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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
Org Biomol Chem ; 17(17): 4326-4334, 2019 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-30976765

RESUMO

An unnatural monosaccharide with a C6-azide, Ac36AzGalNAc, has been developed as a potent and selective probe for O-GlcNAc-modified proteins. Combined with click chemistry, we demonstrate that Ac36AzGalNAc can robustly label O-GlcNAc glycosylation in a wide range of cell lines. Meanwhile, cell imaging and LC-MS/MS proteomics verify its selective activity on O-GlcNAc. More importantly, the protocol presented here provides a general methodology for tracking, capturing and identifying unnatural monosaccharide modified proteins in cells or cell lysates.


Assuntos
Galactosamina/química , Sondas Moleculares/química , N-Acetilglucosaminiltransferases/análise , beta-N-Acetil-Hexosaminidases/análise , Animais , Células Cultivadas , Galactosamina/análogos & derivados , Galactosamina/síntese química , Humanos , Camundongos , Sondas Moleculares/síntese química , Estrutura Molecular , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
12.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1690-1700, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30978390

RESUMO

Mast cell (MC) deficiency in KitW-sh/W-sh mice and inhibition with disodium chromoglycate (DSCG) or ketotifen reduced obesity and diabetes in mice on a high-cholesterol (1.25%) Western diet. Yet, Kit-independent MC-deficient mice and mice treated with DSCG disproved MC function in obesity and diabetes when mice are fed a high-fat diet (HFD) that contains no cholesterol. This study reproduced the obesity and diabetes inhibitory activities of DSCG and ketotifen from mice on a Western diet. Yet, such inhibitory effects were diminished in mice on the HFD. DSCG and ketotifen MC inhibitory activities were recovered from mice on the HFD supplemented with the same amount of cholesterol (1.25%) as that in the Western diet. DSCG and ketotifen effectively blunted the high-cholesterol diet-induced elevations of blood histamine and adipose tissue MC degranulation. Pearson's correlation test demonstrated significant and positive correlations between plasma histamine and total cholesterol or low-density lipoprotein-cholesterol (LDL). In cultured bone marrow-derived MCs, plasma from mice following a Western diet or a cholesterol-supplemented HFD, but not those from HFD-fed mice, induced MC degranulation and the release of ß-hexosaminidase, histamine, and serotonin. IgE, LDL, very low-density lipoprotein, and high-density lipoprotein also induced MC activation, which can be inhibited by DSCG and ketotifen depending on the doses and types of MC inhibitors and cholesterol, and also the MC granule molecules of interest. DSCG or ketotifen lost their activities in inhibiting LDL-induced activation of MCs from LDL receptor-deficient mice. These results indicate that dietary cholesterol critically influences the function of mouse MCs.


Assuntos
Degranulação Celular/efeitos dos fármacos , Colesterol na Dieta/efeitos adversos , Diabetes Mellitus Experimental/sangue , Mastócitos/efeitos dos fármacos , Obesidade/sangue , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Animais , Antialérgicos/farmacologia , Antiasmáticos/farmacologia , LDL-Colesterol/metabolismo , Cromolina Sódica/farmacologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica/efeitos adversos , Expressão Gênica , Histamina/sangue , Cetotifeno/farmacologia , Masculino , Mastócitos/metabolismo , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/patologia , Receptores de LDL/deficiência , Receptores de LDL/genética , Serotonina/metabolismo , Transdução de Sinais , Estreptozocina , beta-N-Acetil-Hexosaminidases/metabolismo
13.
Neurosci Lett ; 705: 251-258, 2019 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-30928480

