistance, whereas subsequent studies have demonstrated that this post­translational modification could also be protective against acute injuries. These studies suggest that diverse types of insults result in dynamic changes to O­GlcNAcylation patterns, which fluctuate with cellular metabolism to promote or inhibit inflammation. In this review, the current understanding of O­GlcNAcylation and its adaptive modulation in immune and inflammatory responses is summarized.


Assuntos
Acetilglucosamina/imunologia , Imunidade , Inflamação/imunologia , Animais , Humanos , Inflamação/patologia , N-Acetilglucosaminiltransferases/imunologia , Proteínas/imunologia , beta-N-Acetil-Hexosaminidases/imunologia
19.
Cardiovasc Diabetol ; 18(1): 66, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151453

RESUMO

BACKGROUND: The mechanisms underlying increased mortality in patients with diabetes and admission hyperglycemia after an acute coronary syndrome may involve reduced capacity for cardioprotection. We investigated the impact of hyperglycemia on exogenously activated cardioprotection by ischemic preconditioning (IPC) in hearts from rats with type 2 diabetes mellitus (T2DM) that were endogenously cardioprotected by an inherent mechanism, and the involvement of myocardial glucose uptake (MGU) and myocardial O-linked ß-N-acetylglucosamine (O-GlcNAc). METHODS AND RESULTS: In isolated, perfused rat hearts subjected to ischemia-reperfusion, infarct size (IS) was overall larger during hyper- ([Glucose] = 22 mmol/L]) than normoglycemia ([Glucose] = 11 mmol/L]) (p < 0.001). IS was smaller in 12-week old Zucker diabetic fatty rats with recent onset T2DM (fa/fa) than in rats without T2DM (fa/+) (n = 8 in each group) both during hyperglycemia (p < 0.05) and normoglycemia (p < 0.05). IPC (2 × 5 min cycles) reduced IS during normo- (p < 0.01 for both groups) but not during hyperglycemia independently of the presence of T2DM. During hyperglycemia, an intensified IPC stimulus (4 × 5 min cycles) reduced IS only in hearts from animals with T2DM (p < 0.05). IPC increased MGU and O-GlcNAc levels during reperfusion in animals with and without T2DM at normoglycemia (MGU: p < 0.05, O-GlcNAc: p < 0.01 for both groups) but not during hyperglycemia. Intensified IPC at hyperglycemia increased MGU (p < 0.05) and O-GlcNAc levels (p < 0.05) only in hearts from animals with T2DM. CONCLUSION: While the effect of IPC is reduced during hyperglycemia in rats without T2DM, endogenous cardioprotection in animals with T2DM is not influenced by hyperglycemia and the capacity for exogenous cardioprotection by IPC is preserved. MGU and O-GlcNAc levels are increased by exogenously induced cardioprotection by IPC but not by endogenous cardioprotection in animals with T2DM reflecting different underlying mechanisms by exogenous and endogenous cardioprotection.


Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Precondicionamento Isquêmico Miocárdico , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Acetilglucosamina/metabolismo , Animais , Biomarcadores/sangue , Diabetes Mellitus Tipo 2/complicações , Modelos Animais de Doenças , Preparação de Coração Isolado , Infarto do Miocárdio/sangue , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Ratos Zucker , beta-N-Acetil-Hexosaminidases/metabolismo
20.
J Zhejiang Univ Sci B ; 20(5): 437-448, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31090269

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification occurring on myriad proteins in the cell nucleus, cytoplasm, and mitochondria. The donor sugar for O-GlcNAcylation, uridine-diphosphate N-acetylglucosamine (UDP-GlcNAc), is synthesized from glucose through the hexosamine biosynthetic pathway (HBP). The recycling of O-GlcNAc on proteins is mediated by two enzymes in cells-O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and removal of O-GlcNAc, respectively. O-GlcNAcylation is involved in a number of important cell processes including transcription, translation, metabolism, signal transduction, and apoptosis. Deregulation of O-GlcNAcylation has been reported to be associated with various human diseases such as cancer, diabetes, neurodegenerative diseases, and cardiovascular diseases. A better understanding of the roles of O-GlcNAcylation in physiopathological processes would help to uncover novel avenues for therapeutic intervention. The aim of this review is to discuss the recent updates on the mechanisms and impacts of O-GlcNAcylation on these diseases, and its potential as a new clinical target.


Assuntos
Acetilglucosamina/química , Doenças Cardiovasculares/metabolismo , Doenças Neurodegenerativas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apoptose , Catálise , Núcleo Celular/metabolismo , Proliferação de Células , Citoplasma/metabolismo , Complicações do Diabetes/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hexosaminas/química , Humanos , Insulina , Mitocôndrias/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias/metabolismo , Fosforilação , Transdução de Sinais , beta-N-Acetil-Hexosaminidases/metabolismo
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