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1.
J Agric Food Chem ; 68(10): 3195-3202, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32075368

RESUMO

d-Tagatose is a rare monosaccharide that is used in products in the food industry as a low-calorie sweetener. To facilitate biological conversion of d-tagatose, the agarolytic enzyme complexes based on the principle of the cellulosome structure were constructed through dockerin-cohesin interaction with the scaffoldin. The construction of agarolytic complexes composed of l-arabinose isomerase caused efficient isomerization activity on the agar-derived sugars. In a trienzymatic complex, the chimeric ß-agarase (cAgaB) and anhydro-galactosidase (cAhgA) from Zobellia galactanivorans could synergistically hydrolyze natural agar substrates and l-arabinose isomerase (LsAraA Doc) from Lactobacillus sakei 23K could convert d-galactose into d-tagatose. The trienzymatic complex increased the concentration of d-tagatose from the agar substrate to 4.2 g/L. Compared with the monomeric enzyme, the multimeric enzyme showed a 1.4-fold increase in tagatose production, good thermostability, and reusability. A residual activity of 75% remained, and 52% of conversion was noted after five recycles. These results indicated that the dockerin-fused chimeric enzymes on the scaffoldin successfully isomerized d-galactose into d-tagatose with synergistic activity. Thus, the results demonstrated the possibility of advancing efficient strategies for utilizing red algae as a biomass source to produce d-tagatose in the industrial food field that uses marine biomass as the feedstock.


Assuntos
Aldose-Cetose Isomerases/química , Proteínas de Bactérias/química , Galactose/química , Galactosidases/química , Glicosídeo Hidrolases/química , Hexoses/química , Edulcorantes/química , Biocatálise , Flavobacteriaceae/enzimologia , Isomerismo , Lactobacillus sakei/enzimologia
2.
Molecules ; 24(17)2019 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-31450608

RESUMO

BACKGROUND: Liver cancer is a common malignant tumor worldwide, and its morbidity and mortality increase each year. The disease has a short course and high mortality, making it a serious threat to human health. PURPOSE: The objective of this study was to create novel liver-targeting nanoliposomes to encapsulate cantharidin (CTD) as a potential treatment for hepatic carcinoma. METHODS: 3-Galactosidase-30-stearyl deoxyglycyrrhetinic acid (11-DGA-3-O-Gal)-modified liposomes (11-DGA-3-O-Gal-CTD-lip) for the liver-targeted delivery of CTD were prepared via the film-dispersion method and characterized. In vitro analyses of the effects on cellular cytotoxicity, cell migration, cell cycle, and cell apoptosis were carried out and an in vivo pharmacokinetics study and tissue distribution analysis were performed. RESULTS: Compared with unmodified liposomes (CTD-lip), 11-DGA-3-O-Gal-CTD-lip showed higher cytotoxicity and increased the inhibition of HepG2 cell migration, but they did not increase the apoptotic rate of cells. The inhibition mechanism of 11-DGA-3-O-Gal-CTD-lip on hepatocellular carcinoma was partly through cell cycle arrest at the S phase. Analysis of pharmacokinetic parameters indicated that 11-DGA-3-O-Gal-CTD-lip were eliminated more rapidly than CTD-lip. Regarding tissue distribution, the targeting efficiency of 11-DGA-3-O-Gal-CTD-lip to the liver was (41.15 ± 3.28)%, relative targeting efficiency was (1.53 ± 0.31)%, relative uptake rate was( 1.69 ± 0.37)%, and peak concentration ratio was (2.68 ± 0.12)%. CONCLUSION: 11-DGA-3-O-Gal-CTD-lip represent a promising nanocarrier for the liver-targeted delivery of antitumor drugs to treat hepatocellular carcinoma.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/química , Cantaridina/administração & dosagem , Cantaridina/química , Galactosidases/química , Ácido Glicirretínico/química , Lipossomos , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Cantaridina/síntese química , Cantaridina/farmacocinética , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Química Sintética , Portadores de Fármacos , Composição de Medicamentos , Feminino , Células Hep G2 , Humanos , Neoplasias Hepáticas , Masculino , Estrutura Molecular , Ratos , Distribuição Tecidual
3.
J Biol Chem ; 294(31): 11701-11711, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31186348