RESUMO

BACKGROUND AND PURPOSE: Studies demonstrated that oxidative damage decreased intracellular ATP level in astrocytes. However, the pathway mediated ATP level decrease is obscure. Our previous study found intracellular ATP could be released via lysosome exocytosis in astrocytes. Here, we explored whether lysosome exocytosis was involved in ATP release during oxidative stress induced by H2O2 in astrocytes. METHODS: Astrocytes were isolated from the cortex of neonatal rats. Intracellular lysosomes and calcium signals were stained in astrocytes before and after H2O2 stimulation. ATP molecules location and ATP level were detected by immunostaining and bioluminescence method, respectively. Extracellular ß-Hexosaminidase and LDH were examined by colorimetric method. RESULTS: We found that ATP located in lysosome of astrocytes. H2O2 stimulation resulted in the decrease of lysosomes staining and the increase of extracellular ATP, compared to the control (p < 0.05). At the same time, intracellular Fluo4 signals and ß-Hexosaminidase level were also increased (p < 0.05). Extracellular LDH level did not show an increase, suggesting that there is no cell membrane damage after H2O2 stimulation. Glycyl-phenylalanine 2-naphthylamide blocked lysosome exocytosis and inhibited ATP release in astrocytes after H2O2-treatment (p < 0.05). CONCLUSION: Our results indicated that H2O2 induced ATP release from intracellular to extracellular via lysosome exocytosis. The increase of intracellular Ca2+ was necessary for lysosome release under oxidative stress induced by H2O2.


Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/metabolismo , Exocitose/fisiologia , Peróxido de Hidrogênio/farmacologia , Lisossomos/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Exocitose/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Lisossomos/efeitos dos fármacos , Cultura Primária de Células , Ratos , beta-N-Acetil-Hexosaminidases/metabolismo
14.
Int J Biol Macromol ; 133: 1029-1034, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31004644

RESUMO

Defatted krill powder (DKP), the byproduct of krill oil industry, is a resource of biological macromolecules. Here, one bacterial protease, three bacterial chitinases and one insect N-acetyl-d-hexosaminidase were integratively used to produce peptide, N,N'-diacetylchitobiose [(GlcNAc)2] and N-acetyl-d-glucosamine (GlcNAc) from DKP. First, alkaline protease was found to outperform neutral protease in deproteinizing DKP and the resultant krill peptides were rich in essential amino acids (41.4%). Second, the mutant of chitinase A from Serratia marcescens [SmChiA-F232W/F396W (SmChiA-M)] was found to be 32% faster than wild-type SmChiA in hydrolyzing the deproteinized DKP (DDKP) and showed significant synergy with chitinase B from S. marcescens (SmChiB) and chitinase C from S. marcescens (SmChiC). Then two SmChiA-M-based enzyme combinations [SmChiA-M + SmChiB + SmChiC and SmChiA-M + SmChiB + SmChiC + OfHex1 (an insect N-acetyl-d-hexosaminidase from Ostrinia furnacalis)] were designed to produce (GlcNAc)2 and GlcNAc, respectively, from DDKP. A yield of 2.04 g/L (GlcNAc)2 or 2.71 g/L GlcNAc (each with 95% purity) could be obtained from 20 g/L DDKP in 24 h.


Assuntos
Acetilglucosamina/metabolismo , Quitinases/metabolismo , Dissacarídeos/metabolismo , Euphausiacea/química , Resíduos , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Aspergillus niger/metabolismo , Quitina/metabolismo , Hidrólise , Lepidópteros/enzimologia , Pós , Serratia marcescens/enzimologia
15.
Contact Dermatitis ; 81(3): 184-193, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31006867

RESUMO

BACKGROUND: Retinoic acid (RA)-induced dermatitis is the most frequent side-effect limiting its widespread use. However, the exact mechanisms triggering dermatitis are not fully understood, including the role of skin mast cells. The newly discovered Mas-related G-protein-coupled receptor-X2 (MRGPRX2) in mast cells mediates pseudoallergic drug reactions in several types of dermatitis. A possible contribution of MRGPRX2 to contact dermatitis induced by RA has hitherto not been examined. OBJECTIVES: To investigate whether all-trans-RA (ATRA) activates mast cells via MRGPRX2/MrgprB2 (the mouse orthologue), contributing to the pathogenesis of retinoid-induced dermatitis. METHODS: Wild-type (WT) and MrgprB2-/- mice were treated with topical ATRA to observe local inflammation and mast cell degranulation in vivo by the use of haematoxylin and eosin and immunofluorescence staining. Release of histamine and release of ß-hexosaminidase were measured and calcium influx was detected in Laboratory of Allergic Disease 2 (LAD2) cells with specific knockdown targeting MRGPRX2 by small interfering RNA (siRNA) and in primary cells from MrgprB2-/- mice. RESULTS: As compared with WT mice, MrgprB2-/- mice showed resistance to ATRA-triggered contact dermatitis and local inflammatory reactions in the paws. ATRA activated mast cells via the MrgprB2 pathway in murine cells, and via the MRGPRX2 pathway in human mast cells. CONCLUSIONS: ATRA-induced dermatitis could be achieved by activating mast cells via MRGPRX2/MrgprB2, which may provide a potential therapy target to reduce the side-effect.


Assuntos
Degranulação Celular/efeitos dos fármacos , Dermatite de Contato/etiologia , Mastócitos/fisiologia , Receptores Acoplados a Proteínas-G/genética , Tretinoína/farmacologia , Animais , Cálcio/metabolismo , Linhagem Celular , Dermatite , Dermatite de Contato/genética , Técnicas de Silenciamento de Genes , Histamina/metabolismo , Humanos , Masculino , Mastócitos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Tretinoína/toxicidade , beta-N-Acetil-Hexosaminidases/metabolismo
16.
Am J Physiol Cell Physiol ; 316(6): C862-C875, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30865517

RESUMO

The attachment of O-linked ß-N-acetylglucosamine (O-GlcNAc) to the serine and threonine residues of proteins in distinct cellular compartments is increasingly recognized as an important mechanism regulating cellular function. Importantly, the O-GlcNAc modification of mitochondrial proteins has been identified as a potential mechanism to modulate metabolism under stress with both potentially beneficial and detrimental effects. This suggests that temporal and dose-dependent changes in O-GlcNAcylation may have different effects on mitochondrial function. In the current study, we found that acutely augmenting O-GlcNAc levels by inhibiting O-GlcNAcase with Thiamet-G for up to 6 h resulted in a time-dependent decrease in cellular bioenergetics and decreased mitochondrial complex I, II, and IV activities. Under these conditions, mitochondrial number was unchanged, whereas an increase in the protein levels of the subunits of several electron transport complex proteins was observed. However, the observed bioenergetic changes appeared not to be due to direct increased O-GlcNAc modification of complex subunit proteins. Increases in O-GlcNAc were also associated with an accumulation of mitochondrial ubiquitinated proteins; phosphatase and tensin homolog induced kinase 1 (PINK1) and p62 protein levels were also significantly increased. Interestingly, the increase in O-GlcNAc levels was associated with a decrease in the protein levels of the mitochondrial Lon protease homolog 1 (LonP1), which is known to target complex IV subunits and PINK1, in addition to other mitochondrial proteins. These data suggest that impaired bioenergetics associated with short-term increases in O-GlcNAc levels could be due to impaired, LonP1-dependent, mitochondrial complex protein turnover.


Assuntos
Proteases Dependentes de ATP/metabolismo , Acetilglucosamina/metabolismo , Regulação para Baixo/fisiologia , Metabolismo Energético/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Proteases Dependentes de ATP/antagonistas & inibidores , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Proteínas Mitocondriais/antagonistas & inibidores
17.
Int Immunopharmacol ; 71: 1-6, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30861392

RESUMO

The anti-allergic effect of berberine was evaluated in cellular and animal models of allergic responses. In this study, the results of the in vitro model of immunoglobulin (Ig) E-mediated mast cell degranulation showed that berberine significantly inhibited the release of ß-hexosaminidase (ß-HEX), histamine, IL-4 and TNF-α in rat basophilic leukemia cells (RBL-2H3 cells). Pretreatment with berberine prevented morphological changes in IgE-stimulated RBL-2H3 cells such as the recovery of an elongated shape. Pretreatment with berberine also suppressed the phosphorylation of antigen-induced Lyn, Syk, and Gab2, thus suppressing the downstream MAPK pathways. In the in vivo model of allergic responses, administration of berberine inhibited passive cutaneous anaphylaxis (PCA) in mice. The above results indicate berberine could suppress mast cell activation and allergic responses.