RESUMO

Bifidobacteria are exposed to substantial amounts of dietary ß-galactosides. Distinctive preferences for growth on different ß-galactosides are observed within Bifidobacterium members, but the basis of these preferences remains unclear. We previously described the first ß-(1,6)/(1,3)-galactosidase from Bifidobacterium animalis subsp. lactis Bl-04. This enzyme is relatively promiscuous, exhibiting only 5-fold higher efficiency on the preferred ß-(1,6)-galactobiose than the ß-(1,4) isomer. Here, we characterize the solute-binding protein (Bal6GBP) that governs the specificity of the ABC transporter encoded by the same ß-galactoside utilization locus. We observed that although Bal6GBP recognizes both ß-(1,6)- and ß-(1,4)-galactobiose, Bal6GBP has a 1630-fold higher selectivity for the former, reflected in dramatic differences in growth, with several hours lag on less preferred ß-(1,4)- and ß-(1,3)-galactobiose. Experiments performed in the presence of varying proportions of ß-(1,4)/ß-(1,6)-galactobioses indicated that the preferred substrate was preferentially depleted from the culture supernatant. This established that the poor growth on the nonpreferred ß-(1,4) was due to inefficient uptake. We solved the structure of Bal6GBP in complex with ß-(1,6)-galactobiose at 1.39 Å resolution, revealing the structural basis of this strict selectivity. Moreover, we observed a close evolutionary relationship with the human milk disaccharide lacto-N-biose-binding protein from Bifidobacterium longum, indicating that the recognition of the nonreducing galactosyl is essentially conserved, whereas the adjacent position is diversified to fit different glycosidic linkages and monosaccharide residues. These findings indicate that oligosaccharide uptake has a pivotal role in governing selectivity for distinct growth substrates and have uncovered evolutionary trajectories that shape the diversification of sugar uptake proteins within Bifidobacterium.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Bifidobacterium animalis/crescimento & desenvolvimento , Galactosidases/metabolismo , Galactosídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Bifidobacterium animalis/enzimologia , Bifidobacterium animalis/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Evolução Molecular , Galactosidases/química , Galactosídeos/química , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Especificidade por Substrato
4.
J Dairy Sci ; 102(7): 6027-6031, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31056324

RESUMO

To date, most studies of lactose utilization have focused on the genetic diversity of lactic acid bacteria or its influence on product quality, but phenotypic evaluation has rarely been based on metabolic characteristics. In the present study, we investigated the growth, acid production, ß-galactosidase, and 6-phospho-ß-galactosidase activities of 16 Lactococcus lactis strains obtained from various habitats with lactose as the sole carbon source. The 15 L. lactis strains obtained from various habitats exhibited significant differences in growth and acid production characteristics in the de Man, Rogosa, and Sharpe-lactose broth, and 4 strains consumed more lactose when cultured in skim milk than the type strain ATCC 19435. Among these strains, DQHXNQ38-12 mainly produced acetoin and diacetyl when cultured in skim milk, whereas the strains 15M2 and 5G2 produced high levels of acid and formed curd rapidly. We concluded that the use of lactose is necessary for strain adaptation to the dairy niche. Comprehensive studies of lactose use and the fermentation characteristics of L. lactis are of significant importance.


Assuntos
Proteínas de Bactérias/metabolismo , Galactosidases/metabolismo , Lactococcus lactis/metabolismo , Lactose/metabolismo , Leite/microbiologia , Animais , Fermentação , Lactococcus lactis/enzimologia , Lactococcus lactis/crescimento & desenvolvimento
5.
J Agric Food Chem ; 67(19): 5486-5495, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31012315

RESUMO

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.


Assuntos
Cannabis/microbiologia , Proteínas Fúngicas/metabolismo , Pleurotus/enzimologia , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Galactosidases/química , Galactosidases/genética , Galactosidases/metabolismo , Glicosilação , Lacase/química , Lacase/genética , Lacase/metabolismo , Lignina/metabolismo , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Peroxidases/química , Peroxidases/genética , Peroxidases/metabolismo , Caules de Planta/microbiologia , Pleurotus/classificação , Pleurotus/genética , Pleurotus/crescimento & desenvolvimento , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Transporte Proteico
6.
Appl Biochem Biotechnol ; 188(4): 1077-1095, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30788710

RESUMO

Agar, a major polysaccharide of red algal cells, is degraded by ß-agarases into neoagarobiose, which is further hydrolyzed into the monomers, D-galactose and 3,6-anhydro-L-galactose, by 1,3-α-3,6-anhydro-L-galactosidases including α-1,3-L-neoagarooligasaccharide hydrolase (α-NAOSH). A novel cold-adapted alkaline α-NAOSH, Ahg558, consisting of 359 amino acids (40.8 kDa) was identified from Gayadomonas joobiniege G7. It was annotated as a glycosyl hydrolase family 43 based on genomic sequence analysis, showing 84% and 74% identities with the characterized α-NAOSHs from Agarivorans gilvus WH0801 and Saccharophagus degradans 2-40, respectively. The recombinant Ahg558 (rAhg558) purified from Escherichia coli formed dimers and cleaved α-1,3 glycosidic bonds at the non-reducing ends of the neoagarobiose, neoagarotetraose, and neoagarohexaose, which was confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for rAhg558 activity were 9.0 and 30 °C, respectively. Unusually, it retained over 93% activity in a broad range of temperatures between 0 and 40 °C and over 73% in a broad range of pH between pH 6.0 and pH 9.0, indicating it is a unique cold-adapted alkaline exo-acting α-NAOSH. Its enzymatic activity was dependent on Mn2+ ions. Km and Vmax values toward neoagarobiose were 2.6 mg/mL (8.01 mM) and 133.33 U/mg, respectively.


Assuntos
Galactosidases/metabolismo , Cromatografia em Camada Delgada , Clonagem Molecular , Dissacarídeos/metabolismo , Galactosídeos/metabolismo , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Oligossacarídeos/metabolismo
7.
Carbohydr Res ; 473: 99-103, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30658252

RESUMO

ß-d-Galactofuranose (Galf) is a component of polysaccharides and glycoconjugates. There are few reports about the involvement of galactofuranosyltransferases and galactofuranosidases (Galf-ases) in the synthesis and degradation of galactofuranose-containing glycans. The cell walls of filamentous fungi in the genus Aspergillus include galactofuranose-containing polysaccharides and glycoconjugates, such as O-glycans, N-glycans, and fungal-type galactomannan, which are important for cell wall integrity. In this study, we investigated the synthesis of p-nitrophenyl ß-d-galactofuranoside and its disaccharides by chemo-enzymatic methods including use of galactosidase. The key step was selective removal of the concomitant pyranoside by enzymatic hydrolysis to purify p-nitrophenyl ß-d-galactofuranoside, a promising substrate for ß-d-galactofuranosidase from Streptomyces species.


Assuntos
Aspergillus/química , Dissacarídeos/química , Dissacarídeos/síntese química , Galactosidases/metabolismo , Mananas/química , Técnicas de Química Sintética , Hidrólise , Especificidade por Substrato
8.
Nat Chem Biol ; 15(2): 151-160, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30598545

RESUMO

The spatiotemporal generation of nitric oxide (NO), a versatile endogenous messenger, is precisely controlled. Despite its therapeutic potential for a wide range of diseases, NO-based therapies are limited clinically due to a lack of effective strategies for precisely delivering NO to a specific site. In the present study, we developed a novel NO delivery system via modification of an enzyme-prodrug pair of galactosidase-galactosyl-NONOate using a 'bump-and-hole' strategy. Precise delivery to targeted tissues was clearly demonstrated by an in vivo near-infrared imaging assay. The therapeutic potential was evaluated in both rat hindlimb ischemia and mouse acute kidney injury models. Targeted delivery of NO clearly enhanced its therapeutic efficacy in tissue repair and function recovery and abolished side effects due to the systemic release of NO. The developed protocol holds broad applicability in the targeted delivery of important gaseous signaling molecules and offers a potent tool for the investigation of relevant molecular mechanisms.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Óxido Nítrico/administração & dosagem , Óxido Nítrico/metabolismo , Animais , Compostos Azo , Galactosidases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Óxido Nítrico/fisiologia , Pró-Fármacos , Ratos , Ratos Sprague-Dawley , beta-Galactosidase/metabolismo , beta-Galactosidase/fisiologia
9.
Nat Chem ; 11(2): 161-169, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30532014

RESUMO

Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N-linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch-selective manner to give asymmetrical structures commonly found in nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in ten or fewer chemical and enzymatic steps into multi-antennary N-glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N-glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart, allowing each arm to be elaborated into a unique appendage.