Assuntos
Antialérgicos/uso terapêutico , Berberina/uso terapêutico , Hipersensibilidade/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Animais , Degranulação Celular , Linhagem Celular , Modelos Animais de Doenças , Histamina/metabolismo , Humanos , Imunoglobulina E/metabolismo , Interleucina-4/metabolismo , Masculino , Mastócitos/fisiologia , Camundongos , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Ratos , Fator de Necrose Tumoral alfa/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
18.
Biochem Biophys Res Commun ; 511(4): 833-839, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30846208

RESUMO

ß-N-acetylhexosaminidases from Akkermansia muciniphila was reported to perform the crystal structure with GlcNAc complex, which proved to be the substrate of Am2301. Domain II of Am2301 is consisted of amino acid residues 111-489 and is folded as a (ß/α)8 barrel with the active site combined of the glycosyl hydrolases. Crystallographic evidence showed that Asp-278 and Glu-279 could be the catalytic site and Tyr-373 may plays a role on binding the substrate. Moreover, Am2301 prefers to bind Zn ion, which similar to other GH 20 family. Enzyme activity and kinetic parameters of wild-type Am2301 and mutants proved that Asp-278 and Glu-279 are the catalytic sites and they play a critical role on the catalytic process. Overall, our results demonstrate that Am2301 and its complex with GlcNAC provide obvious structural evidence for substrate-assisted catalysis. Obviously, this expands our understanding on the mode of substrate-assisted reaction for this enzyme family in Akkermansia muciniphila.


Assuntos
Acetilglucosamina/metabolismo , Proteínas de Bactérias/metabolismo , Verrucomicrobia/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Proteínas de Bactérias/química , Domínio Catalítico , Cristalografia por Raios X , Cinética , Modelos Moleculares , Especificidade por Substrato , Verrucomicrobia/química , Verrucomicrobia/enzimologia , beta-N-Acetil-Hexosaminidases/química
19.
Int J Mol Sci ; 20(6)2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917577

RESUMO

ß-N-Acetyl-d-hexosaminidase from Ostrinia furnacalis (OfHex1) is a new target for the design of insecticides. Although some of its inhibitors have been found, there is still no commercial drug available at present. The residence time of the ligand may be important for its pharmacodynamic effect. However, the unbinding routes of ligands from OfHex1 still remain largely unexplored. In the present study, we first simulated the six dissociation routes of N,N,N-trimethyl-d-glucosamine-chitotriomycin (TMG-chitotriomycin, a highly selective inhibitor of OfHex1) from the active pocket of OfHex1 by steered molecular dynamics simulations. By comparing the potential of mean forces (PMFs) of six routes, Route 1 was considered as the most possible route with the lowest energy barrier. Furthermore, the structures of six different states for Route 1 were snapshotted, and the key amino acid residues affecting the dissociated time were analyzed in the unbinding pathway. Moreover, we also analyzed the "open⁻close" mechanism of Glu368 and Trp448 and found that their conformational changes directly affected the dissociation of TMG-chitotriomycin. Our findings would be helpful to understanding and identifying novel inhibitors against OfHex1 from virtual screening or lead-optimization.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas de Insetos/química , Inseticidas/farmacologia , Simulação de Dinâmica Molecular , Álcoois Açúcares/farmacologia , beta-N-Acetil-Hexosaminidases/química , Animais , Sítios de Ligação , Inibidores Enzimáticos/química , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/metabolismo , Inseticidas/química , Lepidópteros/efeitos dos fármacos , Lepidópteros/enzimologia , Ligação Proteica , Álcoois Açúcares/química , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/metabolismo
20.
Int J Mol Sci ; 20(5)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871033

RESUMO

An unstudied ß-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and a robust thermal stability at 50 °C within 24 h. The addition of urea (1 M) or sodium dodecyl sulfate (1% w/v) reduced the activity of the enzyme by 44% and 58%, respectively, whereas the addition of divalent metal ions had no effect on the enzymatic activity. PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) strongly inhibited the enzyme in sub-micromolar concentrations. The ß-N-acetylhexosaminidase was able to hydrolyze ß1,2-linked, ß1,3-linked, ß1,4-linked, and ß1,6-linked GlcNAc residues from the non-reducing end of various tested glycan standards, including bisecting GlcNAc from one of the tested hybrid-type N-glycan substrates. A mutational study revealed that the amino acids D306 and E307 bear the catalytically relevant side acid/base side chains. When coupled with a chitinase, the ß-N-acetylhexosaminidase was able to generate GlcNAc directly from colloidal chitin, which showed the potential of this enzyme for biotechnological applications.


Assuntos
Actinomycetales/metabolismo , Dissacarídeos/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/metabolismo , Aminoácidos/metabolismo , Quitina/metabolismo , Quitinases/metabolismo , Escherichia coli/metabolismo , Oximas/metabolismo , Fenilcarbamatos/metabolismo , Microbiologia do Solo
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