Assuntos
Materiais Biomiméticos/metabolismo , Polissacarídeos/metabolismo , Asparagina/metabolismo , Sequência de Carboidratos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Galactosidases/metabolismo , Glicopeptídeos/metabolismo , Glicosilação , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/química , Sialiltransferases/metabolismo
10.
Genet Med ; 21(1): 44-52, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29543226

RESUMO

PURPOSE: Plasma globotriaosylsphingosine (lyso-Gb3) is a promising secondary screening biomarker for Fabry disease. Here, we examined its applicability as a primary screening biomarker for classic and late-onset Fabry disease in males and females. METHODS: Between 1 July 2014 and 31 December 2015, we screened 2,359 patients (1,324 males) referred from 168 Japanese specialty clinics (cardiology, nephrology, neurology, and pediatrics), based on clinical symptoms suggestive of Fabry disease. We used the plasma lyso-Gb3 concentration, α-galactosidase A (α-Gal A) activity, and analysis of the α-Gal A gene (GLA) for primary and secondary screens, respectively. RESULTS: Of 8 males with elevated lyso-Gb3 levels (≥2.0 ng ml-1) and low α-Gal A activity (≤4.0 nmol h-1 ml-1), 7 presented a GLA mutation (2 classic and 5 late-onset). Of 14 females with elevated lyso-Gb3, 7 displayed low α-Gal A activity (5 with GLA mutations; 4 classic and 1 late-onset) and 7 exhibited normal α-Gal A activity (1 with a classic GLA mutation and 3 with genetic variants of uncertain significance). CONCLUSION: Plasma lyso-Gb3 is a potential primary screening biomarker for classic and late-onset Fabry disease probands.


Assuntos
Biomarcadores/sangue , Doença de Fabry/sangue , Testes Genéticos , Glicolipídeos/sangue , Esfingolipídeos/sangue , Idoso , Doença de Fabry/genética , Doença de Fabry/patologia , Feminino , Galactosidases/sangue , Galactosidases/genética , Glicolipídeos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Seleção de Pacientes , Fatores de Risco , Esfingolipídeos/genética
11.
Nanoscale ; 10(44): 20702-20716, 2018 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-30398279

RESUMO

The present study compares for the first time the effects of h-MoO3 and α-MoO3 against two fungal strains: Aspergillus niger and Aspergillus flavus. The h-MoO3 nanoparticles were more toxic to both fungi than α-MoO3. The toxic effects of h-MoO3 were more pronounced toward A. flavus, which presented a growth inhibition of 67.4% at 200 mg L-1. The presence of the nanoparticles affected drastically the hyphae morphology by triggering nuclear condensation and compromising the hyphae membrane. Further analysis of the volatile organic compounds (VOCs) produced by both fungi in the presence of the nanomaterials indicated important metabolic changes related to programmed cell death. These nanomaterials induced the production of specific antifungal VOCs, such as ß-Elemene and t-Cadinol, by the fungi. The production of essential enzymes involved in fungal metabolism, such as acid phosphatase, naphthol-As-BI-phosphohydrolase, ß-galactosidase, ß-glucosidase and N-acetyl-ß-glucosaminidase, reduced significantly in the presence of the nanomaterials. The changes in enzymatic production and VOCs corroborate the fact that these nanoparticles, especially h-MoO3, exert changes in the fungal metabolism, triggering apoptotic-like cell death responses in these fungi.


Assuntos
Aspergillus flavus/metabolismo , Aspergillus niger/metabolismo , Enzimas/metabolismo , Nanopartículas Metálicas/química , Molibdênio/química , Óxidos/química , Compostos Orgânicos Voláteis/metabolismo , Fosfatase Ácida/metabolismo , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/crescimento & desenvolvimento , Aspergillus niger/efeitos dos fármacos , Aspergillus niger/crescimento & desenvolvimento , Biomassa , Galactosidases/metabolismo , Nanopartículas Metálicas/toxicidade , Microscopia Eletrônica de Varredura , Espectroscopia Fotoeletrônica , Análise de Componente Principal , Compostos Orgânicos Voláteis/química
12.
Food Microbiol ; 76: 69-77, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30166192

RESUMO

Naturally fermented tofu whey (NFTW) has been used as traditional tofu coagulant in China for hundreds of years. In this study, the microbial diversity in NFTW was firstly analyzed with high-throughput sequencing and its effect on chemical contents of tofu whey (TW) was investigated. Lactobacillus with 95.31% was the predominant genus in the microbial community of NFTW while Picha, Enterococcus, Bacillus and Acetobacter occupied about only 0.90%, 0.04%, 0.02% and 0.09%, respectively. Besides, Lactobacillus amylolyticus were determined to be one of the dominated species with metagenomic analysis and culture method. Lactobacillus with α-galactosidase activities played leading role in metabolizing the soybean oligosaccharides of TW to produce lactic acid. And acetic acid produced by genus of Acetobacter was another main organic acid attributed to the acidification of TW except lactic acid. Meanwhile, the bioconversion of isoflavone glucosides into aglycones could also be promoted by Lactobacillus with the help of ß-glucosidase activity. Moreover, the production of equol in NFTW was confirmed, which might be jointly converted from daidzein by several strains. Therefore, our results indicated that Lactobacillus was the dominated microorganism and mainly affected the chemical changes of NFTW. This study help provide basic theory and technical references for the production of tofu and its derivative products (like sufu) with NFTW as coagulator.


Assuntos
Alimentos e Bebidas Fermentados/microbiologia , Lactobacillus/isolamento & purificação , Alimentos de Soja/microbiologia , Proteínas do Soro do Leite/análise , Bactérias/classificação , Bactérias/isolamento & purificação , Bactérias/metabolismo , Técnicas Bacteriológicas , China , Contagem de Colônia Microbiana , Fermentação , Galactosidases/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Isoflavonas/metabolismo , Lactobacillus/metabolismo
13.
Appl Microbiol Biotechnol ; 102(20): 8855-8866, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30128580

RESUMO

Agar is a major polysaccharide of red algal cells and is mainly decomposed into neoagarobiose by the co-operative effort of ß-agarases. Neoagarobiose is hydrolyzed into monomers, D-galactose and 3,6-anhydro-L-galactose, via a microbial oxidative process. Therefore, the enzyme, 1,3-α-3,6-anhydro-L-galactosidase (α-neoagarobiose/neoagarooligosaccharide hydrolase) involved in the final step of the agarolytic pathway is crucial for bioindustrial application of agar. A novel cold-adapted α-neoagarooligosaccharide hydrolase, Ahg786, was identified and characterized from an agarolytic marine bacterium Gayadomonas joobiniege G7. Ahg786 comprises 400 amino acid residues (45.3 kDa), including a 25 amino acid signal peptide. Although it was annotated as a hypothetical protein from the genomic sequencing analysis, NCBI BLAST search showed 57, 58, and 59% identities with the characterized α-neoagarooligosaccharide hydrolases from Saccharophagus degradans 2-40, Zobellia galactanivorans, and Bacteroides plebeius, respectively. The signal peptide-deleted recombinant Ahg786 expressed and purified from Escherichia coli showed dimeric forms and hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into 3,6-anhydro-L-galactose and other compounds by cleaving α-1,3-glycosidic bonds from the non-reducing ends of neoagarooligosaccharides, as confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for Ahg786 activity were 7.0 and 15 °C, respectively, indicative of its unique cold-adapted features. The enzymatic activity severely inhibited with 0.5 mM ethylenediaminetetraacetic acid was completely restored or remarkably enhanced by Mn2+ in a concentration-dependent manner, suggestive of the dependence of the enzyme on Mn2+ ions. Km and Vmax values for neoagarobiose were 4.5 mM and 1.33 U/mg, respectively.


Assuntos
Alteromonadaceae/enzimologia , Proteínas de Bactérias/química , Galactosidases/química , Alteromonadaceae/química , Alteromonadaceae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Galactosidases/genética , Galactosidases/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Alinhamento de Sequência , Temperatura
14.
Kidney Blood Press Res ; 43(2): 406-421, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29558749

RESUMO

Fabry disease (FD) is a rare, X-linked disorder caused by mutations in the GLA gene encoding the enzyme α-galactosidase A. Complete or partial deficiency in this enzyme leads to intracellular accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids in many cell types throughout the body, including the kidney. Progressive accumulation of Gb3 in podocytes, endothelial cells, epithelial cells, and tubular cells contribute to the renal symptoms of FD, which manifest as proteinuria and reduced glomerular filtration rate leading to renal insufficiency. A correct diagnosis of FD, although challenging, has considerable implications regarding treatment, management, and counseling. The diagnosis may be confirmed by demonstrating the enzyme deficiency in males and by identifying the specific GLA gene mutation in male and female patients. Treatment with enzyme replacement therapy, as part of the therapeutic strategy to prevent complications of the disease, may be beneficial in stabilizing renal function or slowing its decline, particularly in the early stages of the disease. Emergent treatments for FD include the recently approved chaperone molecule migalastat for patients with amenable mutations. The objective of this report is to provide an updated overview on Fabry nephropathy, with a focus on the most relevant aspects of its epidemiology, diagnosis, pathophysiology, and treatment options.


Assuntos
Doença de Fabry/diagnóstico , Nefropatias/diagnóstico , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/uso terapêutico , Terapia de Reposição de Enzimas , Doença de Fabry/tratamento farmacológico , Doença de Fabry/patologia , Doença de Fabry/fisiopatologia , Feminino , Galactosidases/genética , Humanos , Nefropatias/patologia , Masculino , Triexosilceramidas
15.
PLoS One ; 13(3): e0193749, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29538431

RESUMO

OBJECTIVE: Endothelial dysfunction is central to the pathogenesis of many rheumatic diseases, typified by vascular inflammation and damage. Immunosuppressive drugs induce disease remission and lead to improved patient survival. However, there remains a higher incidence of cardiovascular disease in these patients even after adequate disease control. The purpose of this study was to determine the effect of mycophenolic acid (MPA), a commonly used immunosuppressive drug in rheumatology, on blood vessel or circulating endothelial colony forming cell number and function. METHODS: We tested whether mycophenolic acid exerts an inhibitory effect on proliferation, clonogenic potential and vasculogenic function of endothelial colony forming cell. We also studied potential mechanisms involved in the observed effects. RESULTS: Treatment with MPA decreased endothelial colony forming cell proliferation, clonogenic potential and vasculogenic function in a dose-dependent fashion. MPA increased senescence-associated ß-galactosidase expression, p21 gene expression and p53 phosphorylation, indicative of activation of cellular senescence. Exogenous guanosine supplementation rescued diminished endothelial colony forming cell proliferation and indices of senescence, consistent with the known mechanism of action of MPA. CONCLUSION: Our findings show that clinically relevant doses of MPA have potent anti-angiogenic and pro-senescent effects on vascular precursor cells in vitro, thus indicating that treatment with MPA can potentially affect vascular repair and regeneration. This warrants further studies in vivo to determine how MPA therapy contributes to vascular dysfunction and increased cardiovascular disease seen in patients with inflammatory rheumatic disease.


Assuntos
Senescência Celular/efeitos dos fármacos , Ácido Micofenólico/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Galactosidases/metabolismo , Guanosina/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Cordão Umbilical/citologia
16.
Am J Reprod Immunol ; 79(6): e12826, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29446169

RESUMO

PROBLEM: We investigated the effect of oxygen concentrations on cellular senescence and autophagy and examined the role of autophagy in human trophoblast cells. METHOD OF STUDY: Human first-trimester trophoblast cells (Sw.71) were incubated under 21%, 5%, or 1% O2 concentrations for 24 hours. We examined the extent of senescence caused using senescence-associated ß-galactosidase (SA-ß-Gal) and senescence-associated secretory phenotype (SASP) as markers. Moreover, we examined the role of autophagy in causing cellular senescence using an autophagy inhibitor (3-methyladenine, 3MA). RESULTS: Physiological normoxia (5% O2 ) decreased SA-ß-Gal-positive cells and SASP including interleukin-6 (IL-6) and IL-8 compared with cultured cells in 21% O2 . Pathophysiological hypoxia (1% O2 ) caused cytotoxicity, including extracellular release of ATP and lactate dehydrogenase, and decreased senescence phenotypes. 3MA-treated trophoblast cells significantly suppressed senescence markers (SA-ß-Gal-positive cells and SASP secretion) in O2 -independent manner. CONCLUSION: We conclude that O2 concentration modulates cellular senescence phenotypes regulating autophagy in the human trophoblast cells. Moreover, inhibiting autophagy suppresses cellular senescence, suggesting that autophagy contributes to oxygen stress-induced cellular senescence.


Assuntos
Autofagia/fisiologia , Senescência Celular/fisiologia , Oxigênio/metabolismo , Trofoblastos/metabolismo , Trofoblastos/fisiologia , Trifosfato de Adenosina/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Feminino , Galactosidases/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , L-Lactato Desidrogenase/metabolismo , Gravidez , Primeiro Trimestre da Gravidez/metabolismo , Primeiro Trimestre da Gravidez/fisiologia
17.
Appl Biochem Biotechnol ; 185(4): 1060-1074, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29417378

RESUMO

Lactobacilli have several attributes that provide health benefits to the host. The aim of this study was to screen indigenous lactobacilli from human gut and fermented foods for such attributes as production of ß- and α-galactosidase and also their ability to reduce serum cholesterol. Lactobacilli were cultured on MRS broth and ß-galactosidase activity was determined using o-nitrophenyl-ß-D-galactopyranoside (ONPG) as a substrate. Three isolates Lactobacillus fermentum GPI-3 and L. fermentum GPI-6 and Lactobacillus salivarius GPI-1(S) showed better ß-galactosidase activity than the standard strains Lactobacillus rhamnosus GG (LGG) and Lactobacillus plantarum ATCC 8014. The isolates showed variability in assimilating cholesterol during growth. Several isolates showed excellent cholesterol-lowering ability compared to standard strains LGG and L. plantarum ATCC 8014. Isolate L. rhamnosus SCB being the highest acid producer (pH 4.38) also showed the highest cholesterol reduction compared to other strains including standard strains. The ability of these isolates to produce α-galactosidase was also studied and the maximum α-galactosidase activity was found in isolate L. salivarius GPI-1(S) followed by L. fermentum FA-5 and Lactobacillus helveticus FA-7. This study therefore reports Lactobacillus isolates that have superior probiotic properties when compared to the standard strains; hence, they could be considered as potential probiotic strains, which can provide health benefits to the Indian population.


Assuntos
Proteínas de Bactérias/metabolismo , Galactosidases/metabolismo , Lactobacillus/enzimologia , Probióticos , Concentração de Íons de Hidrogênio , Lactobacillus/isolamento & purificação
18.
J Bacteriol ; 200(5)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29229702

RESUMO

Bacillus anthracis, the causative agent of anthrax disease, elaborates a secondary cell wall polysaccharide (SCWP) that is essential for bacterial growth and cell division. B. anthracis SCWP is comprised of trisaccharide repeats with the structure, [→4)-ß-ManNAc-(1→4)-ß-GlcNAc(O3-α-Gal)-(1→6)-α-GlcNAc(O3-α-Gal, O4-ß-Gal)-(1→]6-12 The genes whose products promote the galactosylation of B. anthracis SCWP are not yet known. We show here that the expression of galE1, encoding a UDP-glucose 4-epimerase necessary for the synthesis of UDP-galactose, is required for B. anthracis SCWP galactosylation. The galE1 mutant assembles surface (S) layer and S layer-associated proteins that associate with ketal-pyruvylated SCWP via their S layer homology domains similarly to wild-type B. anthracis, but the mutant displays a defect in γ-phage murein hydrolase binding to SCWP. Furthermore, deletion of galE1 diminishes the capsulation of B. anthracis with poly-d-γ-glutamic acid (PDGA) and causes a reduction in bacterial virulence. These data suggest that SCWP galactosylation is required for the physiologic assembly of the B. anthracis cell wall envelope and for the pathogenesis of anthrax disease.IMPORTANCE Unlike virulent Bacillus anthracis isolates, B. anthracis strain CDC684 synthesizes secondary cell wall polysaccharide (SCWP) trisaccharide repeats without galactosyl modification, exhibits diminished growth in vitro in broth cultures, and is severely attenuated in an animal model of anthrax. To examine whether SCWP galactosylation is a requirement for anthrax disease, we generated variants of B. anthracis strains Sterne 34F2 and Ames lacking UDP-glucose 4-epimerase by mutating the genes galE1 and galE2 We identified galE1 as necessary for SCWP galactosylation. Deletion of galE1 decreased the poly-d-γ-glutamic acid (PDGA) capsulation of the vegetative form of B. anthracis and increased the bacterial inoculum required to produce lethal disease in mice, indicating that SCWP galactosylation is indeed a determinant of anthrax disease.


Assuntos
Antraz/microbiologia , Bacillus anthracis/metabolismo , Bacillus anthracis/patogenicidade , Proteínas de Bactérias/genética , Galactose/metabolismo , Polissacarídeos Bacterianos/metabolismo , Animais , Bacillus anthracis/genética , Bacillus anthracis/crescimento & desenvolvimento , Proteínas de Bactérias/metabolismo , Divisão Celular , Parede Celular/química , Parede Celular/genética , Parede Celular/fisiologia , Feminino , Galactose/genética , Galactosidases/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Trissacarídeos/química , Trissacarídeos/metabolismo , UDPglucose 4-Epimerase/genética , Uridina Difosfato Galactose/biossíntese , Uridina Difosfato Galactose/metabolismo
19.
Physiol Plant ; 163(2): 259-266, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29286539

RESUMO

To understand the action mechanism of yieldin (YLD) on the regulation of the yield threshold (Y), one of the critical parameters of cell wall extension, YLD was extracted from the cell walls of cowpea (Vigna unguiculata L.) hypocotyls and the hemagglutinin activity (HA) as well as the glycosidase activity of the protein was measured. Sedimentation assays using trypsinated rabbit erythrocytes showed that YLD possessed HA at pH 7. The digestion assays using 4-nitrophenyl (pNP) glycopyranosides as artificial substrates showed that YLD liberated galactose residues from pNP alpha-d-galactopyranoside mainly at pH 4.0, i.e. the pH level where Y was decreased at most. These results show that YLD is a bifunctional protein that switches between the HA and the galactosidase activities depending on the surrounding pH. Since D-galactose at concentration of 0.03 g l-1 perfectly inhibited the HA, YLD was suggested to associate with galactose residues. However, the galactose application ten times concentrated was necessary to inhibit both the galactosidase activity of YLD and the acid-induced shift of Y regulated by YLD. In addition, the specific inhibitor of alpha-d-galactosidase (deoxygalactonojirimycin) inhibited both the galactosidase activity of YLD and the shift of Y at the same concentration, but not the HA. On the basis of these results, it is suggested the galactosidase activity of YLD plays a central role in the mechanism of Y-regulation at acidic pH.


Assuntos
Parede Celular/metabolismo , Hipocótilo/enzimologia , Vigna/enzimologia , Galactose/metabolismo , Galactosidases/genética , Galactosidases/metabolismo , Hipocótilo/fisiologia , Vigna/fisiologia
20.
Nat Commun ; 8(1): 1685, 2017 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-29162826

RESUMO

Macroalgae contribute substantially to primary production in coastal ecosystems. Their biomass, mainly consisting of polysaccharides, is cycled into the environment by marine heterotrophic bacteria using largely uncharacterized mechanisms. Here we describe the complete catabolic pathway for carrageenans, major cell wall polysaccharides of red macroalgae, in the marine heterotrophic bacterium Zobellia galactanivorans. Carrageenan catabolism relies on a multifaceted carrageenan-induced regulon, including a non-canonical polysaccharide utilization locus (PUL) and genes distal to the PUL, including a susCD-like pair. The carrageenan utilization system is well conserved in marine Bacteroidetes but modified in other phyla of marine heterotrophic bacteria. The core system is completed by additional functions that might be assumed by non-orthologous genes in different species. This complex genetic structure may be the result of multiple evolutionary events including gene duplications and horizontal gene transfers. These results allow for an extension on the definition of bacterial PUL-mediated polysaccharide digestion.


Assuntos
Carragenina/metabolismo , Flavobacteriaceae/genética , Flavobacteriaceae/metabolismo , Regulon , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroidetes/genética , Bacteroidetes/metabolismo , Cristalografia por Raios X , Evolução Molecular , Galactosidases/química , Galactosidases/genética , Galactosidases/metabolismo , Genes Bacterianos , Redes e Vias Metabólicas/genética , Modelos Moleculares , Família Multigênica , Filogenia , Conformação Proteica , RNA Bacteriano/genética , Análise de Sequência de RNA , Especificidade da Espécie
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